The present application is a continuation of U.S. application Ser. No. 14/943,612, filed Nov. 17, 2015, which is a continuation of U.S. application Ser. No. 14/282,769, filed May 20, 2014, which is a continuation of U.S. application Ser. No. 13/626,196, filed Sep. 25, 2012, which is a continuation of U.S. application Ser. No. 12/721,413, filed Mar. 10, 2010, which is a continuation of U.S. application Ser. No. 10/548,611, filed Aug. 22, 2006, which is the National Stage of International Application No. PCT/US2004/007070, filed Mar. 8, 2004, which claims the benefit of Application No. 60/452,682, filed Mar. 7, 2003; Application No. 60/462,894, filed Apr. 14, 2003; and Application No. 60/465,665, filed Apr. 25, 2003; Application No. 60/463,772, filed Apr. 17, 2003; Application No. 60/465,802, filed Apr. 25, 2003; Application No. 60/493,986, filed Aug. 8, 2003; Application No. 60/494,597, filed Aug. 11, 2003; Application No. 60/506,341, filed Sep. 26, 2003; Application No. 60/518,453, filed Nov. 7, 2003; Application No. 60/454,265, filed Mar. 12, 2003; Application No. 60/454,962, filed Mar. 13, 2003; Application No. 60/455,050, filed Mar. 13, 2003; Application No. 60/469,612, filed May 9, 2003; Application No. 60/510,246, filed Oct. 9, 2003; Application No. 60/510,318, filed Oct. 10, 2003. The contents of the above applications are hereby incorporated by reference in their entirety.

›TECHNICAL FIELD

The invention relates to RNAi and related methods, e.g., methods of making and using iRNA agents.

›BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al . (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.

›SUMMARY · 1 of 19

A number of advances related to the application of RNAi to the treatment of subjects are disclosed herein. For example, the invention features iRNA agents targeted to specific genes; palindromic iRNA agents; iRNA agents having non canonical monomer pairings; iRNA agents having particular structures or architectures e.g., the Z-X-Y or asymmetrical iRNA agents described herein; drug delivery conjugates for the delivery of iRNA agents; amphipathic substances for the delivery of iRNA agents, as well as iRNA agents having chemical modifications for optimizing a property of the iRNA agent. The invention features each of these advances broadly as well as in combinations. For example, an iRNA agent targeted to a specific gene can also include one or more of a palindrome, non canonical, Z-X-Y, or asymmetric structure. Other nonlimiting examples of combinations include an asymmetric structure combined with a chemical modification, or formulations or methods or routes of delivery combined with, e.g., chemical modifications or architectures described herein. The iRNA agents of the invention can include any one of these advances, or pairwise or higher order combinations of the separate advances.

In one aspect, the invention features iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents.

In one embodiment, the first and second sequences of the iRNA agent are on different RNA strands, and the mismatch between the first and second sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

In another embodiment, the first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent is in bimolecular form.

In another embodiment, the first and second sequences of the iRNA agent are fully complementary to each other.

In one embodiment, the first target RNA region is encoded by a first gene and the second target RNA region is encoded by a second gene, and in another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene. In another embodiment, the first and second sequences differ by at least 1 and no more than 6 nucleotides.

In certain embodiments, the first and second target RNA regions are on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example.

In certain embodiments, the first target RNA region includes a nucleotide substitution, insertion, or deletion relative to the second target RNA region.

In other embodiments, the second target RNA region is a mutant or variant of the first target RNA region.

In certain embodiments, the first and second target RNA regions comprise viral, e.g., HCV, or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In one embodiment, the oncogene, or tumor suppressor gene is expressed in the liver. In addition, the first and second target RNA regions correspond to hot-spots for genetic variation.

In another aspect, the invention features a mixture of varied iRNA agent molecules, including one iRNA agent that includes a first sequence and a second sequence sufficiently complementary to each other to hybridize, and where the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture also includes at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence is sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. In one embodiment, at least one, two, three or all four of the target RNA regions are expressed in the liver. Exemplary RNAs are transcribed from the apoB-100 gene, glucose-6-phosphatase gene, beta catenin gene, or an HCV gene.

In certain embodiments, the first and second sequences are on the same or different RNA strands, and the third and fourth sequences are on same or different RNA strands.

In one embodiment, the mixture further includes a third iRNA agent that is composed of the first or second sequence and the third or fourth sequence.

In one embodiment, the first sequence is identical to at least one of the second, third and fourth sequences, and in another embodiment, the first region differs by at least 1 but no more than 6 nucleotides from at least one of the second, third and fourth regions.

In certain embodiments, the first target RNA region comprises a nucleotide substitution, insertion, or deletion relative to the second, third or fourth target RNA region.

The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. In one embodiment, the oncogene or tumor suppressor gene is expressed in the liver.

In certain embodiments, at least two of the target RNA regions correspond to hot-spots for genetic variation.

In one embodiment, the iRNA agents of the invention are formulated for pharmaceutical use. In one aspect, the invention provides a container (e.g., a vial, syringe, nebulizer, etc) to hold the iRNA agents described herein.

Another aspect of the invention features a method of making an iRNA agent. The method includes constructing an iRNA agent that has a first sequence complementary to a first target RNA region, and a second sequence complementary to a second target RNA region. The first and second target RNA regions have been identified as being sufficiently complementary to each other to be capable of hybridizing. In one embodiment, the first and second target RNA regions are on transcripts expressed in the liver.

›SUMMARY · 2 of 19

In certain embodiments, the first and second target RNA regions can correspond to two different regions encoded by one gene, or to regions encoded by two different genes.

Another aspect of the invention features a method of making an iRNA agent composition. The method includes obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region is obtained or provided, where each RNA target corresponds to a different variant or mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent is constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S). The first and second sequences are sufficiently complementary to hybridize. In certain embodiments, the target gene can be a viral or human gene expressed in the liver.

In one embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of apoB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In another embodiment, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a target gene, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region.

In one embodiment, the iRNA agent is administered to a subject, e.g., a human.

In another embodiment, the first and second sequences are between 15 and 30 nucleotides in length.

In one embodiment, the method of modulating expression of the target gene further includes providing a second iRNA agent that has a third sequence complementary to a third target RNA region. The third sequence can be sufficiently complementary to the first or second sequence to be capable of hybridizing to either the first or second sequence.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a plurality of target RNAs, each of the plurality of target RNAs corresponding to a different target gene. The method includes providing an iRNA agent selected by identifying a first region in a first target RNA of the plurality and a second region in a second target RNA of the plurality, where the first and second regions are sufficiently complementary to each other to be capable of hybridizing.

In another aspect of the invention, an iRNA agent molecule includes a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region, and the first and second variant RNA target regions correspond to first and second variants or mutants of a target gene. In certain embodiments, the target gene is an apoB-100, beta catenin, or glucose-6 phosphatase gene.

In one embodiment, the target gene is a viral gene (e.g., an HCV gene), tumor suppressor or oncogene.

In another embodiment, the first and second variant target RNA regions include allelic variants of the target gene.

In another embodiment, the first and second variant RNA target regions comprise mutations (e.g., point mutations) or polymorphisms of the target gene.

In one embodiment, the first and second variant RNA target regions correspond to hot-spots for genetic variation.

Another aspect of the invention features a plurality (e.g., a panel or bank) of iRNA agents. Each of the iRNA agents of the plurality includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, where the first and second variant target RNA regions correspond to first and second variants of a target gene. In certain embodiments, the variants are allelic variants of the target gene.

Another aspect of the invention provides a method of identifying an iRNA agent for treating a subject. The method includes providing or obtaining information, e.g., a genotype, about a target gene, providing or obtaining information about a plurality (e.g., panel or bank) of iRNA agents, comparing the information about the target gene to information about the plurality of iRNA agents, and selecting one or more of the plurality of iRNA agents for treating the subject. Each of the plurality of iRNA agents includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, and the first and second variant target RNA regions correspond to first and second variants of the target gene. The target gene can be an endogenous gene of the subject or a viral gene. The information about the plurality of iRNA agents can be the sequence of the first or second sequence of one or more of the plurality.

In certain embodiments, at least one of the selected iRNA agents includes a sequence capable of hybridizing to an RNA region corresponding to the target gene, and at least one of the selected iRNA agents comprises a sequence capable of hybridizing to an RNA region corresponding to a variant or mutant of the target gene.

›SUMMARY · 3 of 19

In one aspect, the invention relates to compositions and methods for silencing genes expressed in the liver, e.g., to treat disorders of or related to the liver. An iRNA agent composition of the invention can be one which has been modified to alter distribution in favor of the liver.

In another aspect, the invention relates to iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents. In one embodiment, the RNA is from a gene that is active in the liver, e.g., apoB-100, glucose-6-phosphatase, beta-catenin, or Hepatitis C virus (HCV).

In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. For example, the first sequence can be complementary to a first target apoB-100 RNA region and the second sequence can be complementary to a second target apoB-100 RNA region.

In one embodiment, the first target RNA region is encoded by a first gene, e.g., a gene expressed in the liver, and the second target RNA region is encoded by a second gene, e.g., a second gene expressed in the liver. In another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene, e.g., a single gene that is at least expressed in the liver. In another embodiment, the first and second sequences differ by at least one and no more than six nucleotides.

In another embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of aopB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In particular, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

In another aspect, the invention features a method for reducing apoB-100 levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets apoB-100. The iRNA agent can be one described here, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the apoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Tables 5 and 6. In a preferred embodiment it targets both sequences of a palindromic pair provided in Tables 5 and 6. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Tables 5 and 6.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Tables 5 and 6. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Tables 5 and 6 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Tables 5 and 6 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The iRNA agent that targets apoB-100 can be administered in an amount sufficient to reduce expression of apoB-100 mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%). Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA or protein. This can be effected, e.g., by selection of an iRNA agent which specifically targets the nucleotides subject to RNA editing in the apoB-100 transcript.

The iRNA agent that targets apoB-100 can be administered to a subject, wherein the subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of lipid metabolism. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder. These disorders include HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia; hypercholestorolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis. In one embodiment, the iRNA that targets apoB-100 is administered to a subject suffering from statin-resistant hypercholesterolemia.

The apoB-100 iRNA agent can be administered in an amount sufficient to reduce levels of serum LDL-C and/or HDL-C and/or total cholesterol in a subject. For example, the iRNA is administered in an amount sufficient to decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in the subject. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce the risk of myocardial infarction the subject.

›SUMMARY · 4 of 19

In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and apoB expression level are decreased in the liver following administration of the apoB iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to a lipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, or cholesteryl residue (see Table 1). Other lipophilic moieties that can be associated, e.g., conjugated with the iRNA agent include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric carrier complex with sugar residues.

In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a liposome complexed with sugar residues. A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver (see Table 1). In a preferred embodiment, the targeting moiety includes more than one galactose moiety, preferably two or three. Preferably, the targeting moiety includes 3 galactose moieties, e.g., spaced about 15 angstroms from each other. The targeting moiety can be lactose. A lactose is a glucose coupled to a galactose. Preferably, the targeting moiety includes three lactoses.

The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety can be used for macrophage targeting. The targeting agent can be linked directly, e.g., covalently or non covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to a lipophilic molecule to conjugate to an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In one embodiment, the iRNA agent has been modified, or is associated with a delivery agent, e.g., a delivery agent described herein, e.g., a liposome, which has been modified to alter distribution in favor of the liver. In one embodiment, the modification mediates association with a serum albumin (SA), e.g., a human serum albumin (HSA), or a fragment thereof.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to an SA molecule, e.g., an HSA molecule, or a fragment thereof. In one embodiment, the iRNA agent or composition thereof has an affinity for an SA, e.g., HSA, which is sufficiently high such that its levels in the liver are at least 10, 20, 30, 50, or 100% greater in the presence of SA, e.g., HSA, or is such that addition of exogenous SA will increase delivery to the liver. These criteria can be measured, e.g., by testing distribution in a mouse in the presence or absence of exogenous mouse or human SA.

The SA, e.g., HSA, targeting agent can be linked directly, e.g., covalently or non-covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to an SA, e.g., an HSA, molecule wherein the iRNA agent is an apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In another aspect, the invention features, a method for reducing glucose-6-phosphatase levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets glucose-6-phosphatase. The iRNA agent can be a dsRNA that has a sequence that is substantially identical to a sequence of the glucose-6-phosphatase gene.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 7. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 7. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 7.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 7. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 7 as a duplex region.

›SUMMARY · 5 of 19

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 7 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

Table 7 refers to sequences from human glucose-6-phosphatase. Table 8 refers to sequences from rat glucose-6-phosphatase. The sequences from table 8 can be used, e.g., in experiments with rats or cultured rat cells.

In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

The iRNA that targets glucose-6-phosphatase can be administered in an amount sufficient to reduce expression of glucose-6-phosphatase mRNA.

The iRNA that targets glucose-6-phosphatase can be administered to a subject to inhibit hepatic glucose production, for the treatment of glucose-metabolism-related disorders, such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder.

In other embodiments, iRNA agents having sequence similarity to the following genes can also be used to inhibit hepatic glucose production. These other genes include “forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphate locator; glucokinase inhibitory regulatory protein; and phosphoenolpyruvate carboxykinase (PEPCK).

In one embodiment, the iRNA agent can be targeted to the liver, and RNA expression levels of the targeted genes are decreased in the liver following administration of the iRNA agent.

The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of a target gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length

In another aspect, the invention features a method for reducing beta-catenin levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent that targets beta-catenin. The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the beta-catenin gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 9. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 9as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 9 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

The iRNA agent that targets beta-catenin can be administered in an amount sufficient to reduce expression of beta-catenin mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).

The iRNA agent that targets beta-catenin can be administered to a subject, wherein the subject is suffering from a disorder characterized by unwanted cellular proliferation in the liver or of liver tissue, e.g., metastatic tissue originating from the liver. Examples include , a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma.

›SUMMARY · 6 of 19

The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder

In one embodiment, the iRNA agent can be targeted to the liver, and beta-catenin expression level are decreased in the liver following administration of the beta-catenin iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

In another aspect, the invention provides methods to treat liver disorders, e.g., disorders characterized by unwanted cell proliferation, hematological disorders, disorders characterized by inflammation disorders, and metabolic or viral diseases or disorders of the liver. A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g, a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver can include dyslipidemias, and irregularities in glucose regulation. Viral diseases of the liver can include hepatitis C or hepatitis B. In one embodiment, a liver disorder is treated by administering one or more iRNA agents that have a sequence that is substantially identical to a sequence in a gene involved in the liver disorder.

In one embodiment an iRNA agent to treat a liver disorder has a sequence which is substantially identical to a sequence of the beta-catenin or c-jun gene. In another embodiment, such as for the treatment of hepatitis C or hepatitis B, the iRNA agent can have a sequence that is substantially identical to a sequence of a gene of the hepatitis C virus or the hepatitis B virus, respectively. For example, the iRNA agent can target the 5′ core region of HCV. This region lies just downstream of the ribosomal toe-print straddling the initiator methionine. Alternatively, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 10. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 10. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 10.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 10. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 10 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 10, but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In another aspect, an iRNA agent can be administered to modulate blood clotting, e.g., to reduce the tendency to form a blood clot. In a preferred embodiment the iRNA agent targets Factor V expression, preferably in the liver. One or more iRNA agents can be used to target a wild type allele, a mutant allele, e.g., the Leiden Factor V allele, or both. Such administration can be used to treat or prevent venous thrombosis, e.g., deep vein thrombosis or pulmonary embolism, or another disorder caused by elevated or otherwise unwanted expression of Factor V, in, e.g., the liver. In one embodiment the iRNA agent can treat a subject, e.g., a human who has Factor V Leiden or other genetic trait associated with an unwanted tendency to form blood clots.

In a preferred embodiment administration of an iRNA agent which targets Factor V is with the administration of a second treatment, e.g, a treatment which reduces the tendency of the blood to clot, e.g., the administration of heparin or of a low molecular weight heparin.

In one embodiment, the iRNA agent that targets Factor V can be used as a prophylaxis in patients, e.g., patients with Factor V Leiden, who are placed at risk for a thrombosis, e.g., those about to undergo surgery, in particular those about to undergo high-risk surgical procedures known to be associated with formation of venous thrombosis, those about to undergo a prolonged period of relative inactivity, e.g., on a motor vehicle, train or airplane flight, e.g., a flight or other trip lasting more than three or five hours. Such a treatment can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.

›SUMMARY · 7 of 19

In another embodiment, the iRNA agent that targets Factor V can be administered to patients with Factor V Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE). Such a treatment can be an adjunct to (or can replace) therapeutic uses of heparin or coumadin. The treatment can be administered by inhalation or generally by pulmonary routes.

In a preferred embodiment, an iRNA agent administered to treat a liver disorder is targeted to the liver. For example, the iRNA agent can be complexed with a targeting moiety, e.g., an antibody or ligand that recognizes a liver-specific receptor.

The invention also includes preparations, including substantially pure or pharmaceutically acceptable preparations of iRNA agents which silence any of the genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genes discussed herein.

The methods and compositions of the invention, e.g., the methods and compositions to treat diseases and disorders of the liver described herein, can be used with any of the iRNA agents described. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.

In another aspect, the invention features, a method of selecting two sequences or strands for use in an iRNA agent. The method includes:

providing a first candidate sequence and a second candidate sequence; determining the value of a parameter which is a function of the number of palindromic pairs between the first and second sequence, wherein a palindromic pair is a nucleotide on said first sequence which, when the sequences are aligned in anti-parallel orientation, will hybridize with a nucleotide on said second sequence; comparing the number with a predetermined reference value, and if the number has a predetermined relationship with the reference, e.g., if it is the same or greater, selecting the sequences for use in an iRNA agent. In most cases each of the two sequences will be completely complementary with a target sequence (though as described elsewhere herein that may not always be the case, there may not be perfect complementarity with one or both of the target sequences) and will have sufficient complementarity with each other to form a duplex. The parameter can be derived e.g., by directly determining the number of palindromic pairs, e.g., by inspection or by the use of a computer program which compares or analyses sequence. The parameter can also be determined less directly, and include e.g., calculation of or measurement of the Tm or other value related to the free energy of association or dissociation of a duplex.

In a preferred embodiment the determination can be performed on a target sequence, e.g., a genomic sequence. In such embodiments the selected sequence is converted to its complement in the iRNA agent.

In a preferred embodiment the first and second sequences are selected from the sequence of a single target gene. In other embodiments the first sequence is selected from the sequence of a first target gene and the second sequence is selected from the target of a second target gene.

In a preferred embodiment the method includes comparing blocks of sequence, e.g., blocks which are between 15 and 25 nucleotides in length, and preferably 19, 20, or 21, and most preferably 19 nucleotides in length, to determine if they are suitable for use, e.g., if they possess sufficient palindromic pairs.

In a preferred embodiment the first and second sequences are divided into a plurality of regions, e.g., terminal regions and a middle region disposed between the terminal regions and where in the reference value, or the predetermined relationship to the reference value, is different for at least two regions. E.g., the first and second sequences, when aligned in anti-parallel orientation, are divided into terminal regions each of a selected number of base pairs, e.g., 2, 3, 4, 5, or 6, and a middle region, and the reference value for the terminal regions is higher than for the middle regions. In other words, a higher number or proportion of palindromic pairs is required in the terminal regions.

In a preferred embodiment the first and second sequences are gene sequences thus the complements of the sequences will be used in a iRNA agent.

In a preferred embodiment hybridize means a classical Watson-Crick pairing. In other embodiments hybridize can include non-Watson-Crick paring, e.g., parings seen in micro RNA precursors.

In a preferred embodiment the method includes the addition of nucleotides to form overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined , e.g., by additional bases to form a hairpin, or by other non-base linkers.

In a preferred embodiment the method is used to select all or part of a iRNA agent. The selected sequences can be incorporated into an iRNA agent having any architecture, e.g., an architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

Preferred iRNA agents of this method will target genes expressed in the liver, e.g., one of the genes disclosed herein, e.g., apo B, Beta catenin, an HVC gene, or glucose 6 phosphatase.

In another aspect, the invention features, an iRNA agent, determined, made, or selected by a method described herein.

The methods and compositions of the invention, e.g., the methods and iRNA compositions to treat liver-based diseases described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

›SUMMARY · 8 of 19

The invention also provides for the use of an iRNA agent which includes monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand.

The use of “other than canonical Watson-Crick pairing” between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).

In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can be, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.

Thus, in one aspect, the invention features, an iRNA agent which includes: a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA duplex.

The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

›SUMMARY · 9 of 19

In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.

The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer in one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick pairing.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.

By way of example:

the monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

the monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

In another embodiment a non-naturally occurring or modified monomer or monomers are chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

›SUMMARY · 10 of 19

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 1 .

The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An example of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:

the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);

the monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;

optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.

›SUMMARY · 11 of 19

By way of example:

the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;

the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G;

the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A cis , A trans , or U; or

the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.

In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes:

a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differ in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.

In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.

An embodiment is illustrated in the FIGS. 2 and 3 .

In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained position in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a target sequence. The determinations can be compared. An iRNA agent thus analyzed can be used in the development of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or design optimized iRNA agents.

In another aspect, the invention features, a kit which includes one or more of the following an iRNA described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.

In another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.

iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.

›SUMMARY · 12 of 19

The term “other than canonical Watson-Crick pairing” as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or (3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to a sequence other than the sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

(1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self-pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal

complexes. Weizman, H ; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209)

(Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside. J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K ; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K ; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001, 11, 335-337)

Other universal bases can have the following formulas:

wherein:

Q is N or CR 44 ;

Q′ is N or CR 45 ;

Q″ is N or CR 47 ;

Q′″ is N or CR 49 ;

Q iv is N or CR 50 ;

R 44 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, or when taken together with R 45 forms —OCH 2 O—;

R 45 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, or when taken together with R 44 or R 46 forms —OCH 2 O—;

R 46 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, or when taken together with R 45 or R 47 forms —OCH 2 O—;

›SUMMARY · 13 of 19

R 47 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, or when taken together with R 46 or R 48 forms —OCH2O—;

R 48 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, or when taken together with R 47 forms —OCH2O—;

R 49 , R 50 , R 51 , R 52 , R 53 , R 54 , R 57 , R 58 , R 59 , R 60 , R 61 , R 62 , R 63 , R 64 , R 65 , R 66 , R 67 , R 68 , R 69 , R 70 , R 71 , and R 72 are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 17 , or NC(O)R o ;

R 55 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 17 , or NC(O)R o , or when taken together with R 56 forms a fused aromatic ring which may be optionally substituted;

R 56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 17 , or NC(O)R o , or when taken together with R 55 forms a fused aromatic ring which may be optionally substituted;

R 17 is halo, NH 2 , NHR b , or NR b R c ;

R b is C 1 -C 6 alkyl or a nitrogen protecting group;

R c is C 1 -C 6 alkyl; and

R o is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH 2 , NHR b , or NR b R c , C 1 -C 6 alkyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 6 -C 10 heteroaryl, C 3 -C 8 heterocyclyl, NC(O)R 17 , or NC(O)R o .

Examples of universal bases include:

In one aspect, the invention features methods of producing iRNA agents, e.g., sRNA agents, e.g. an sRNA agent described herein, having the ability to mediate RNAi. These iRNA agents can be formulated for administration to a subject.

In another aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10 16 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose. Selected modes of delivery are discussed in more detail below.

›SUMMARY · 14 of 19

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

In some embodiments the medication is delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

In one embodiment, the rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution.

In another embodiment, the rectal administration is by means of a suppository. The suppository can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

The invention also provides methods, compositions, and kits for oral delivery of iRNA agents described herein.

Oral administration can be in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. In a preferred embodiment the composition is applied topically to a surface of the oral cavity.

The invention also provides methods, compositions, and kits for buccal delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the buccal cavity. The medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

The invention also provides methods, compositions, and kits for ocular delivery of iRNA agents described herein.

The medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. It can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose.

The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

The invention also provides methods, compositions, and kits for delivery of iRNA agents described herein to or through the skin.

The medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue.

In one embodiment, the administration of the iRNA agent composition is topical. In another embodiment, topical administration delivers the composition to the dermis or epidermis of a subject. In other embodiments the topical administration is in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly.

›SUMMARY · 15 of 19

In another embodiment, the transdermal administration is applied with at least one penetration enhancer. In other embodiments, the penetration can be enhanced with iontophoresis, phonophoresis, and sonophoresis. In another aspect, the invention provides methods, compositions, devices, and kits for pulmonary delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40, preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

In another aspect, the invention provides methods, compositions, devices, and kits for nasal delivery of iRNA agents described herein. Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of nasal delivery is by spray, aerosol, liquid, e.g., by drops, of by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser which delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

In another embodiment, the iRNA agent is packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

In one aspect, of the invention, the dosage of a pharmaceutical composition including a iRNA agent is administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease.

In another aspect, gene expression in a subject is modulated by administering a pharmaceutical composition including a iRNA agent. In other embodiments, a subject is treated with the pharmaceutical composition by any of the methods mentioned above. In another embodiment, the subject has cancer.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition can be administered as a liposome. For example, the composition can be prepared by a method that includes: (1) contacting a iRNA agent with an amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-ϑ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. The iRNA agent can be an sRNA agent. The method can include preparing a composition that includes a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, a subject is treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form. In one embodiment, the powder is a collection of microparticles. In one embodiment, the powder is a collection of crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In one aspect, a subject is treated by administering a defined amount of a iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

›SUMMARY · 16 of 19

In one aspect, the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), is provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. In one embodiment, this includes providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

In another aspect, a subject is treated for a condition treatable by inhalation. In one embodiment, this method includes aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, the invention features a method of treating a subject that includes: administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

›SUMMARY · 17 of 19

In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof

In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one aspect, the invention features a method for making a pharmaceutical composition, the method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex.

In another aspect, the invention features a pharmaceutical composition produced by a method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-ϑ-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. In another embodiment, the amphipathic cationic lipid conjugate is biodegradable. In yet another embodiment the pharmaceutical composition includes a targeting ligand.

›SUMMARY · 18 of 19

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, a-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

›SUMMARY · 19 of 19

In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The materials, methods, and examples are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. Other features and advantages of the invention will be apparent from the accompanying drawings and description, and from the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. In case of conflict, the present specification, including definitions, will control.

›BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural representation of base pairing in psuedocomplementary siRNA 2 .

FIG. 2 is a schematic representation of dual targeting siRNAs (SEQ ID NOs 3952, 3953, 3954, and 3955 respectively) designed to target the HCV genome.

FIG. 3 is a schematic representation of pseudocomplementary, bifunctional siRNAs (SEQ ID NOs 3956, 3957, 3958, and 4087 respectively) designed to target the HCV genome.

FIG. 4 is a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.

FIG. 5 is a table of representative RRMS carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based

RRMSs; panel 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 is succinate or phosphoramidate and R2 is H or a conjugate ligand.

FIG. 6A is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen) following intervenous injection (iv) of buffer or siRNA into the tail vein. Each bar represents data from one mouse. RNA levels were quantified by QuantiGene Assay (Genospectra, Inc.; Fremont, Calif.)). The Y axis represents chemiluminescence values in counts per second (CPS).

FIG. 6B is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen). The values are averaged from the data depicted in FIG. 6A .

FIG. 7 is a graph depicting the pharmacokinetics of cholesterol-conjugated and unconjugated siRNA. The diamonds represent the amount of unconjugated 33 P-labeled siRNA (ALN-3000) in mouse plasma over time; the squares represent the amount of cholesterol-conjugated 33 P-labeled siRNA (ALN-3001) in mouse plasma over time. “L1163” is equivalent to ALN3000; “L1163Chol” is equivalent to ALN-3001.

›DETAILED DESCRIPTION · 1 of 24

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA agent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.

In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.

Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., once per week or once per month.

A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.

iRNA Agent Structure

Described herein are isolated iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi. The iRNA agents preferably mediate RNAi with respect to an endogenous gene of a subject or to a gene of a pathogen.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.

›DETAILED DESCRIPTION · 2 of 24

Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.

Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2- 3 nucleotides.

In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below; (2) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group; (3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an anti sense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA; (4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C 3 ′-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C 3′ -endo pucker in all, or at least 50, 75,80, 85, 90, or 95% of its sugars; exhibit a C 3′ -endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a C 3′ -endo pucker structure. These limitations are particularly preferably in the antisense strand; (5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.

›DETAILED DESCRIPTION · 3 of 24

A “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). (These modifications can also be used with the antisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents

It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5′-end. Antisense strand modifications include 5′ phosphorylation as well as any of the other 5′ modifications discussed herein, particularly the 5′ modifications discussed above in the section on single stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.

›DETAILED DESCRIPTION · 4 of 24

The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” (excluding the RRMS containing subunit(s))to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

›DETAILED DESCRIPTION · 5 of 24

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5′ end of a nucleic acid and the terminal Z position at the 3′ end of a nucleic acid, are within the term “linking phosphate oxygens” as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with “dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone (bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X═O and Y═N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3′) or position Z (5′). Replacement of W with carbon or Z with nitrogen is preferred.

›DETAILED DESCRIPTION · 6 of 24

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2′ alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH 2 CH 2 O) n CH 2 CH 2 OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH 2 ) n AMINE, (e.g., AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH 2 CH 2 OCH 3 , a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH 2 CH 2 NH)—CH 2 CH 2 -AMINE (AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C- allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.

Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called “chimeric” oligonucleotides are those that contain two or more different modifications.

The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end, 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH 2 ) n —, —(CH 2 ) n N—, —(CH 2 ) n O—, —(CH 2 ) n S—, O(CH 2 CH 2 O) n CH 2 CH 2 OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′ end can be an —OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

›DETAILED DESCRIPTION · 7 of 24

Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).

Terminal modifications useful for increasing resistance to degradation include

Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety; modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.

Alternatively, substituted or modified analogs of any of the above bases, e.g., “unusual bases” and “universal bases,” can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNA's

One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.

›DETAILED DESCRIPTION · 8 of 24

A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.

In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.

References

General References

The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can be used herein.

The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.

›DETAILED DESCRIPTION · 9 of 24

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.

Bases References

N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R 1 , R 2 , and R 3 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 5 -halouracil, 5 -(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methyl cytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R 4 , R 5 , and R 6 are each, independently, OR 8 , O(CH 2 CH 2 O) m CH 2 CH 2 OR 8 ; O(CH 2 ) n R 9 ; O(CH 2 ) n OR 9 , H; halo; NH 2 ; NHR 2 ; N(R 8 ) 2 ; NH(CH 2 CH 2 NH) m CH 2 CH 2 NHR 9 ; NHC(O)R 8 ; cyano; mercapto, SR 8 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R 4 , R 5 , or R 6 together combine with R 7 to form an [—O—CH 2 —] covalently bound bridge between the sugar 2′ and 4′ carbons.

A 1 is:

H; OH; OCH 3 ; W 1 ; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosen from 5′-monophosphate ((HO) 2 (O)P—O-5′), 5′-diphosphate ((HO) 2 (O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO) 2 (O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate (phosphorothioate; (HO) 2 (S)P—O—5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO) 2 (O)P—S-5′); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO) 2 (O)P—NH-5′, (HO)(NH 2 )(O)P—O—5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH) 2 (O)P-5′-CH 2 —), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH 2 —), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-)).

A 2 is:

A 3 is:

and

A 4 is:

H; Z 4 ; an inverted nucleotide; an abasic nucleotide; or absent.

W 1 is OH, (CH 2 ) n R 10 , (CH 2 ) n NHR 10 , (CH 2 ) n OR 10 , (CH 2 ) n SR 10 ; O(CH 2 ) n R 10 ; O(CH 2 ) n OR 10 , O(CH 2 ) n NR 10 , O(CH 2 ) n SR 10 ; O(CH 2 ) n SS(CH 2 ) n OR 10 , O(CH 2 ) n C(O)OR 10 , NH(CH 2 ) n R 10 ; NH(CH 2 ) n NR 10 ; NH(CH 2 ) n OR 10 , NH(CH 2 ) n SR 10 ; S(CH 2 ) n R 10 , S(CH 2 ) n NR 10 , S(CH 2 ) n R 10 , S(CH 2 ) n SR 10 O(CH 2 CH 2 O) m CH 2 CH 2 OR 10 ; O(CH 2 CH 2 O) m CH 2 CH 2 NHR 10 , NH(CH 2 CH 2 NH) m CH 2 CH 2 NHR 10 ; Q-R 10 , O-Q-R′° S-Q-R 10 or —O—. W 4 is O, CH 2 , NH, or S.

X 1 , X 2 , X 3 , and X 4 are each, independently, O or S.

Y 1 , Y 2 , Y 3 , and Y 4 are each, independently, OH, O − , OR 8 , S, Se, BH 3 − , H, NHR 9 , N(R 9 ) 2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

Z 1 , Z 2 , and Z 3 are each independently O, CH 2 , NH, or S. Z 4 is OH, (CH 2 ) n R 10 , (CH 2 ) n NHR 10 , (CH 2 ) n OR 10 , (CH 2 ) n SR 10 ; O(CH 2 ) n R 10 ; O(CH 2 ) n NR 10 , O(CH 2 ) n NR 10 , O(CH 2 ) n SR 10 , O(CH 2 ) n SS(CH 2 ) n OR 10 , O(CH 2 ) n C(O)OR 10 ; NH(CH 2 ) n R 10 ; NH(CH 2 ) n NR 10 ; NH(CH 2 ) n OR 10 , NH(CH 2 ) n SR 10 ; S(CH 2 ) n R 10 , S(CH 2 ) n NR 10 , S(CH 2 ) n OR 10 , S(CH 2 ) n SR 10 O(CH 2 CH 2 O) n CH 2 CH 2 OR 10 , O(CH 2 CH 2 O) n CH 2 CH 2 NHR 10 . NH(CH 2 CH 2 NH) m CH 2 CH 2 NHR 10 ; Q-R 10 ; O-Q-R 10 N-Q-R 10 , S-Q-R 10 .

›DETAILED DESCRIPTION · 10 of 24

x is 5-100, chosen to comply with a length for an RNA agent described herein.

R 7 is H; or is together combined with R 4 , R 5 , or R 6 to form an [—O—CH 2 —] covalently bound bridge between the sugar 2′ and 4′ carbons.

R 8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R 9 is NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R 10 is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

Referring to Formula 3, A 10 -A 40 is L-G-L; A 10 and/or A 40 may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH 2 (CH 2 ) g ; N(CH 2 ) g ; O(CH 2 ) g ; S(CH 2 ) g . G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethylhydrazo and methyleneoxymethylimino.

R 10 , R 20 , and R 30 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4 -acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R 40 , R 50 , and R 60 are each, independently, OR 8 , O(CH 2 CH 2 O) m CH 2 CH 2 OR 8 ; O(CH 2 ) n R 9 ; O(CH 2 ) n OR 9 , H; halo; NH 2 ; NHR 8 ; N(R 8 ) 2 ; NH(CH 2 CH 2 NH) m CH 2 CH 2 R 9 ; NHC(O)R 8 ; cyano; mercapto, SR 7 ; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R 40, R 50 , or R 60 together combine with R 70 to form an [—O—CH 2 —] covalently bound bridge between the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent described herein.

R 70 is H; or is together combined with R 40 , R 50 or R 60 to form an [—O—CH 2 —] covalently bound bridge between the sugar 2′ and 4′ carbons.

R 8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R 9 is NH 2 , alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (see Formula 4 below):

SLR 100 -(M-SLR 200 ) x -M-SLR 300 FORMULA 4

S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH 2 (CH 2 ) g ; N(CH 2 ) g ; O(CH 2 ) g ; S(CH 2 ) g ; —C(O)(CH 2 ) n — or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

›DETAILED DESCRIPTION · 11 of 24

R 100 , R 200 , and R 300 are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4 -acetyl cytosine, 2-thioicytosine, N6-methyladenine, N6-isopentyl adenine, 2-methylthio-N6-isopentenyl adenine, N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.

Nuclease Resistant Monomers

In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May 9, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a NMR and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.

An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3′ and/or 5′ end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any double-standed RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3′ end and/or the 5′ end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., S P or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr 2 , or Br 3 . When X is S, enriched or chirally pure S P linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;

›DETAILED DESCRIPTION · 12 of 24

(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5′ end of an antisense sequence should have a terminal —OH or phosphate group this NRM is preferraly not used at th 5′ end of an anti-sense sequence. The group should be attached at a position on the base which minimizes intererence with H bond formation and hybridization, e.g., away form the face which intereacts with the complementary base on the other strand, e.g, at the 5′ position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3′ CH2-NCH 3 -O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′.;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus, preferred NRM's can inlcuded these structures;

(v) L-RNA, 2′-5′ likages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages; monomers having an alpha linkage at the 1′ site on the sugar, e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5′ position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an uprotected phosphate.

One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintainted.

In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.

Chiral S P Thioates

A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SB 1 and SB 2 .

In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR 3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR 2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (herein R P ) or the “S” configuration (herein S P ). Thus if 60% of a population of stereogenic phosphorus atoms have the R P configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the S P configuration.

In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the S P configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the R P configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the S P configuration and may be substantially free of stereogenic phosphorus atoms having the R P configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the R P configuration and may be substantially free of stereogenic phosphorus atoms having the S P configuration. As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the R P configuration” means that moieties containing stereogenic phosphorus atoms having the R P configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1 H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the S P configuration” means that moieties containing stereogenic phosphorus atoms having the S P configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1 H NMR analysis using chiral shift reagents, etc.).

›DETAILED DESCRIPTION · 13 of 24

In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the S P configuration and be substantially free of stereogenic phosphorus atoms having the R P configuration.

Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate

at the 3′ end of a strand, while the latter can be used to introduce this modification at the 5′ end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R 2 can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH 3 ), DMT is dimethoxytrityl, and “BASE” can be a natural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the R P configuration (i.e., being substantially free of the Sp configuration) or only the S P configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the R P configuration and about 50% of the atoms have the S P configuration. Dimers having stereogenic phosphorus atoms with the R P configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the S P configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2′ position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH 2 ) n AMINE, (e.g., AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH 2 CH 2 NH) n CH 2 CH 2 -AMINE (AMINE=NH 2 ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphate linkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethyl hydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.

3′-bridging Thiophosphates and 5′-bridging Thiophosphates; Locked-RNA, 2′-5′ Likages, Inverted Linkages, α-nucleosides; Conjugate Groups; Abasic Linkages; and 5′-phosphonates and 5′-phosphate Prodrugs

Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.

Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2′ position of a first sugar and the 5′ position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3′ positions. Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the α-configuration. Modifcations can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g., P(O)(O − ) 2 —X—C 5′ -sugar (X═CH2, CF2, CHF and 5′-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R] 2 CH 2 C 5 ′-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular S N 2 displacement can depart as episulfide to afford the underivatized phosphate group.

Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene,as it can minimize off-target silencing.

›DETAILED DESCRIPTION · 14 of 24

In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent—it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.

In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.

An iRNA agent can have a first and a second strand chosen from the following:

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

An iRNA agent can also target two sequences and can have a first and second strand chosen from:

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand) and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

›DETAILED DESCRIPTION · 15 of 24

a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

Ribose Mimics

In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co-owned U.S. Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3′ ribose hydroxyl group, thereby causing it to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to “W” of the phosphate group. These spacers or linkers can include e.g., —(CH 2 ) n —, —(CH 2 ) n N—, —(CH 2 ) n O—, —(CH 2 ) n S—, O(CH 2 CH 2 O) n CH 2 CH 2 OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH 3 − , C 1 -C 6 alkyl, C 6 -C 10 aryl, H, O, O − , alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.

R 1 and R 3 are each, independently, hydrogen; or C 1 -C 100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R 2 is hydrogen; C 1 -C 100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 2 may be taken together with with R 4 or R 6 to form a ring of 5-12 atoms.

R 4 is hydrogen; C 1 -C 100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 4 may be taken together with with R 2 or R 5 to form a ring of 5-12 atoms.

R 5 is hydrogen, C 1 -C 100 alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R 5 may be taken together with with R 4 to form a ring of 5-12 atoms.

R 6 is hydrogen, C 1 -C 100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R 6 may be taken together with with R 2 to form a ring of 6-10 atoms;

R 7 is hydrogen, C 1 -C 100 alkyl, or C(O)(CH 2 ) q C(O)NHR 9 ; T is hydrogen or a functional group; n and q are each independently 1-100; R 8 is C 1 -C 10 alkyl or C 6 -C 10 aryl; and R 9 is hydrogen, C 1 -C 10 alkyl, C 6 -C 10 aryl or a solid support agent.

›DETAILED DESCRIPTION · 16 of 24

Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3′ or 5′ ribose sugar carbon of the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.

In still yet another subset of RNAi agent delivery modules, n is 1, and R 2 and R 6 are taken together to form a ring containing six atoms and R 4 and R 5 are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):

The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of U.S. Provisional Application Ser. No. 60/493,986, filed on Aug. 8, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/494,597, filed on Aug. 11, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/506,341, filed on Sep. 26, 2003, which is hereby incorporated by reference; and in U.S. Provisional Application Ser. No. 60/158,453, filed on Nov. 7, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an archtecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.

The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).

›DETAILED DESCRIPTION · 17 of 24

Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.

Referring to formula (R-1), X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CR 9 R 10 , or absent; and Z is CR 11 R 12 or absent.

Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , OR b , (CH 2 ) n OR a , or (CH 2 ) n OR b , provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is OR a or OR b and that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is (CH 2 ) n OR a , or (CH 2 ) n OR b (when the RRMS is terminal, one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 will include R a and one will include R b ; when the RRMS is internal, two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 will each include an R b ); further provided that preferably OR a may only be present with (CH 2 ) n OR b and (CH 2 ) n OR a may only be present with OR b .

Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 .

R 7 is C 1 -C 20 alkyl substituted with NR c R d ; R 8 is C 1 -C 6 alkyl; R 13 is hydroxy, C 1 -C 4 alkoxy, or halo; and R 14 is NR c R 7 .

R a is:

and

R b is:

Each of A and C is, independently, O or S.

B is OH, O − , or

R c is H or C1-C6 alkyl; R d is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 .

In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR 11 R 12 .

In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R 7 or NR 7 , Y is O, and Z is CR 11 R 12 .

In other preferred embodiments the ribose is replaced with a decalin scaffold, and X isCH 2 ; Y is CR 9 R 10 ; and Z is CR 11 R 12 ; and R 5 and together are C 6 cycloalkyl.

In other preferred embodiments the ribose is replaced with a decalin/indane scafold and , and X is CH 2 ; Y is CR 9 R 10 ; and Z is CR 11 R 12 ; and R 5 and R 11 together are C 5 cycloalkyl.

In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.

In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

›DETAILED DESCRIPTION · 18 of 24

An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4 . The carrier includes two “backbone attachment points” (hydroxyl groups), a “tethering attachment point,” and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the RRMS may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA molecule, e.g., an iRNA agent.

The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG. 4 ) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.

The RRMS monomers generally include two differently functionalized hydroxyl groups (OFG 1 and OFG 2 above), which are linked to the carrier molecule (see A in FIG. 4 ), and a tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFG 1 ) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG 2 ) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the strand.

The (OFG 1 ) protecting group may be selected as desired, e.g., from T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, —OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A preferred protecting group is the dimethoxytrityl group.

When the OFG 2 in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG 1 is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG 2 . Subsequent to this operation, OFG 1 can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1 ). R′ can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C 1 -C 10 alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

OFG 2 in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be —C(O)(CH 2 ) q C(O)—, or —C(O)(CH 2 ) q S—, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5′ silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-stable polystyrene based supports or PEG are preferred.

Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R 1 or R 2 ; R 3 or R 4 ; or R 9 and R 10 if Y is CR 9 R 10 (two positions are chosen to give two backbone attachment points, e.g., R 1 and R 4 , or R 4 and R 9 . Preferred tethering attachment points include R 7 ; R 5 or R 6 when X is CH 2 . The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., le or R 2 ; R 3 or R 4 ; or R 9 or R 10 (when Y is CR 9 R 10 ), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be —CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)

›DETAILED DESCRIPTION · 19 of 24

X is N(CO)R 7 , NR 7 or CH 2 ; Y is NR 8 , O, S, CR 9 R 10 ; and Z is CR 11 R 12 or absent.

Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , or (CH 2 ) n OR b , provided that at least two of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 are OR a and/or (CH 2 ) n OR b .

Each of R 5 , R 6 , R 11 , and R 12 is, independently, a ligand, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 .

R 7 is H, a ligand, or C 1 -C 20 alkyl substituted with NR c R d ; R 8 is H or C 1 -C 6 alkyl; R 13 is hydroxy, C 1 -C 4 alkoxy, or halo; R 14 is NR c R 7 ; R 15 is C 1 -C 6 alkyl optionally substituted with cyano, or C 2 -C 6 alkenyl; R 16 is C 1 -C 10 alkyl; and R 17 is a liquid or solid phase support reagent.

L is —C(O)(CH 2 ) q C(O)—, or —C(O)(CH 2 ) q S—; R a is CAr 3 ; R b is P(O)(O − )H, P(OR 15 )N(R 16 ) 2 or L-R 17 ; R c is H or C 1 -C 6 alkyl; and R d is H or a ligand.

Each Ar is, independently, C 6 -C 10 aryl optionally substituted with C 1 -C 4 alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent; or X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 , or X is N(CO)R 7 or NR 7 , Y is NR 8 , and Z is CR 11 R 12 , or X is N(CO)R 7 or NR 7 , Y is O, and Z is CR 11 R 12 , or X is CH 2 ; Y is CR 9 R 10 ; Z is CR 11 R 12 , and R 5 and R 11 together form C 6 cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH 2 ; Y is CR 9 R 10 ; Z is CR 11 R 12 , and R 5 and R 11 together form C 5 cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent (D). OFG 1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (—CH 2 OFG 1 in D). OFG 2 is preferably attached directly to one of the carbons in the five-membered ring (—OFG 2 in D). For the pyrroline-based carriers, —CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; or —CH 2 OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4. In certain embodiments, CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.For the 3-hydroxyproline-based carriers, —CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH 2 OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is CR 11 R 12 . OFG 1 is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [—(CH 2 ) n OFG 1 in E]. OFG 2 is preferably attached directly to one of the carbons in the six-membered ring (—OFG 2 in E). —(CH 2 ) n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; —(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; —(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or -(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R 7 or NR 7 , Y is N 8 , and Z is CR 11 R 12 , or the morpholine ring system (G), e.g., X is N(CO)R 7 or NR 7 , Y is O, and Z is CR 11 R 12 . OFG 1 is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (—CH 2 OFG 1 in F or G). OFG 2 is preferably attached directly to one of the carbons in the six-membered rings (—OFG 2 in F or G). For both F and G, —CH 2 OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3;

or vice versa. In certain embodiments, CH 2 OFG 1 and OFG 2 may be geminally substituted to one of the above-referenced carbons.The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH 2 OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R′″ can be, e.g., C 1 -C 6 alkyl, preferably CH 3 . The tethering attachment point is preferably nitrogen in both F and G.

›DETAILED DESCRIPTION · 20 of 24

In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH 2 ; Y is CR 9 R 10 ; Z is CR 11 R 12 , and R 5 and R 11 together form C 6 cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH 2 ; Y is CR 9 R 10 ; Z is CR 11 R 12 , and R 5 and together form C 5 cycloalkyl (H, z=1). OFG 1 is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [—(CH 2 ) n OFG 1 in H]. OFG 2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG 2 in H). —(CH 2 ) n OFG 1 and OFG 2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, —(CH 2 ) n OFG 1 and OFG 2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH 2 ) n OFG 1 may be attached to C-2 and OFG 2 may be attached to C-3; —(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-2; —(CH 2 ) n OFG 1 may be attached to C-3 and OFG 2 may be attached to C-4; or —(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-3; —(CH 2 ) n OFG 1 may be attached to C-4 and OFG 2 may be attached to C-5; or -(CH 2 ) n OFG 1 may be attached to C-5 and OFG 2 may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus, —(CH 2 ) n OFG 1 and OFG 2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

Representative carriers are shown in FIG. 5 .

In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C 1 -C 100 carbon-containing moiety, (e.g. C 1 -C 75 , C 1 -C 50 , C 1 -C 20 , C 1 -C 10 , C 1 -C 6 ), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP- (CH 2 ) n NH 2 ; TAP- C(O)(CH 2 ) n NH 2 ; or TAP- NR′″″(CH 2 ) n NH 2 , in which n is 1-6 and R′″″ is C 1 -C 6 alkyl, and R d is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., —ONH 2 , or hydrazino group, —NHNH 2 . The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP- (CH 2 ) n NH(LIGAND) , TAP- C(O)(CH 2 ) n NH(LIGAND) , or TAP- NR′″″(CH 2 ) n NH(LIGAND) ; TAP- (CH 2 ) n ONH(LIGAND) , TAP- C(O)(CH 2 ) n ONH(LIGAND) , or TAP- NR′″″(CH 2 ) n ONH(LIGAND) ; TAP- (CH 2 ) n NHNH 2 (LIGAND) , TAP- C(O)(CH 2 ) n NHNH 2 (LIGAND) , or TAP- NR′″″(CH 2 ) n NHNH 2 (LIGAND) .

In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP- (CH 2 ) n CHO ; TAP- C(O)(CH 2 ) n CHO ; or TAP- NR″″(CH 2 ) n CHO , in which n is 1-6 and R″″ is C 1 -C 6 alkyl; or TAP- (CH 2 ) n C(O)ONHS ; TAP- C(O)(CH 2 ) n ONHS ; or TAP- NR″″(CH 2 ) n C(O)ONHS , in which n is 1-6 and R″″ is C 1 -C 6 alkyl; TAP- (CH 2 ) n C(O)OC 6 F 5 ; TAP- C(O)(CH 2 ) n C(O)OC 6 F 5 ; or TAP- NR″″(CH 2 ) n C(O)OC 6 F 5 , in which n is 1-6 and R″″ is C 1 -C 6 alkyl; or —(CH 2 ) n CH 2 LG ; TAP- C(O)(CH 2 ) n CH 2 LG ; or TAP- NR″″(CH 2 ) n CH 2 LG , in which n is 1-6 and R″″ is C 1 -C 6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH 2 ) n NH 2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.

›DETAILED DESCRIPTION · 21 of 24

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.

›DETAILED DESCRIPTION · 22 of 24

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as that or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, for example).

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:6751). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6752)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:6753)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:6754)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing a v B 3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an I v ϑ 3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the I v -ϑ 3 integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

›DETAILED DESCRIPTION · 23 of 24

In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a-helical peptide. Exemplary amphipathic a-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H 2 A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, which is hereby incorporated by reference and/or in copending and coowned U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in U.S. Provisional Application Ser. No. 60/463,772, filed on Apr. 17, 2003, and/or in U.S. Provisional Application Ser. No. 60/465,802, filed on Apr. 25, 2003.

In addition, the invention includes iRNA agents having a modified or non-naturally occuring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.

The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the a-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. β-peptides are resistant to degradation by proteolytic enzymes. β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).

›DETAILED DESCRIPTION · 24 of 24

The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.

A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

Targeting

The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.

An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the a-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase; glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.

›Definitions · 1 of 36

The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C 1 -C 12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp 2 and sp 3 carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.

The term “alkoxy” refers to an -O-alkyl radical. The term “aminoalkyl” refers to an alkyl substituted with an aminoThe term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an -S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH 2 —, —CH 2 CH 2 —, and —CH 2 CH 2 CH 2 —. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by sub stituents.

The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO 3 H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O) n alkyl (where n is 0-2), S(O) n aryl (where n is 0-2), S(O) n heteroaryl (where n is 0-2), S(O) n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned sub stituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.

As used herein, an “unusual” nucleobase can include any one of the following:

2-methyladeninyl,

N6-methyladeninyl,

2-methylthio-N6-methyladeninyl,

N6-isopentenyladeninyl,

2-methylthio-N6-isopentenyladeninyl,

N6-(cis-hydroxyisopentenyl)adeninyl,

2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,

N6-glycinylcarbamoyladeninyl,

N6-threonylcarbamoyladeninyl,

2-methylthio-N6-threonyl carbamoyladeninyl,

N6-methyl-N6-threonyl carbamoyladeninyl,

›Definitions · 2 of 36

N6-hydroxynorvalylcarbamoyladeninyl,

2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,

N6,N6-dimethyladeninyl,

3-methylcytosinyl,

5-methylcytosinyl,

2-thiocytosinyl,

5-formylcytosinyl,

N4-methylcytosinyl,

5-hydroxymethylcytosinyl,

1-methylguaninyl,

N2-methylguaninyl,

7-methylguaninyl,

N2,N2-dimethylguaninyl,

N2,N2,7-trimethylguaninyl,

1-methylguaninyl,

7-cyano-7-deazaguaninyl,

7-aminomethyl-7-deazaguaninyl,

pseudouracilyl,

dihydrouracilyl,

5-methyluracilyl,

1-methylpseudouracilyl,

2-thiouracilyl,

4-thiouracilyl,

2-thiothyminyl

5-methyl-2-thiouracilyl,

3-(3-amino-3-carboxypropyl)uracilyl,

5-hydroxyuracilyl,

5-methoxyuracilyl,

uracilyl 5-oxyacetic acid,

uracilyl 5-oxyacetic acid methyl ester,

5-(carboxyhydroxymethyl)uracilyl,

5-(carboxyhydroxymethyl)uracilyl methyl ester,

5-methoxycarbonylmethyluracilyl,

5-methoxycarbonylmethyl-2-thiouracilyl,

5-aminomethyl-2-thiouracilyl,

5-methylaminomethyluracilyl,

5-methylaminomethyl-2-thiouracilyl,

5-methylaminomethyl-2-selenouracilyl,

5 -carb amoylmethyluracilyl,

5-carboxymethyl aminomethyluracilyl,

5-carboxymethyl aminomethyl-2-thiouracilyl,

3-methyluracilyl,

1-methyl-3 -(3 -amino-3 -carboxypropyl) pseudouracilyl,

5-carb oxym ethyluracilyl,

5-methyldihydrouracilyl, or

3-methylpseudouracilyl.

Asymmetrical Modifications

In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.

In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.

iRNA agents of the invention can be asymmetrically modified. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or “asymmetrical” functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand . In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2′ modification, e.g., 2′ OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5′ or 3′ end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2′OMe is present in the sense strand, and a targeting moiety is added to either the 5′ or 3′ end of the sense strand of the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.

Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2′ modification, e.g., a 2′ OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2′ OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2′-O alkyl, preferably, 2′-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2′-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3′ overhangs of about 2 subunits in length.

›Definitions · 3 of 36

Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2′, 6′, 7′, or 8′ position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.

Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.

Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids ,2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.

iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5′ end of the sense strand and the 3′ end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5′ end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5′ exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a “tight” structure at this end of the molecule.

›Definitions · 4 of 36

The end region of the iRNA agent defined by the 3′ end of the sense strand and the 5′ end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an “open” structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the 3′ end); e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5′ end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a “loose” structure in the 5′ region, additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.

The 5′-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3′ end, particularly in the 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.

The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50% activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.

›Definitions · 5 of 36

In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.

This table shows examples having strand I with a selected modification and strand II with a selected modification.

Z-X-Y Architecture

In one aspect, the invention features an iRNA agent which can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/510,246, filed on Oct. 9, 2003, which is hereby incorporated by reference, and in copending, co-owned U.S. Provisional Application Ser. No. 60/510,318, filed on Oct. 10, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a Z-X-Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z-X-Y architecture.

The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:

Z—X—Y.

It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.

The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3′ or 5′ terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P═O; P═S; S—P═S; P—NR 2 ; and P—BR 2 . Chiral P═X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3′-OR, 3′ SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alpha configuration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X′s will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2′-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2′OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2′OH subunits.

The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and infact, one will usually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2′-OH remains unsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided bewteen the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided.

In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2′-OH remains unsubstituted.

›Definitions · 6 of 36

As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.

Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2′ moieties.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2′ moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Yand /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.

An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.

›Definitions · 7 of 36

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5′ end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5′ end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5′ end of the antisense strand.

The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3′ end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5′ end.

Modifications which increase the tendency of the 5′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3′ end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5′ end of the duplex to dissociate.

Decreasing the stability of the AS 5′ end of the duplex

Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C;

G:U is preferred over G:C;

I:C is preferred over G:C (I=inosine);

mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;

pairings which include a universal base are preferred over canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R 1 N 1 N 2 N 3 N 4 N 5 [N] N −5 N −4 N −3 N −2 N −1 R 2 3′

AS 3′ R 3 N 1 N 2 N 3 N 4 N 5 [N] N −5 N −4 N −3 N −2 N −1 R 4 5′

S:AS P 1 P 2 P 3 P 4 P 5 [N] P −5 P −4 P −3 P −2 P −1 5′

S indicates the sense strand; AS indicates antisense strand; R 1 indicates an optional (and nonpreferred) 5′ sense strand overhang; R 2 indicates an optional (though preferred) 3′ sense overhang; R 3 indicates an optional (though preferred) 3′ antisense sense overhang; R 4 indicates an optional (and nonpreferred) 5′ antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P, indicates a paring of sense N x and antisense N x . Overhangs are not shown in the P diagram. In some embodiments a 3′ AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3′ S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5′ end of the AS strand. The terminal pair (the most 5′ pair in terms of the AS strand) is designated as P −1 , and the subsequent pairing positions (going in the 3′ direction in terms of the AS strand) in the duplex are designated, P −2 , P −3 , P −4 , P −5 , and so on. The preferred region in which to modify to modulate duplex formation is at P −5 through P −1 , more preferably P −4 through P −1 , more preferably P −3 through P −1 . Modification at P −1 , is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are pairs which promote disociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are G:U.

›Definitions · 8 of 36

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through R −4 , are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:

G:C is preferred over A:U

Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings

analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U is preferred over A:U

2-thio U or 5 Me-thio-U:A are preferred over U:A

G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G

guanadinium-G-clamp:G is preferred over C:G

psuedo uridine:A is preferred over U:A

sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3′ end of the AS strand. The terminal pair (the most 3′ pair in terms of the AS strand) is designated as P 1 , and the subsequent pairing positions (going in the 5′ direction in terms of the AS strand) in the duplex are designated, P 2 , P 3 , P 4 , P 5 , and so on. The preferred region in which to modify to modulate duplex formation is at P 5 through P 1 , more preferably P 4 through P 1 , more preferably P 3 through P 1 . Modification at P 1 , is particularly preferred, alone or with mdification(s) at other position(s), e.g.,any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P −1 , through P −4 , are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are a pair having an analog that increases stability over Watson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P 1 , through P 4 , are a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhances binding.

G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., “Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., “Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Raj eev, et al., “High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (”G-clamp“) and origins of their nuclease resistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al., “A cytosine analog that confers enhanced potency to antisense oligonucleotides,” Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′ end of the Duplex and Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5′ end of the duplex and increase the stability of the AS 3′ end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5′ end of the duplex with one or more of the stability increasing modifications in the AS 3′ end of the duplex. Accordingly a preferred embodiment includes modification in P −5 through P −1 , more preferably P −4 through P −1 and more preferably P −3 through P −1 . Modification at P −1 , is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5′ end of the duplex region be chosen independently from the group of:

›Definitions · 9 of 36

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and

a modification in P 5 through P 1 , more preferably P 4 through P 1 and more preferably P 3 through P 1 . Modification at P 1 , is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3′ end of the duplex region be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P −5 through P −1 and/or at least one P in P 5 through P 1 to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) which self associate to form intrastrand duplex structure.

Preferably these iRNA agents will have an architecture described herein.

Other Embodiments

In vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems , as described in U.S. Pat. No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

A composition for iRNA delivery can employ an “inclusion complex,” a molecular compound having the characteristic structure of an adduct. In this structure, the “host molecule” spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the “guest molecule”) is situated in the cavity of the host molecule without affecting the framework structure of the host. A “host” is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of “host-guest” complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.

›Definitions · 10 of 36

Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.

The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.

Delivery Module

In one aspect, the invention features a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar. 12, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent hying a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.

The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.

The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

Carrier Agents

The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.

›Definitions · 11 of 36

The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.

Fusogenic Agents

A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.

The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.

A fusogenic agent can have one or more of the following characteristics: (a) is at least 1 Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.

Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.

Condensing Agent

The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.

›Definitions · 12 of 36

A condensing agent can have the following characteristics: (a) at least 1Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.

Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.

Amphipathic Delivery Agents

In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/455,050, filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention include an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent hying a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) Protein design and folding: template trapping of self - assembled helical bundles J Pept Sci 7(3):146-51; Chen et al. (2002) Determination of stereochemistry stability coefficients of amino acid side - chains in an amphipathic alpha - helix J Pept Res 59(1):18-33; Iwata et al. (1994) Design and synthesis of amphipathic 3(10)- helical peptides and their interactions with phospholipid bilayers and ion channel formation J Biol Chem 269(7):4928-33; Cornut et al. (1994) The amphipathic alpha - helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin FEBS Lett 349(1):29-33; Negrete et al. (1998) Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha - helices. Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.

›Definitions · 13 of 36

An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

Referring to Formula M, R 1 is C 1 -C 100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C 1 -C 100 perfluoroalkyl; or OR 5 .

R 2 is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR 6 ; or C 1 -C 100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO 2 , alkenyl, or alkynyl.

R 3 is hydrogen, or when taken together with R 4 froms a fused phenyl ring.

›Definitions · 14 of 36

R 4 is hydrogen, or when taken together with R 3 froms a fused phenyl ring.

R 5 is C 1 -C 100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C 1 -C 100 perfluoroalkyl; and R 6 is C 1 -C 100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO 2 , alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug can have a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand of a double-stranded RNA molecule. The component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

dsRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribeTM RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.

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Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.

Formulation

The iRNA agents described herein can be formulated for administration to a subject

A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA composition is formulated in a manner that is compatible with the intended method of administration (see, below).

In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg 2− ), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.

In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

Exemplary formulations are discussed below:

Liposomes

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g. , by dialysis, to yield a liposomal preparation of iRNA.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes.

›Definitions · 16 of 36

Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

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Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.

Surfactants

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and other Membranous Formulations

For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C 8 to C 22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.

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In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.

Particles

For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.

Sustained -Release Formulations. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 μm rather than 10 82 m. The time of mixing also can be altered.

Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.

Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.

Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a 1/8″ microtip probe, at setting #3.

›Definitions · 19 of 36

Spray-Drying. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.

Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.

Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.

The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By “associated with or comprise” it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.

›Definitions · 20 of 36

Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C 6 -C 22 ) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, di steroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic.®. F68), poloxamer 407 (Pluronic.®. F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include “Parubisu [phonetic rendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”, manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray Drier L-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,” manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50° C. to about 200° C., preferably between about 50° C. and 100° C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0° C. and about 150° C., preferably between 0° C. and 90° C., and even more preferably between 0° C. and 60° C.

The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 μm or less with about 90% of the mass being in particles having a diameter less than 5 μm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 μm with about 80% of the mass of the particles having a diameter of less than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 μm to 100 μm. A preferred carrier material is crystalline lactose having a size in the above-stated range.

›Definitions · 21 of 36

Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components are described in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Pharm. Acta Hely 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP 806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP 4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870.

Lyophilization.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

Targeting

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen (e.g., CA15-3, CA19-9, CEA, or HER2/neu.) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).

Table 3 provides a number of antigens which can be used to target selected cells.

In one embodiment, the targeting moiety is attached to a liposome. For example, U.S. Pat. No. 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.

Genes and Diseases

In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;

administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject,

thereby treating the subject.

In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.

›Definitions · 22 of 36

In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer

In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.

In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.

In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia.

In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted INK expression, e.g., pancreatic or breast cancers.

In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In a preferred embodiment the iRNA agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.

In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.

In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.

In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.

In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.

In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.

In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers.

In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.

In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.

In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.

In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.

›Definitions · 23 of 36

In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.

In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.

In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.

In a preferred embodiment the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAF1/CIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the PPM1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1D expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).

In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.

In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.

In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.

In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.

In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.

In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.

In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.

In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TEL/AML1 fusion gene expression, e.g., childhood acute leukemia.

In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma.

In another preferred embodiment the iRNA agent silences the TLS/FUS1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.

In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:

›Definitions · 24 of 36

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin.

In a preferred embodiment the iRNA agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis.

In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.

In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection.

The method includes:

providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.

In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced.

The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).

In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCR5, Gag, or Rev.

In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.

In preferred embodiment the expression of a human gene that is required for HBV replication is reduced.

The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.

In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced.

The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis

In a preferred embodiment, the expression of a HCV gene is reduced.

In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced.

The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.

In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.

In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.

In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.

In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced.

Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.

In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.

In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced.

The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.

In a preferred embodiment, the expression of a CMV gene is reduced.

›Definitions · 25 of 36

In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced.

Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.

In a preferred embodiment, the expression of a EBV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced.

Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.

In a preferred embodiment, the expression of a KSHV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced.

The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).

In a preferred embodiment, the expression of a JCV gene is reduced.

In preferred embodiment the expression of a human gene that is required for JCV replication is reduced.

Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.

In a preferred embodiment, the expression of a myxovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.

In a preferred embodiment, the expression of a rhinovirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.

In a preferred embodiment, the expression of a coronavirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.

In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5.

In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.

In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.

In a preferred embodiment, the expression of a dengue virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.

In a preferred embodiment, the expression of a SV40 gene is reduced.

In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced.

The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.

In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV1 gene is the Tax transcriptional activator.

In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.

›Definitions · 26 of 36

In a preferred embodiment, the expression of a EMCV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced.

The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.

In a preferred embodiment, the expression of a MV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for MV replication is reduced.

The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).

In a preferred embodiment, the expression of a VZV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced.

The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.

In a preferred embodiment, the expression of an adenovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.

The invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.

In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.

In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced.

Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.

In a preferred embodiment, the expression of a poliovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox

In a preferred embodiment, the expression of a poxvirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes:

providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA is homologous to and can silence, e.g., by cleavage of a pathogen gene;

administering the iRNA agent to a subject, prefereably a human subject,

thereby treating the subject.

The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.

Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.

In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA1).

In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.

In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.

In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.

In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.

In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.

In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.

In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

›Definitions · 27 of 36

In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder.

The method includes:

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplantated organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.

In a prefered embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.

In a prefered embodiment the disease or disorder is inflammation associated with an infection or injury.

In a prefered embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.

In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1).

In particularly preferred embodiments the iRNA agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.

In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, I-309.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes:

providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain;

administering the iRNA to a subject,

thereby treating the subject.

In particularly preferred embodiments the iRNA agent silences a component of an ion channel.

In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder;

administering the to a subject,

thereby treating the subject.

In a prefered embodiment the disease or disorder is Alzheimer Disease or Parkinson Disease.

In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein.

In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCAT or SCA8.

In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCAT, SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.

E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR 2 A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.

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Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes:

optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell;

providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;

administerning the iRNA to the subject,

thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene

In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:6736) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.

The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.

Route of Delivery

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. “Topical administration” refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

›Definitions · 29 of 36

The term “skin,” as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μm and 0.2 mm thick, depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.

One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.

The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

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The term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be “respirable.” Preferably the average particle size is less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

The term “dry” means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.

The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

Oral or Nasal Delivery

In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.

›Definitions · 31 of 36

Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.

In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.

In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide.

Dosage

In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10 16 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent,. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

›Definitions · 32 of 36

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a viral disease, e.g. an HIV associated disease (e.g., AIDS), may be administered a iRNA agent specific for a target gene essential to the virus in conjunction with a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a patient being treated for cancer may be administered a iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent , or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

›Definitions · 33 of 36

The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

›Definitions · 34 of 36

The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques.

›Definitions · 35 of 36

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

›Definitions · 36 of 36

›Tables in the description — 13

TABLE 1 — Exemplary Cell Permeation Peptides Cell Permeation

Peptide	Amino acid Sequence	Reference
Penetratin	RQIKIWFQNRRMKWKK (SEQ ID NO: 6737)	Derossi et al., J. Biol.
		Chem. 269:10444,
		1994
Tat fragment	GRKKRRQRRRPPQC (SEQ ID NO: 6738)	Vives et al., J. Biol.
(48-60)		Chem., 272:16010,
		1997
Signal	GALFLGWLGAAGSTMGAWSQPKKKRKV	Chaloin et al.,
Sequence-	(SEQ ID NO: 6738)	Biochem. Biophys.
based peptide		Res. Commun.,
		243:601, 1998
PVEC	LLIILRRRIRKQAHAHSK (SEQ ID NO: 6739)	Elmquist et al., Exp.
		Cell Res., 269:237,
		2001
Transportan	GWTLNSAGYLLKINLKALAALAKKIL	Pooga et al., FASEB
	(SEQ ID NO: 6740)	J., 12:67, 1998
Amphiphilic	KLALKLALKALKAALKLA (SEQ ID	Oehlke et al., Mol.
model peptide	NO: 6741)	Ther., 2:339, 2000
Arg 9	RRRRRRRRR (SEQ ID NO: 6742)	Mitchell et al., J.
		Pept. Res., 56:318,
		2000
Bacterial cell	KFFKFFKFFK (SEQ ID NO: 6743)
wall
permeating
LL-37	LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN
	LVPRTES (SEQ ID NO: 6744)
Cecropin P1	SWLSKTAKKLENSAKKRISEGIAIAIQGGP
	R (SEQ ID NO: 6745)
α-defensin	ACYCRIPACIAGERRYGTCIYQGRLWAFC
	C (SEQ ID NO: 6746)
b-defensin	DHYNCVSSGGQCLYSACPIFTKIQGTCYR
	GKAKCCK (SEQ ID NO: 6747)
Bactenecin	RKCRIVVIRVCR (SEQ ID NO: 6748)
PR-39	RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP
	RFPPRFPGKR-NH2 (SEQ ID NO: 6749)
Indolicidin	ILPWKWPWWPWRR-NH2 (SEQ ID
	NO: 6750)

TABLE 2 — Some examples of Asymmetric Modification

Strand I	Strand II
Nuclease Resistance (e.g. 2′-OMe)	Biodistribution (e.g., P═S)
Biodistribution conjugate	Protein Binding Functionality
(e.g. Lipophile)	(e.g. Naproxen)
Tissue Distribution Functionality	Cell Targeting Functionality
(e.g. Carbohydrates)	(e.g. Folate for cancer cells)
Tissue Distribution Functionality	Fusogenic Functionality
(e.g. Liver Cell Targeting	(e.g. Polyethylene imines)
Carbohydrates)
Cancer Cell Targeting (e.g. RGD	Fusogenic Functionality
peptides and imines)	(e.g. peptides)
Nuclease Resistance (e.g. 2′-OMe)	Increase in binding Affinity
	(5-Me—C, 5-Me—U, 2-thio-U,
	2-amino-A, G-clamp, LNA)
Tissue Distribution Functionality	RISC activity improving
	Functionality
Helical conformation changing	Tissue Distribution Functionality
Functionalities	(P═S; lipophile, carbohydrates)

TABLE 3

ANTIGEN	Exemplary tumor tissue
CEA (carcinoembryonic antigen)	colon, breast, lung
PSA (prostate specific antigen)	prostate cancer
CA-125	ovarian cancer
CA 15-3	breast cancer
CA 19-9	breast cancer
HER2/neu	breast cancer
α-feto protein	testicular cancer,
	hepatic cancer
β-HCG (human chorionic	testicular cancer,
gonadotropin)	choriocarcinoma
MUC-1	breast cancer
Estrogen receptor	breast cancer, uterine cancer
Progesterone receptor	breast cancer, uterine cancer
EGFr (epidermal growth	bladder cancer
factor receptor)

TABLE 5 — iRNA molecules targeted to mouse ApoM

	iRNA targeted	iRNA targeted	iRNA targeted
	to 5′ end	to middle ORF	to middle ORF	iRNA targeted
	of ORF	(5′ proximal)	(3′ proximal)	to 3′UTS
Class	1	2	3	4
I
Class	11	13	15	17
II
Class	19	21	23	25
III

TABLE 6 — Reference Range Lipid level in the Blood

Lipid	Range (mmols/L)
Plasma Cholesterol	3.5-6.5
Low density lipoprotein	1.55-4.4
Very low density lipoprotein	0.128-0.645
High density lipoprotein/triglycerides	0.5-2.1
Total lipid	4.0-10 g/L

TABLE 8

		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 1	ggccattccagaagggaag	509	528	SEQ ID NO: 1004	cttccgttctgtaatggcc	5795	5814	1	9
SEQ ID NO: 2	tgccatctcgagagttcca	4099	4118	SEQ ID NO: 1005	tggaactctctccatggca	10876	10995	1	8
SEQ ID NO: 3	catgtcaaacactttgtta	7068	7075	SEQ ID NO: 1006	taacaaattccttgacatg	7358	7377	1	8
SEQ ID NO: 4	tttgttataaatcttattg	7068	7087	SEQ ID NO: 1007	caataagatcaatagcaaa	8990	9009	1	8
SEQ ID NO: 5	tctggaaaaagggtcatga	8880	8699	SEQ ID NO: 1008	tccatgtcccatttacaga	11358	11375	1	8
SEQ ID NO: 6	cagctcttgttcaggtcca	10900	10919	SEQ ID NO: 1009	tggacctgcaccaaagctg	13952	13971	1	8
SEQ ID NO: 7	ggaggttccccagctctgc	356	375	SEQ ID NO: 1010	gcagccctgggaaaactcc	8447	6466	1	7
SEQ ID NO: 8	ctgttttgaagactctcca	1081	1100	SEQ ID NO: 1011	tggagggtagtcataacag	10327	10346	1	7
SEQ ID NO: 9	agtggctgaaacgtgtgca	1297	1316	SEQ ID NO: 1012	tgcagagctttctgccact	13508	13527	1	7
SEQ ID NO: 10	ccaaaatagaagggaatct	2068	2087	SEQ ID NO: 1013	agattcctttctttttcaa	4000	4019	1	7
SEQ ID NO: 11	tgaagagaagattgaattt	3620	3638	SEQ ID NO: 1014	aaattctcttttattttca	9212	9231	1	7
SEQ ID NO: 12	agtggtggcaacaccagca	4230	4249	SEQ ID NO: 1015	tgctagtgaggccaacact	10649	10668	1	7
SEQ ID NO: 13	aaggctccacaagtcatca	5950	5989	SEQ ID NO: 1016	tgatgatatctggaacctt	10724	10743	1	7
SEQ ID NO: 14	gtccgccaggtttctagca	7725	7744	SEQ ID NO: 1017	tgctaagaaccttactgac	7781	7800	1	7
SEQ ID NO: 15	tgatatctggaaccttgga	10727	10745	SEQ ID NO: 1018	ttcactgttcctgaaatca	7863	7882	1	7
SEQ ID NO: 16	gtcaagttgagcaatttct	13423	13442	SEQ ID NO: 1019	agaaaaggcacaccttgac	11072	11091	1	7
SEQ ID NO: 17	atccagatggaaaagggaa	13480	13499	SEQ ID NO: 1020	ttccaatttccctgtggat	3680	3698	1	7
SEQ ID NO: 18	atttgtttgtcaaagaagt	4543	4562	SEQ ID NO: 1021	acttcagagagaaatacat	11401	11420	4	6
SEQ ID NO: 19	ctggaaaatgtcagcctgg	204	223	SEQ ID NO: 1022	ccagacttcagttaccagc	8235	8264	2	6
SEQ ID NO: 20	accaggaggttcttcttca	1729	1748	SEQ ID NO: 1023	tgaagtgtgatctcctggt	5089	5108	2	6
SEQ ID NO: 21	aaagaagttctgaaaagat	1956	1975	SEQ ID NO: 1024	attccatcacaaaatcctt	9661	9680	2	6
SEQ ID NO: 22	gctacagcttatggctcca	3570	3589	SEQ ID NO: 1025	tggatctaaatgcagtagc	11623	11642	2	6
SEQ ID NO: 23	atcaatatgatcaatttgt	6414	3433	SEQ ID NO: 1026	caaagaaatcaagattgat	4553	4572	2	6
SEQ ID NO: 24	gaattatctttaaaacatt	7326	7345	SEQ ID NO: 1027	atgtggacaaatataccgg	11494	11513	2	6
SEQ ID NO: 25	cgaggcccgcgctgctggc	130	149	SEQ ID NO: 1028	gccagaagtgagatcctcg	3507	3526	1	6
SEQ ID NO: 26	acaactatgaggctgagag	271	290	SEQ ID NO: 1029	ctctgagcaacaaattttt	10309	10326	1	6
SEQ ID NO: 27	gctgagagttccagtggag	282	301	SEQ ID NO: 1030	ctccatggcaaatgtcagg	10885	10904	1	6
SEQ ID NO: 28	tgaagaaaaaccaagaact	448	467	SEQ ID NO: 1031	gagtcattgaggttcttca	4929	494	1	6
SEQ ID NO: 29	cctacttacatcctgaaca	558	577	SEQ ID NO: 1032	tgttcataagggaggtagg	12766	12785	1	6
SEQ ID NO: 30	ctacttacatcctgaacat	559	578	SEQ ID NO: 1033	atgttcataagggaggtag	12785	12784	1	6
SEQ ID NO: 31	gagacagaagccaagagcc	615	634	SEQ ID NO: 1034	gcttggttttgcccagtct	2459	2478	1	6
SEQ ID NO: 32	cactcactttaccgtcaag	671	690	SEQ ID NO: 1035	cttgaacaccaaagtcact	6000	6019	1	6
SEQ ID NO: 33	ctgatcagcagcagccagt	822	641	SEQ ID NO: 1036	actgggaagtgcttatcag	5237	5256	1	6
SEQ ID NO: 34	actggacgctaagaggaag	854	873	SEQ ID NO: 1037	cttccccaaagagaggagt	2890	2909	1	6
SEQ ID NO: 35	agaggaagcatgtggcaga	865	884	SEQ ID NO: 1038	tctggcatttactttctct	5921	5940	1	6
SEQ ID NO: 36	tgaagactctccaggaact	1087	1106	SEQ ID NO: 1039	agttgaaggagactattca	7216	7235	1	6
SEQ ID NO: 37	ctctgagcaaaatatccag	1121	1140	SEQ ID NO: 1040	ctggttactgagctgagag	1151	1180	1	6
SEQ ID NO: 38	atgaagcagtcacatctct	1189	1208	SEQ ID NO: 1041	agagctgccagtcttcatt	10016	10035	1	6
SEQ ID NO: 39	ttgccacagctgattgagg	1209	1228	SEQ ID NO: 1042	cctcctacagtggtggcaa	4222	4241	1	6
SEQ ID NO: 40	agctgattgaggtgtccag	1216	1235	SEQ ID NO: 1043	ctggattccacatgcagct	11847	11866	1	6
SEQ ID NO: 41	tgctccactcacatcctcc	1276	1297	SEQ ID NO: 1044	ggaggctttaagttcagca	7601	7620	1	6
SEQ ID NO: 42	tgaaacgtgtgcatgtcaa	1303	1322	SEQ ID NO: 1045	ttgggagagacaagtttca	6500	6519	1	6
SEQ ID NO: 43	gacattgctaattacctga	1503	1522	SEQ ID NO: 1046	tcagaagctaagcaatgtc	7282	7251	1	6
SEQ ID NO: 44	ttcttcttcagactttcct	1735	1757	SEQ ID NO: 1047	aggagagtccaaatttaga	8498	8517	1	6
SEQ ID NO: 45	ccaatatcttgaaactcag	1903	1922	SEQ ID NO: 1048	tctgaattcattcaattgg	8485	6504	1	6
SEQ ID NO: 46	aaagttagtgaaaagaagt	1946	1965	SEQ ID NO: 1049	aactaccctcactgccttt	2132	2151	1	6
SEQ ID NO: 47	aagttagtgaaagaagttc	1947	1966	SEQ ID NO: 1050	gaacctctggcattttact	5918	5935	1	6
SEQ ID NO: 48	aaagaagttctgaaagaat	1956	1975	SEQ ID NO: 1051	attctctggtaactacttt	5482	5501	1	6
SEQ ID NO: 49	tttggctataccaaagatg	2322	2341	SEQ ID NO: 1052	catcttaggcactgacaaa	4997	5016	1	6
SEQ ID NO: 50	tgttgagaagctgattaaa	2381	2400	SEQ ID NO: 1053	tttagccatcggctcaaca	5700	5719	1	6
SEQ ID NO: 51	caggaagggctcaaagaat	2561	2580	SEQ ID NO: 1054	attcctttaacaattcctg	9492	9511	1	6
SEQ ID NO: 52	aggaagggctcaaagaatg	2562	2581	SEQ ID NO: 1055	cattcctttaacaattcct	9491	9510	1	6
SEQ ID NO: 53	gaagggctcaaagaatgac	2564	2583	SEQ ID NO: 1056	gtcagtcttcaggctcttc	7914	7933	1	6
SEQ ID NO: 54	caaagaatgacttttttct	2572	2591	SEQ ID NO: 1057	agaaggatggcattttttg	14000	14019	1	6
SEQ ID NO: 55	catggagaatgcctttgaa	2603	2622	SEQ ID NO: 1058	ttcagagccaaagtccatg	7119	7138	1	6
SEQ ID NO: 56	ggagccaaggctggagtaa	2679	2698	SEQ ID NO: 1059	ttactccaacgccagctcc	3050	3069	1	6
SEQ ID NO: 57	tcattccttccccaaagag	2884	2903	SEQ ID NO: 1060	ctctctggggcatctatgc	5139	5155	1	6
SEQ ID NO: 58	acctatgagctccagagag	3165	3184	SEQ ID NO: 1061	ctctcaagaccacagaagg	12976	12995	1	6
SEQ ID NO: 59	gggcaaaaacgtcttacag	3355	3384	SEQ ID NO: 1062	tctgaaagacaacgtgccc	12317	12336	1	6
SEQ ID NO: 60	accctggaccttcagaaca	3387	3406	SEQ ID NO: 1063	tgttgctaaggttcagggt	5675	5694	1	6
SEQ ID NO: 61	atgggcgacctaagttgtg	3429	3448	SEQ ID NO: 1064	cacaaattagtttcaccat	8941	8960	1	6
SEQ ID NO: 62	gatgaagagaagattgaat	3618	3637	SEQ ID NO: 1065	atccagcttccccacactt	8330	8349	1	6
SEQ ID NO: 63	caatgtgataccaaaaaaa	3556	3675	SEQ ID NO: 1066	tttttggaaatgccattgt	8643	8662	1	6
SEQ ID NO: 64	gtagataccaaaaaaatga	3660	3679	SEQ ID NO: 1067	tcatgtgatgggtctctac	4371	4390	1	6
SEQ ID NO: 65	gcttcagttcatttggact	4509	4528	SEQ ID NO: 1068	agtcaagaaggacttaagc	5304	5323	1	6
SEQ ID NO: 66	tttgtttgtcaaagaagtc	4544	4563	SEQ ID NO: 1069	gacttcagagaaatacaaa	11400	11419	1	6
SEQ ID NO: 67	ttgtttgtcaaagaagtca	4545	4564	SEQ ID NO: 1070	tgacttcagagaaatacaa	11399	11418	1	6
SEQ ID NO: 68	tggcaatgggaaactcgct	5846	5865	SEQ ID NO: 1071	agcgagagtcccctgccat	8219	8238	1	6
SEQ ID NO: 69	aacctctggcatttacttt	5917	5936	SEQ ID NO: 1072	aaaggagatgtcaagggtt	10599	10618	1	6
SEQ ID NO: 70	catttactttctctcatga	5926	5945	SEQ ID NO: 1073	tcatttgaaagaataaatg	7026	7045	1	6
SEQ ID NO: 71	aaagtcagtgccctgctta	6009	6028	SEQ ID NO: 1074	taagaaccttactgacttt	7784	7803	1	6
SEQ ID NO: 72	tcccattttttgagacctt	6322	6341	SEQ ID NO: 1075	aaggacttcaggaatggga	12004	12023	1	6
SEQ ID NO: 73	catcaatattgatcaattt	6413	6432	SEQ ID NO: 1076	aaattaaaaagtcttgatg	6732	6751	1	6
SEQ ID NO: 74	taaagatagttatgattta	6665	6684	SEQ ID NO: 1077	taaaccaaaaacttggtta	9019	9038	1	6
SEQ ID NO: 75	tattgatgaaatcattgaa	6713	6732	SEQ ID NO: 1078	ttcaaagacttaaaaaata	8007	8026	1	6
SEQ ID NO: 76	atgatctacatttgtttat	6790	5809	SEQ ID NO: 1079	ataaagaaattaaagtcat	7380	7399	1	6
SEQ ID NO: 77	agagacacatacagaatat	6919	6936	SEQ ID NO: 1080	atatattgtcagtgcctct	13385	13401	1	6
SEQ ID NO: 78	gacacatacagaatataga	6922	6941	SEQ ID NO: 1081	tctaaattcagttcttgtc	11327	11349	1	6
SEQ ID NO: 79	agcatgtcaaacactttgt	7054	7073	SEQ ID NO: 1082	acaaagtcagtgccctgct	6007	8026	1	6
SEQ ID NO: 80	tttttagaggaaaccaagg	7515	7534	SEQ ID NO: 1083	ccttgtgtacaccaaaaac	11230	11249	1	6
SEQ ID NO: 81	ttttagaggaaaccaaggc	7516	7535	SEQ ID NO: 1084	gcctttgtgtacaccaaaa	11229	11248	1	6
SEQ ID NO: 82	ggaagatagacttcctgaa	9307	9326	SEQ ID NO: 1085	ttcagaaatactgtttttc	12824	12843	1	6
SEQ ID NO: 83	cactgtttctgagtcccag	9334	9353	SEQ ID NO: 1086	ctgggacctaccaagagtg	12523	12542	1	6
SEQ ID NO: 84	cacaaatcctttggctgtg	9668	9687	SEQ ID NO: 1087	cacatttcaaggaattgtg	10063	10082	1	6
SEQ ID NO: 85	ttcctggatacactgttcc	9853	9872	SEQ ID NO: 1088	ggaactgttgactcaggac	12569	12588	1	6
SEQ ID NO: 86	gaaatctcaagctttctct	10042	10061	SEQ ID NO: 1089	agagccaggtcgagctttc	11044	11063	1	6
SEQ ID NO: 87	tttcttcatcttcatctgt	10210	10229	SEQ ID NO: 1090	acagctgaaagagatgaaa	13055	13074	1	6
SEQ ID NO: 88	tctaccgctaaaggagcag	10521	10540	SEQ ID NO: 1091	ctgcacgctttgaggtaga	11761	11780	1	6
SEQ ID NO: 89	ctaccgctaaaggagcagt	10522	10541	SEQ ID NO: 1092	actgcacgctttgaggtcg	11760	11779	1	6
SEQ ID NO: 90	aggggcctcttttcaccaa	10631	10650	SEQ ID NO: 1093	ttggccaggaagtggccct	10957	10976	1	6
SEQ ID NO: 91	tctccatccctgtaaaaag	11285	11284	SEQ ID NO: 1094	ctttttcaccaacggagaa	10838	10857	1	6
SEQ ID NO: 92	gaaaaacaaagcagattat	11816	11635	SEQ ID NO: 1095	ataaactgcaagatttttc	13600	13619	1	6
SEQ ID NO: 93	actcactcattgattttct	12682	12701	SEQ ID NO: 1096	agaaaatcaggatcgtagt	14027	14046	1	6
SEQ ID NO: 94	taaactaatagatgtaatc	12890	12909	SEQ ID NO: 1097	gattaccaccagcagttta	13578	13597	1	6
SEQ ID NO: 95	caaaaacgagcttcggaag	13200	13219	SEQ ID NO: 1098	cttcgtgagaatatttgaa	13260	13279	1	6
SEQ ID NO: 96	tggaataatgctcagtgtt	2366	2385	SEQ ID NO: 1099	aacacttaacttgaattca	10562	10681	3	5
SEQ ID NO: 97	gatttgaaatccaaagaag	2400	2419	SEQ ID NO: 1100	cttcagagaaatacaaatc	11402	11421	3	5
SEQ ID NO: 98	atttgaaatccaaagaagt	2401	2420	SEQ ID NO: 1101	acttcagagaaatacaaat	11401	11420	3	5
SEQ ID NO: 99	atcaacagccgcttctttg	990	1009	SEQ ID NO: 1102	caaagaagtcaagattgat	4553	4572	2	5
SEQ ID NO: 100	tgttttgaagactctccag	1082	1101	SEQ ID NO: 1103	ctggaaagttaaaacaaac	6955	6974	2	5
SEQ ID NO: 101	cccttctgatagatgtggt	1324	1343	SEQ ID NO: 1104	accaaagctggcaccaggg	13961	13980	2	5
SEQ ID NO: 102	tgagcaagtgaagaacttt	1868	1887	SEQ ID NO: 1105	aaagccattcagtctctca	12963	12982	2	5
SEQ ID NO: 103	atttgaaatccaaagaagt	2401	2420	SEQ ID NO: 1106	actttctaaacttgaaatt	9055	9074	2	5
SEQ ID NO: 104	atccaaagaagtcccggaa	2408	227	SEQ ID NO: 1107	tccggggaaacctgggatt	12721	12740	2	5
SEQ ID NO: 105	agagcctacctccgcatct	2430	2449	SEQ ID NO: 1108	agatggtacgttagcctct	11921	11940	2	5
SEQ ID NO: 106	aatgcctttgaactcccca	2610	2629	SEQ ID NO: 1109	tgggaactacaattcattt	7012	7031	2	5
SEQ ID NO: 107	gaagtccaaattccggatt	3297	3316	SEQ ID NO: 1110	aatcttcaatttattcttc	13815	13834	2	5
SEQ ID NO: 108	tgcaagcagaagccagaag	3496	3515	SEQ ID NO: 1111	cttcaggttccatcgtgca	11376	11395	2	5
SEQ ID NO: 109	gaagagaagattgaatttg	3621	3640	SEQ ID NO: 1112	caaaacctactgtgcttcc	10459	10476	2	5
SEQ ID NO: 110	atgctaaaggcacatatgg	4597	4616	SEQ ID NO: 1113	ccatatgaaagtcaagcat	12656	12675	2	5
SEQ ID NO: 111	tccctcacctccacctctg	4737	4756	SEQ ID NO: 1114	cagattctcagatgaggga	8912	8931	2	5
SEQ ID NO: 112	atttacagctctgacaagt	5427	5446	SEQ ID NO: 1115	acttttctaaacttgaaat	9055	9074	2	5
SEQ ID NO: 113	aggagcctaccaaaataat	5594	5513	SEQ ID NO: 1116	attatgttgaaaacagtct	11830	11849	2	5
SEQ ID NO: 114	aaagctgaagcacatcaat	6401	8420	SEQ ID NO: 1117	attgttgctcatctccttt	10184	10213	2	5
SEQ ID NO: 115	ctgctggaaacaacgagaa	9416	9437	SEQ ID NO: 1118	ttctgattaccaccagcag	13574	13593	2	5
SEQ ID NO: 116	ttgaaggaattcttgaaaa	9582	9601	SEQ ID NO: 1119	ttttaaaagaaatcttcaa	13805	13824	2	5
SEQ ID NO: 117	gaagtaaaagaaaattttg	10743	10762	SEQ ID NO: 1120	caaaacctactgtctcttc	10459	10476	2	5
SEQ ID NO: 118	tgaagaagatggcaaattt	11984	12003	SEQ ID NO: 1121	aaatgtcagctcttgttca	10894	10913	2	5
SEQ ID NO: 119	aggatctgagttatttttc	14035	14054	SEQ ID NO: 1122	gcaagtcagcccagttcct	10920	10939	2	5
SEQ ID NO: 120	gtgcccttctcggttgctg	18	37	SEQ ID NO: 1123	cagccattgacatgagcac	5740	5759	1	5
SEQ ID NO: 121	ggcgcggcctgcgctgctg	146	165	SEQ ID NO: 1124	cagctccacagactccgcc	3062	3061	1	5
SEQ ID NO: 122	ctgcgctgctgctgctgct	154	173	SEQ ID NO: 1125	agcagaaggtgcgaagcag	3224	3243	1	5
SEQ ID NO: 123	gctggtggcgggcgccagg	170	189	SEQ ID NO: 1126	cctggattccacatgcagc	11646	11865	1	5
SEQ ID NO: 124	aagaggaaatgctggaaaa	193	212	SEQ ID NO: 1127	tttttcttcactacatctt	2584	2603	1	5
SEQ ID NO: 125	ctggaaaatgtcagcctgg	204	223	SEQ ID NO: 1128	ccagacttccacatcccag	3915	3934	1	5
SEQ ID NO: 126	tggagtccctgggactgct	296	315	SEQ ID NO: 1129	agcatgcctagtttctcca	9945	9964	1	5
SEQ ID NO: 127	ggagtccctgggactgctg	297	316	SEQ ID NO: 1130	cagcatgcctagtttctcc	9944	9953	1	5
SEQ ID NO: 128	tgggactgctgattcaaga	305	324	SEQ ID NO: 1131	tcttccatcacttgaccca	2042	2061	1	5
SEQ ID NO: 129	ctgctgattcaagaagtgc	310	329	SEQ ID NO: 1132	gcacaccttgacattgcag	11079	11098	1	5
SEQ ID NO: 130	tgccaccaggatcaactgc	326	345	SEQ ID NO: 1133	gcaggctgaactggtggca	2717	2736	1	5
SEQ ID NO: 131	gccaccaggatcaactgca	327	346	SEQ ID NO: 1134	tgcaggctgaactggtggc	2715	2735	1	5
SEQ ID NO: 132	tgcaaggttgagcgtggag	342	361	SEQ ID NO: 1135	cctccatcctctgatctga	4744	4763	1	5
SEQ ID NO: 133	caaggttgagctggaggtt	344	363	SEQ ID NO: 1136	aacccctacatgaagcttg	13755	13774	1	5
SEQ ID NO: 134	ctctgcagcttcatcctga	369	366	SEQ ID NO: 1137	tcaggaagcttctcaagag	13211	13230	1	5
SEQ ID NO: 135	cagcttcatcctgaagaac	374	393	SEQ ID NO: 1138	ggtcttgagttaaatgctg	4977	4996	1	5
SEQ ID NO: 136	gcttcatcctgaagaccag	376	395	SEQ ID NO: 1139	ctggacgctaagaggaagc	855	874	1	5
SEQ ID NO: 137	tcatcctgaagaccagcca	379	398	SEQ ID NO: 1140	tggcatggcattatgatga	3604	3623	1	5
SEQ ID NO: 138	gaaaaccaagaactctgag	452	471	SEQ ID NO: 1141	ctcaaccttaatgattttc	8286	8305	1	5
SEQ ID NO: 139	agaactctgaggagtttgc	460	479	SEQ ID NO: 1142	gcaagctatatcagtattc	8377	8396	1	5
SEQ ID NO: 140	tctgaggagtttgctgcag	465	484	SEQ ID NO: 1143	ctgcagggatcccccagat	2528	2545	1	5
SEQ ID NO: 141	tttgctgcagccatgtcca	474	493	SEQ ID NO: 1144	tggaagtgtcagtggcaaa	10372	10391	1	5
SEQ ID NO: 142	caagaggggcatcatttct	578	597	SEQ ID NO: 1145	agaataaatgacgttcttg	7035	7054	1	5
SEQ ID NO: 143	tcactttaccgtcaagacg	674	693	SEQ ID NO: 1146	cgtctacactatcatgtga	4360	4379	1	5
SEQ ID NO: 144	tttaccgtcaagacgagga	678	697	SEQ ID NO: 1147	tccttgacatgttgataaa	7366	7335	1	5
SEQ ID NO: 145	cactggacgctaagaggaa	653	672	SEQ ID NO: 1148	ttccagaaagcagccagtg	12498	12517	1	5
SEQ ID NO: 146	aggaagcatgtggcagaag	867	886	SEQ ID NO: 1149	cttcatacacattaatcct	9988	10007	1	5
SEQ ID NO: 147	caaggagcaacacctcttc	893	912	SEQ ID NO: 1150	gaagtagtactgcatgttg	6835	6854	1	5
SEQ ID NO: 148	acagactttgaaacttgaa	959	978	SEQ ID NO: 1151	ttcaattcttcaatgctgt	10500	10519	1	5
SEQ ID NO: 149	tgatgaagcagtcacatct	1187	1206	SEQ ID NO: 1152	agatttgaggattccatca	7975	7995	1	5
SEQ ID NO: 150	agcagtcacatctctcttg	1193	1212	SEQ ID NO: 1153	caaggagaaactgactcgt	6524	6543	1	5
SEQ ID NO: 151	ccagccccatcactttaca	1231	1250	SEQ ID NO: 1154	tgtagtctcctggtgctgg	5094	5113	1	5
SEQ ID NO: 152	ctccactccatcctccagg	1280	1299	SEQ ID NO: 1155	ctggagcttagtaatggag	8709	8728	1	5
SEQ ID NO: 153	catgccaacccccttctga	1314	1333	SEQ ID NO: 1156	tcagatgagggaacacatg	8919	8936	1	5
SEQ ID NO: 154	gagagatcttcaacatggc	1390	1409	SEQ ID NO: 1157	gccaccctggaactctctc	10859	10888	1	5
SEQ ID NO: 155	tcaacatggcgagggatca	1398	1418	SEQ ID NO: 1158	tgatcccacctctcattga	2965	2984	1	5
SEQ ID NO: 156	ccaccttgtatgcgctgag	1429	1446	SEQ ID NO: 1159	ctcagggatctgaaggtgg	8187	8205	1	5
SEQ ID NO: 157	gtcaacaactatcataaga	1455	1474	SEQ ID NO: 1160	tcttgagttaaatgctgac	4979	4998	1	5
SEQ ID NO: 158	tggacattgctaattacct	1501	1520	SEQ ID NO: 1161	aggtatattcgaaagtcca	12799	12618	1	5
SEQ ID NO: 159	ggacattgctaattacctg	1502	1521	SEQ ID NO: 1162	caggtatattcgaaagtcc	12798	12817	1	5
SEQ ID NO: 160	ttctgcgggtcattggaaa	1573	1592	SEQ ID NO: 1163	tttcacatgccaaggagaa	6514	6533	1	5
SEQ ID NO: 161	ccagaactcaagtcttcaa	1520	1639	SEQ ID NO: 1164	ttgaagtgtatgtctcctg	5088	5107	1	5
SEQ ID NO: 162	agtcttcaatcctgaaatg	1630	1649	SEQ ID NO: 1165	catttctgattggtggact	7757	7776	1	5
SEQ ID NO: 163	tgagcaagtgaagaacttt	1868	1887	SEQ ID NO: 1166	aaagtgccacttttactca	6183	6202	1	5
SEQ ID NO: 164	agcaagtgaagaactttgt	1870	1889	SEQ ID NO: 1167	acaaagtcagtgccctgct	6007	6026	1	5
SEQ ID NO: 165	tctgaaagaatctcaactt	1964	1983	SEQ ID NO: 1168	aagtccataatggttcaga	12611	12630	1	5
SEQ ID NO: 166	actgtcatggacttcagaa	1986	2005	SEQ ID NO: 1169	ttctgaatatattgtcagt	13376	13395	1	5
SEQ ID NO: 167	acttgacccagcctcagcc	2051	2070	SEQ ID NO: 1170	ggctcaccctgagagaagt	12391	12410	1	5
SEQ ID NO: 168	tccaaataactaccttcct	2096	2115	SEQ ID NO: 1171	aggaagatatgaagatgga	4712	4731	1	5
SEQ ID NO: 169	actaccctcactgcctttg	2133	2152	SEQ ID NO: 1172	caaatttgtggagggtagt	10319	10338	1	5
SEQ ID NO: 170	ttggatttgcttcagctga	2149	2168	SEQ ID NO: 1173	tcagtataagtacaaccaa	9392	9411	1	5
SEQ ID NO: 171	ttggaagctctttttggga	2211	2230	SEQ ID NO: 1174	tcccgattcacgcttccaa	11577	11596	1	5
SEQ ID NO: 172	ggaagctctttttgggaag	2213	2232	SEQ ID NO: 1175	cttcagaaagctaccttcc	7929	7946	1	5
SEQ ID NO: 173	tttttcccagacagtgtca	2238	2257	SEQ ID NO: 1176	tgaccttctctaagcaaaa	4876	4805	1	5
SEQ ID NO: 174	agacagtgtcaacaaagct	2246	2285	SEQ ID NO: 1177	agcttggttttgccagtct	2458	2477	1	5
SEQ ID NO: 175	ctttggctataccaaagat	2321	2340	SEQ ID NO: 1178	atctcgtgtctaggaaaag	5968	5987	1	5
SEQ ID NO: 176	caaagatgataaacatgag	2333	2382	SEQ ID NO: 1179	ctcaaggataacgtgtttg	12609	12628	1	5
SEQ ID NO: 177	gatatggtaaatggaataa	2356	2374	SEQ ID NO: 1180	ttatcttataattatatca	13079	13098	1	5
SEQ ID NO: 178	ggaataatgctcagtgttg	2367	2386	SEQ ID NO: 1181	caaacacttacttgaattc	10661	10680	1	5
SEQ ID NO: 179	tttgaaatccaaagaagtc	2402	2421	SEQ ID NO: 1182	gacttcagagaaatacaaa	11400	11419	1	5
SEQ ID NO: 180	gatcccccagatgattgga	2534	2553	SEQ ID NO: 1183	tccaatttccctgtggatc	3681	3700	1	5
SEQ ID NO: 181	cagatattggagaggtcaa	2541	2560	SEQ ID NO: 1184	tgaccacacaaaacagtct	5363	5382	1	5
SEQ ID NO: 182	agaatgacttttttctctc	2575	2594	SEQ ID NO: 1185	tgaagtccggattcattct	11015	11034	1	5
SEQ ID NO: 183	gaactccccactggagctg	2619	2638	SEQ ID NO: 1186	cagctcaaccgtacagttc	11851	11880	1	5
SEQ ID NO: 184	atatcttcatgggagtcaa	2652	2671	SEQ ID NO: 1187	tgacttcagtgcagaatat	11966	11965	1	5
SEQ ID NO: 185	gtcatgctccccggaggca	2657	2688	SEQ ID NO: 1188	tggccccgtttaccatgac	5809	5828	1	5
SEQ ID NO: 186	gctgaagtttatcattcct	2873	2892	SEQ ID NO: 1189	aggaggctttaagttcagc	7600	7619	1	5
SEQ ID NO: 187	attccttccccaaagagac	2886	2905	SEQ ID NO: 1190	gtctcttcctccatggaat	10470	10489	1	5
SEQ ID NO: 188	ctcattgagaacaggcagt	2976	2995	SEQ ID NO: 1191	actgactgcacgctttgag	11756	11775	1	5
SEQ ID NO: 189	ttgagcagtattctgtcag	3142	3161	SEQ ID NO: 1192	cttagagaagtgtcttcaa	12399	12418	1	5
SEQ ID NO: 190	accttgtccagtgaagtcc	3285	3304	SEQ ID NO: 1193	ggacggtactgtcccaggt	12784	12803	1	5
SEQ ID NO: 191	ccagtgaagtccaaattcc	3292	3311	SEQ ID NO: 1194	ggaaggcagagtttactgg	9148	9167	1	5
SEQ ID NO: 192	acattcagaacaagaaaat	3394	3413	SEQ ID NO: 1195	atttcctaaagctggatgc	11167	11186	1	5
SEQ ID NO: 193	gaaaaatcaagggtgttat	3463	3482	SEQ ID NO: 1196	ataaactgcaagatttttc	13600	13619	1	5
SEQ ID NO: 194	aaatcaagggtgttatttc	3466	3485	SEQ ID NO: 1197	gaaacaatgcattagattt	9745	9764	1	5
SEQ ID NO: 195	tggcattatgatgaagaga	3609	3628	SEQ ID NO: 1198	tctcccgtgtataatgcca	11781	11800	1	5
SEQ ID NO: 196	aagagaagattgaatttga	3622	3641	SEQ ID NO: 1199	tcaaaacctactgtctcct	10458	10477	1	5
SEQ ID NO: 197	aaatgacttccaatttccc	3673	3692	SEQ ID NO: 1200	gggaactacaatttcattt	7013	7032	1	5
SEQ ID NO: 198	atgacttccaatttccctg	3675	3694	SEQ ID NO: 1201	caggctgattacgagtcat	4917	4936	1	5
SEQ ID NO: 199	acttccaatttccctgtgg	3678	3697	SEQ ID NO: 1202	ccacgaaaaatatggaagt	10360	10379	1	5
SEQ ID NO: 200	agttgcaatgagctcatgg	3803	3822	SEQ ID NO: 1203	ccatcagttcagataaact	7969	8000	1	5
SEQ ID NO: 201	tttgcaagaccacctcaat	3860	3879	SEQ ID NO: 1204	attgacctgtccattcaaa	13671	13690	1	5
SEQ ID NO: 202	gaaggagttcaacctccag	388	3903	SEQ ID NO: 1205	ctggaattgtcattccttc	11728	11747	1	5
SEQ ID NO: 203	acttccacatcccagaaaa	43919	3938	SEQ ID NO: 1206	ttttaacaaaaagtggaag	6821	6840	1	5
SEQ ID NO: 204	ctcttcttaaaaagcgatg	3939	3956	SEQ ID NO: 1207	catcactgccaaaggagag	8486	8505	1	5
SEQ ID NO: 205	aaaagcgatggccgggtcc	3948	3967	SEQ ID NO: 1208	tgactcactcattgatttt	12680	12689	1	5
SEQ ID NO: 206	ttcctttgccttttggtgg	4003	4022	SEQ ID NO: 1209	ccacaaaacaatgaaggga	9256	9275	1	5
SEQ ID NO: 207	caagtctgtgggattccat	4079	4098	SEQ ID NO: 1210	atgggaaaaaacaggcttg	9566	9585	1	5
SEQ ID NO: 208	aagtccctacttttaccat	4117	4136	SEQ ID NO: 1211	atgggaagtataagaactt	4834	4853	1	5
SEQ ID NO: 209	tgcctctcctgggtgttct	4159	4176	SEQ ID NO: 1212	agaaaaacaaacaaacgga	9643	9662	1	5
SEQ ID NO: 210	accagcacagaccatttca	4242	4251	SEQ ID NO: 1213	tgaagtgtagtctcctggt	5089	5108	1	5
SEQ ID NO: 211	ccagcacagaccatttcag	4243	4262	SEQ ID NO: 1214	ctgaaatacaatgctctgg	5511	5530	1	5
SEQ ID NO: 212	actatcatgtgatgggtct	4367	4388	SEQ ID NO: 1215	agacacctgattttatagt	7948	7967	1	5
SEQ ID NO: 213	accacagatgtctgcttca	4498	4515	SEQ ID NO: 1216	tgaaggctgactctgtggt	4282	4301	1	5
SEQ ID NO: 214	ccacagagtgtctgcttcg	4497	4516	SEQ ID NO: 1217	ctgagcaacaatttgtgga	10311	10330	1	5
SEQ ID NO: 215	tttggactccaaaaagaaa	4520	4539	SEQ ID NO: 1218	tttctctcatgattacaaa	5933	5952	1	5
SEQ ID NO: 216	tcaaagaagtcaagattga	4552	4571	SEQ ID NO: 1219	tcaaggataactgtttgag	12610	12629	1	5
SEQ ID NO: 217	atgagaactacgagctgac	4798	4817	SEQ ID NO: 1220	gtcagatattgttgctcat	10167	10206	1	5
SEQ ID NO: 218	ttaaactctgacaccaatg	4818	4837	SEQ ID NO: 1221	cattcattgaagatgttaa	7342	7361	1	5
SEQ ID NO: 219	gaagtataagaactttgcc	4838	4857	SEQ ID NO: 1222	ggcaaatttgaaggacttc	11994	12013	1	5
SEQ ID NO: 220	aagtataagaaactttgca	4839	4858	SEQ ID NO: 1223	tggcaaatttgaaggactt	11993	12012	1	5
SEQ ID NO: 221	ttcttcagcctgctttctg	4941	4960	SEQ ID NO: 1224	cagaatccagatacaagaa	6664	6903	1	5
SEQ ID NO: 222	ctggatcactaaattccca	4957	4976	SEQ ID NO: 1225	tgggtctttccagagcccg	11033	11052	1	5
SEQ ID NO: 223	aaattaatagtggtgctca	5014	5033	SEQ ID NO: 1226	tgagaagccccaagaattt	6245	6267	1	5
SEQ ID NO: 224	ggccattccagaagggaag	5073	5092	SEQ ID NO: 1227	cttccgttctgtaatggcc	9848	9867	1	5
SEQ ID NO: 225	tgccatctcgagagttcca	5238	5257	SEQ ID NO: 1228	tggaactctctccatggca	8310	8329	1	5
SEQ ID NO: 226	catgtcaaacactttgtta	5278	5297	SEQ ID NO: 1229	taacaaattccttgacatg	10744	10763	1	5
SEQ ID NO: 227	tttgttataaatcttattg	5280	5299	SEQ ID NO: 1230	caataagatcaatagcaaa	10742	10761	1	5
SEQ ID NO: 228	tctggaaaaagggtcatga	5302	5321	SEQ ID NO: 1231	tccatgtcccatttacaga	13363	13382	1	5
SEQ ID NO: 229	cagctcttgttcaggtcca	5325	5344	SEQ ID NO: 1232	tggacctgcaccaaagctg	6205	6224	1	5
SEQ ID NO: 230	ggaggttccccagctctgc	5367	5388	SEQ ID NO: 1233	gcagccctgggaaaactcc	11219	11238	1	5
SEQ ID NO: 231	ctgttttgaagactctcca	5408	5428	SEQ ID NO: 1234	tggagggtagtcataacag	12835	12854	1	5
SEQ ID NO: 232	agtggctgaaacgtgtgca	5441	5460	SEQ ID NO: 1235	tgcagagctttctgccact	14043	14062	1	5
SEQ ID NO: 233	ccaaaatagaagggaatct	5488	5507	SEQ ID NO: 1236	agattcctttctttttcaa	7512	7531	1	5
SEQ ID NO: 234	tgaagagaagattgaattt	5502	5521	SEQ ID NO: 1237	aaattctcttttattttca	5890	5909	1	5
SEQ ID NO: 235	agtggtggcaacaccagca	5544	5583	SEQ ID NO: 1238	tgctagtgaggccaacact	10933	10952	1	5
SEQ ID NO: 236	aaggctccacaagtcatca	5620	5639	SEQ ID NO: 1239	tgatgatatctggaacctt	11204	11223	1	5
SEQ ID NO: 237	gtccgccaggtttctagca	5652	5671	SEQ ID NO: 1240	tgctaagaaccttactgac	7780	7799	1	5
SEQ ID NO: 238	tgatatctggaaccttgga	5667	5686	SEQ ID NO: 1241	ttcactgttcctgaaatca	11746	11765	1	5
SEQ ID NO: 239	gtcaagttgagcaatttct	5865	5885	SEQ ID NO: 1242	agaaaaggcacaccttgac	8036	8057	1	5
SEQ ID NO: 240	atccagatggaaaagggaa	5934	5953	SEQ ID NO: 1243	ttccaatttccctgtggat	10838	10857	1	5
SEQ ID NO: 241	atttgtttgtcaaagaagt	6034	6053	SEQ ID NO: 1244	acttcagagagaaatacat	7599	7618	1	5
SEQ ID NO: 242	ctggaaaatgtcagcctgg	6066	6085	SEQ ID NO: 1245	ccagacttcagttaccagc	8082	8101	1	5
SEQ ID NO: 243	accaggaggttcttcttca	6140	6159	SEQ ID NO: 1246	tgaagtgtgatctcctggt	10924	10943	1	5
SEQ ID NO: 244	aaagaagttctgaaaagat	6192	6211	SEQ ID NO: 1247	attccatcacaaaatcctt	7827	7846	1	5
SEQ ID NO: 245	gctacagcttatggctcca	6217	6236	SEQ ID NO: 1248	tggatctaaatgcagtagc	13174	13193	1	5
SEQ ID NO: 245	atcaatatgatcaatttgt	6295	6314	SEQ ID NO: 1249	caaagaaatcaagattgat	8630	8649	1	5
SEQ ID NO: 247	gaattatctttaaaacatt	6357	6376	SEQ ID NO: 1250	atgtggacaaatataccgg	8294	8313	1	5
SEQ ID NO: 248	cgaggcccgcgctgctggc	6376	6395	SEQ ID NO: 1251	gccagaagtgagatcctcg	11928	11947	1	5
SEQ ID NO: 249	acaactatgaggctgagag	386	6405	SEQ ID NO: 1252	ctctgagcaacaaattttt	13014	13033	1	5
SEQ ID NO: 250	gctgagagttccagtggag	6387	6406	SEQ ID NO: 1253	ctccatggcaaatgtcagg	10052	10071	1	5
SEQ ID NO: 251	tgaagaaaaaccaagaact	6401	3420	SEQ ID NO: 1254	gagtcattgaggttcttca	6984	7003	1	5
SEQ ID NO: 252	cctacttacatcctgaaca	2402	6421	SEQ ID NO: 1255	tgttcataagggaggtagg	6983	7002	1	5
SEQ ID NO: 253	ctacttacatcctgaacat	6405	6425	SEQ ID NO: 1256	atgttcataagggaggtag	8287	8306	1	5
SEQ ID NO: 254	gagacagaagccaagagcc	6414	6433	SEQ ID NO: 1257	gcttggttttgcccagtct	1660	1679	1	5
SEQ ID NO: 255	cactcactttaccgtcaag	6476	6495	SEQ ID NO: 1258	cttgaacaccaaagtcact	6719	6738	1	5
SEQ ID NO: 256	ctgatcagcagcagccagt	6480	6499	SEQ ID NO: 1259	actgggaagtgcttatcag	7094	7113	1	5
SEQ ID NO: 257	actggacgctaagaggaag	6498	6517	SEQ ID NO: 1260	cttccccaaagagaggagt	9488	9507	1	5
SEQ ID NO: 158	agaggaagcatgtggcaga	6693	6712	SEQ ID NO: 1261	tctggcatttactttctct	6806	6825	1	5
SEQ ID NO: 259	tgaagactctccaggaact	6731	6750	SEQ ID NO: 1262	agttgaaggagactattca	6757	6776	1	5
SEQ ID NO: 260	ctctgagcaaaatatccag	6808	6827	SEQ ID NO: 1263	ctggttactgagctgagag	13171	13190	1	5
SEQ ID NO: 261	atgaagcagtcacatctct	6938	6957	SEQ ID NO: 1264	agagctgccagtcttcatt	2458	2477	1	5
SEQ ID NO: 262	ttgccacagctgattgagg	7021	7040	SEQ ID NO: 1265	cctcctacagtggtggcaa	8082	8101	1	5
SEQ ID NO: 263	agctgattgaggtgtccag	7174	7193	SEQ ID NO: 1266	ctggattccacatgcagct	13147	13166	1	5
SEQ ID NO: 264	tgctccactcacatcctcc	7233	7252	SEQ ID NO: 1267	ggaggctttaagttcagca	12545	12564	1	5
SEQ ID NO: 265	tgaaacgtgtgcatgtcaa	7262	7281	SEQ ID NO: 1268	ttgggagagacaagtttca	13155	13174	1	5
SEQ ID NO: 266	gacattgctaattacctga	7269	7288	SEQ ID NO: 1269	tcagaagctaagcaatgtc	9481	9500	1	5
SEQ ID NO: 267	ttcttcttcagactttcct	7281	7300	SEQ ID NO: 1270	aggagagtccaaatttaga	12962	12981	1	5
SEQ ID NO: 268	ccaatatcttgaaactcag	7295	7314	SEQ ID NO: 1271	tctgaattcattcaattgg	7371	7390	1	5
SEQ ID NO: 269	aaagttagtgaaaagaagt	7326	7345	SEQ ID NO: 1272	aactaccctcactgccttt	7677	7696	1	5
SEQ ID NO: 270	aagttagtgaaagaagttc	7403	7422	SEQ ID NO: 1273	gaacctctggcattttact	10731	10750	1	5
SEQ ID NO: 271	aaagaagttctgaaagaat	7540	7559	SEQ ID NO: 1274	attctctggtaactacttt	8412	8431	1	5
SEQ ID NO: 272	tttggctataccaaagatg	7691	7710	SEQ ID NO: 1275	catcttaggcactgacaaa	12001	12020	1	5
SEQ ID NO: 273	tgttgagaagctgattaaa	7950	7969	SEQ ID NO: 1276	tttagccatcggctcaaca	12505	12625	1	5
SEQ ID NO: 274	caggaagggctcaaagaat	7984	8003	SEQ ID NO: 1277	attcctttaacaattcctg	13116	13135	1	5
SEQ ID NO: 275	aggaagggctcaaagaatg	8104	8123	SEQ ID NO: 1278	cattcctttaacaattcct	12352	12371	1	5
SEQ ID NO: 276	gaagggctcaaagaatgac	8148	8167	SEQ ID NO: 1279	gtcagtcttcaggctcttc	8801	8820	1	5
SEQ ID NO: 277	caaagaatgacttttttct	8399	5418	SEQ ID NO: 1280	agaaggatggcattttttg	11009	11028	1	5
SEQ ID NO: 278	catggagaatgcctttgaa	8524	8543	SEQ ID NO: 1281	ttcagagccaaagtccatg	14015	14034	1	5
SEQ ID NO: 279	ggagccaaggctggagtaa	8525	8544	SEQ ID NO: 1282	ttactccaacgccagctcc	9614	9633	1	5
SEQ ID NO: 280	tcattccttccccaaagag	8526	8547	SEQ ID NO: 1283	ctctctggggcatctatgc	14017	14036	1	5
SEQ ID NO: 281	acctatgagctccagagag	8637	8656	SEQ ID NO: 1284	ctctcaagaccacagaagg	11195	11214	1	5
SEQ ID NO: 282	gggcaaaaacgtcttacag	8638	8657	SEQ ID NO: 1285	tctgaaagacaacgtgccc	11194	11213	1	5
SEQ ID NO: 283	accctggaccttcagaaca	8698	8717	SEQ ID NO: 1286	tgttgctaaggttcagggt	10825	10844	1	5
SEQ ID NO: 284	atgggcgacctaagttgtg	8708	8727	SEQ ID NO: 1287	cacaaattagtttcaccat	1261	1300	1	5
SEQ ID NO: 285	gatgaagagaagattgaat	8878	8897	SEQ ID NO: 1288	atccagcttccccacactt	13751	13770	1	5
SEQ ID NO: 286	caatgtgataccaaaaaaa	8883	8902	SEQ ID NO: 1289	tttttggaaatgccattgt	9274	9298	1	5
SEQ ID NO: 287	gtagataccaaaaaaatga	8902	8921	SEQ ID NO: 1290	tcatgtgatgggtctctac	9432	9451	1	5
SEQ ID NO: 288	gcttcagttcatttggact	8916	8935	SEQ ID NO: 1291	agtcaagaaggacttaagc	12406	12427	1	5
SEQ ID NO: 289	tttgtttgtcaaagaagtc	8922	8941	SEQ ID NO: 1292	gacttcagagaaatacaaa	8330	8349	1	5
SEQ ID NO: 290	ttgtttgtcaaagaagtca	5978	8997	SEQ ID NO: 1293	tgacttcagagaaatacaa	11159	11175	1	5
SEQ ID NO: 291	tggcaatgggaaactcgct	9252	9271	SEQ ID NO: 1294	agcgagagtcccctgccat	10219	10238	1	5
SEQ ID NO: 292	aacctctggcatttacttt	9257	9276	SEQ ID NO: 1295	aaaggagatgtcaagggtt	11480	11499	1	5
SEQ ID NO: 293	catttactttctctcatga	9407	9426	SEQ ID NO: 1296	tcatttgaaagaataaatg	9663	9682	1	5
SEQ ID NO: 294	aaagtcagtgccctgctta	9408	9427	SEQ ID NO: 1297	taagaaccttactgacttt	9662	9681	1	5
SEQ ID NO: 295	tcccattttttgagacctt	9417	9436	SEQ ID NO: 1298	aaggacttcaggaatggga	13221	13240	1	5
SEQ ID NO: 296	catcaatattgatcaattt	9418	9437	SEQ ID NO: 1299	aaattaaaaagtcttgatg	13220	13239	1	5
SEQ ID NO: 297	taaagatagttatgattta	9433	9452	SEQ ID NO: 1300	taaaccaaaaacttggtta	8901	8920	1	5
SEQ ID NO: 298	tattgatgaaatcattgaa	9467	9486	SEQ ID NO: 1301	ttcaaagacttaaaaaata	13813	13832	1	5
SEQ ID NO: 299	atgatctacatttgtttat	9557	95786	SEQ ID NO: 1302	ataaagaaattaaagtcat	14013	14032	1	5
SEQ ID NO: 300	agagacacatacagaatat	9704	9723	SEQ ID NO: 1303	atatattgtcagtgcctct	14025	14044	1	5
SEQ ID NO: 301	gacacatacagaatataga	9743	9762	SEQ ID NO: 1304	tctaaattcagttcttgtc	5625	5644	1	5
SEQ ID NO: 302	agcatgtcaaacactttgt	9993	10012	SEQ ID NO: 1305	acaaagtcagtgccctgct	14081	14100	1	5
SEQ ID NO: 303	tttttagaggaaaccaagg	10186	10205	SEQ ID NO: 1306	ccttgtgtacaccaaaaac	4766	4818	1	5
SEQ ID NO: 304	ttttagaggaaaccaaggc	10328	10347	SEQ ID NO: 1307	gcctttgtgtacaccaaaa	2726	2745	1	5
SEQ ID NO: 305	ggaagatagacttcctgaa	10396	10415	SEQ ID NO: 1308	ttcagaaatactgtttttc	8358	8377	1	5
SEQ ID NO: 306	cactgtttctgagtcccag	10397	10416	SEQ ID NO: 1309	ctgggacctaccaagagtg	8357	8376	1	5
SEQ ID NO: 307	cacaaatcctttggctgtg	10428	10447	SEQ ID NO: 1310	cacatttcaaggaattgtg	13250	13269	1	5
SEQ ID NO: 308	ttcctggatacactgttcc	10570	10589	SEQ ID NO: 1311	ggaactgttgactcaggac	12939	12958	1	5
SEQ ID NO: 309	gaaatctcaagctttctct	10655	10674	SEQ ID NO: 1312	agagccaggtcgagctttc	12658	12687	1	5
SEQ ID NO: 310	tttcttcatcttcatctgt	10664	10683	SEQ ID NO: 1313	acagctgaaagagatgaaa	6000	6019	1	5
SEQ ID NO: 311	tctaccgctaaaggagcag	10743	10762	SEQ ID NO: 1314	ctgcacgctttgaggtaga	5279	6298	1	5
SEQ ID NO: 312	ctaccgctaaaggagcagt	10874	10893	SEQ ID NO: 1315	actgcacgctttgaggtcg	11364	11353	1	5
SEQ ID NO: 313	aggggcctcttttcaccaa	11176	11195	SEQ ID NO: 1316	ttggccaggaagtggccct	11847	11836	1	5
SEQ ID NO: 314	tctccatccctgtaaaaag	11477	11496	SEQ ID NO: 1317	ctttttcaccaacggagaa	6757	8775	1	5
SEQ ID NO: 315	gaaaaacaaagcagattat	11605	11624	SEQ ID NO: 1318	ataaactgcaagatttttc	12416	12435	1	5
SEQ ID NO: 316	actcactcattgattttct	11608	11625	SEQ ID NO: 1319	agaaaatcaggatcgtagt	12415	12434	1	5
SEQ ID NO: 317	taaactaatagatgtaatc	11834	11853	SEQ ID NO: 1320	gattaccaccagcagttta	13095	13114	1	5
SEQ ID NO: 318	caaaaacgagcttcggaag	12221	12240	SEQ ID NO: 1321	cttcgtgagaatatttgaa	13623	13642	1	5
SEQ ID NO: 319	tggaataatgctcagtgtt	12930	12949	SEQ ID NO: 1322	aacacttaacttgaattca	13818	13837	1	5
SEQ ID NO: 320	gatttgaaatccaaagaag	13026	13045	SEQ ID NO: 1323	cttcagagaaatacaaatc	8005	8025	1	5
SEQ ID NO: 321	atttgaaatccaaagaagt	13089	13108	SEQ ID NO: 1324	acttcagagaaatacaaat	10471	10490	1	5
SEQ ID NO: 322	atcaacagccgcttctttg	13210	13229	SEQ ID NO: 1325	caaagaagtcaagattgat	13175	13194	1	5
SEQ ID NO: 323	tgttttgaagactctccag	13429	13448	SEQ ID NO: 1326	ctggaaagttaaaacaaac	12924	12943	1	5
SEQ ID NO: 324	cccttctgatagatgtggt	13704	13723	SEQ ID NO: 1327	accaaagctggcaccaggg	7457	7475	1	5
SEQ ID NO: 325	tgagcaagtgaagaacttt	13711	13730	SEQ ID NO: 1328	aaagccattcagtctctca	7231	7250	1	5
SEQ ID NO: 326	atttgaaatccaaagaagt	13874	13893	SEQ ID NO: 1329	actttctaaacttgaaatt	8372	8391	1	5
SEQ ID NO: 327	atccaaagaagtcccggaa	14003	14022	SEQ ID NO: 1330	tccggggaaacctgggatt	3005	3024	1	5
SEQ ID NO: 328	agagcctacctccgcatct	14012	14031	SEQ ID NO: 1331	agatggtacgttagcctct	9558	9577	1	5
SEQ ID NO: 329	aatgcctttgaactcccca	1619	1638	SEQ ID NO: 1332	tgggaactacaattcattt	8633	8662	3	4
SEQ ID NO: 330	gaagtccaaattccggatt	1948	1967	SEQ ID NO: 1333	aatcttcaatttattcttc	12555	12575	3	4
SEQ ID NO: 331	tgcaagcagaagccagaag	5427	5446	SEQ ID NO: 1334	cttcaggttccatcgtgca	11401	11420	3	4
SEQ ID NO: 332	gaagagaagattgaatttg	6480	8499	SEQ ID NO: 1335	caaaacctactgtgcttcc	7421	7440	3	4
SEQ ID NO: 333	atgctaaaggcacatatgg	18	37	SEQ ID NO: 1336	ccatatgaaagtcaagcat	6031	6050	2	4
SEQ ID NO: 334	tccctcacctccacctctg	245	264	SEQ ID NO: 1337	cagattctcagatgaggga	13176	13195	2	4
SEQ ID NO: 335	atttacagctctgacaagt	308	327	SEQ ID NO: 1338	acttttctaaacttgaaat	13407	13426	2	4
SEQ ID NO: 336	aggagcctaccaaaataat	475	494	SEQ ID NO: 1339	attatgttgaaaacagtct	5881	5900	2	4
SEQ ID NO: 337	aaagctgaagcacatcaat	547	566	SEQ ID NO: 1340	attgttgctcatctccttt	10490	10509	2	4
SEQ ID NO: 338	ctgctggaaacaacgagaa	1087	1106	SEQ ID NO: 1341	ttctgattaccaccagcag	13183	13202	2	4
SEQ ID NO: 339	ttgaaggaattcttgaaaa	1202	1221	SEQ ID NO: 1342	ttttaaaagaaatcttcaa	9229	9248	2	4
SEQ ID NO: 340	gaagtaaaagaaaattttg	1203	1222	SEQ ID NO: 1343	caaaacctactgtctcttc	9226	9247	2	4
SEQ ID NO: 341	tgaagaagatggcaaattt	1223	1242	SEQ ID NO: 1344	aaatgtcagctcttgttca	5208	5227	2	4
SEQ ID NO: 342	aggatctgagttatttttc	1620	1639	SEQ ID NO: 1345	gcaagtcagcccagttcct	5907	5926	2	4
SEQ ID NO: 343	gtgcccttctcggttgctg	1941	1960	SEQ ID NO: 1346	cagccattgacatgagcac	10623	10642	2	4
SEQ ID NO: 344	ggcgcggcctgcgctgctg	2238	2257	SEQ ID NO: 1347	cagctccacagactccgcc	9722	9741	2	4
SEQ ID NO: 345	ctgcgctgctgctgctgct	2239	2258	SEQ ID NO: 1348	agcagaaggtgcgaagcag	9721	9740	2	4
SEQ ID NO: 346	gctggtggcgggcgccagg	3395	3414	SEQ ID NO: 1349	cctggattccacatgcagc	10406	10425	2	4
SEQ ID NO: 347	aagaggaaatgctggaaaa	3620	3639	SEQ ID NO: 1350	tttttcttcactacatctt	10894	10913	2	4
SEQ ID NO: 348	ctggaaaatgtcagcctgg	3636	3655	SEQ ID NO: 1351	ccagacttccacatcccag	11807	11826	2	4
SEQ ID NO: 349	tggagtccctgggactgct	4399	4416	SEQ ID NO: 1352	agcatgcctagtttctcca	7369	7388	2	4
SEQ ID NO: 350	ggagtccctgggactgctg	4404	4423	SEQ ID NO: 1353	cagcatgcctagtttctcc	7154	7173	2	4
SEQ ID NO: 351	tgggactgctgattcaaga	5075	5094	SEQ ID NO: 1354	tcttccatcacttgaccca	11376	11395	2	4
SEQ ID NO: 352	ctgctgattcaagaagtgc	5317	5336	SEQ ID NO: 1355	gcacaccttgacattgcag	7374	7393	2	4
SEQ ID NO: 353	tgccaccaggatcaactgc	6066	6085	SEQ ID NO: 1356	gcaggctgaactggtggca	13558	13887	2	4
SEQ ID NO: 354	gccaccaggatcaactgca	6080	6099	SEQ ID NO: 1357	tgcaggctgaactggtggc	10679	10698	2	4
SEQ ID NO: 355	tgcaaggttgagcgtggag	6413	6432	SEQ ID NO: 1358	cctccatcctctgatctga	11478	11497	2	4
SEQ ID NO: 356	caaggttgagctggaggtt	7051	7070	SEQ ID NO: 1359	aacccctacatgaagcttg	9373	9392	2	4
SEQ ID NO: 357	ctctgcagcttcatcctga	7219	7238	SEQ ID NO: 1360	tcaggaagcttctcaagag	13438	13457	2	4
SEQ ID NO: 358	cagcttcatcctgaagaac	7921	7940	SEQ ID NO: 1361	ggtcttgagttaaatgctg	12304	12323	2	4
SEQ ID NO: 359	gcttcatcctgaagaccag	8779	8798	SEQ ID NO: 1362	ctggacgctaagaggaagc	12525	12544	2	4
SEQ ID NO: 360	tcatcctgaagaccagcca	10159	10178	SEQ ID NO: 1363	tggcatggcattatgatga	13183	13202	2	4
SEQ ID NO: 361	gaaaaccaagaactctgag	12890	12909	SEQ ID NO: 1364	ctcaaccttaatgattttc	13632	13651	2	4
SEQ ID NO: 362	agaactctgaggagtttgc	13672	13691	SEQ ID NO: 1365	gcaagctatatcagtattc	13805	13824	2	4
SEQ ID NO: 363	tctgaggagtttgctgcag	11	30	SEQ ID NO: 1366	ctgcagggatcccccagat	76	95	1	4
SEQ ID NO: 364	tttgctgcagccatgtcca	12	31	SEQ ID NO: 1367	tggaagtgtcagtggcaaa	75	94	1	4
SEQ ID NO: 365	caagaggggcatcatttct	14	33	SEQ ID NO: 1368	agaataaatgacgttcttg	11549	11568	1	4
SEQ ID NO: 366	tcactttaccgtcaagacg	25	44	SEQ ID NO: 1369	cgtctacactatcatgtga	2160	2179	1	4
SEQ ID NO: 367	tttaccgtcaagacgagga	82	101	SEQ ID NO: 1370	tccttgacatgttgataaa	368	367	1	4
SEQ ID NO: 368	cactggacgctaagaggaa	143	162	SEQ ID NO: 1371	ttccagaaagcagccagtg	4244	4263	1	4
SEQ ID NO: 369	aggaagcatgtggcagaag	169	188	SEQ ID NO: 1372	cttcatacacattaatcct	11178	11197	1	4
SEQ ID NO: 370	caaggagcaacacctcttc	219	238	SEQ ID NO: 1373	gaagtagtactgcatgttg	380	399	1	4
SEQ ID NO: 371	acagactttgaaacttgaa	283	302	SEQ ID NO: 1374	ttcaattcttcaatgctgt	3383	3402	1	4
SEQ ID NO: 372	tgatgaagcagtcacatct	291	310	SEQ ID NO: 1375	agatttgaggattccatca	2675	2694	1	4
SEQ ID NO: 373	agcagtcacatctctcttg	348	365	SEQ ID NO: 1376	caaggagaaactgactcgt	4728	4747	1	4
SEQ ID NO: 374	ccagccccatcactttaca	350	369	SEQ ID NO: 1377	tgtagtctcctggtgctgg	9163	9182	1	4
SEQ ID NO: 375	ctccactccatcctccagg	370	389	SEQ ID NO: 1378	ctggagcttagtaatggag	3261	3280	1	4
SEQ ID NO: 376	catgccaacccccttctga	394	413	SEQ ID NO: 1379	tcagatgagggaacacatg	5794	5813	1	4
SEQ ID NO: 377	gagagatcttcaacatggc	464	483	SEQ ID NO: 1380	gccaccctggaactctctc	6340	6359	1	4
SEQ ID NO: 378	tcaacatggcgagggatca	492	511	SEQ ID NO: 1381	tgatcccacctctcattga	12716	12735	1	4
SEQ ID NO: 379	ccaccttgtatgcgctgag	535	554	SEQ ID NO: 1382	ctcagggatctgaaggtgg	2219	2238	1	4
SEQ ID NO: 380	gtcaacaactatcataaga	575	594	SEQ ID NO: 1383	tcttgagttaaatgctgac	2277	2296	1	4
SEQ ID NO: 381	tggacattgctaattacct	601	620	SEQ ID NO: 1384	aggtatattcgaaagtcca	13988	140007	1	4
SEQ ID NO: 382	ggacattgctaattacctg	622	641	SEQ ID NO: 1385	caggtatattcgaaagtcc	14042	14091	1	4
SEQ ID NO: 383	ttctgcgggtcattggaaa	630	649	SEQ ID NO: 1386	tttcacatgccaaggagaa	11042	11061	1	4
SEQ ID NO: 384	ccagaactcaagtcttcaa	644	663	SEQ ID NO: 1387	ttgaagtgtatgtctcctg	11356	11375	1	4
SEQ ID NO: 385	agtcttcaatcctgaaatg	670	689	SEQ ID NO: 1388	catttctgattggtggact	6817	6836	1	4
SEQ ID NO: 386	tgagcaagtgaagaacttt	693	712	SEQ ID NO: 1389	aaagtgccacttttactca	3005	3024	1	4
SEQ ID NO: 387	agcaagtgaagaactttgt	700	719	SEQ ID NO: 1390	acaaagtcagtgccctgct	3480	3499	1	4
SEQ ID NO: 388	tctgaaagaatctcaactt	701	720	SEQ ID NO: 1391	aagtccataatggttcaga	13826	13845	1	4
SEQ ID NO: 389	actgtcatggacttcagaa	706	725	SEQ ID NO: 1392	ttctgaatatattgtcagt	1887	1906	1	4
SEQ ID NO: 390	acttgacccagcctcagcc	729	748	SEQ ID NO: 1393	ggctcaccctgagagaagt	2930	2949	1	4
SEQ ID NO: 391	tccaaataactaccttcct	744	763	SEQ ID NO: 1394	aggaagatatgaagatgga	5210	5229	1	4
SEQ ID NO: 392	actaccctcactgcctttg	745	764	SEQ ID NO: 1395	caaatttgtggagggtagt	5209	5226	1	4
SEQ ID NO: 393	ttggatttgcttcagctga	776	795	SEQ ID NO: 1396	tcagtataagtacaaccaa	8144	8163	1	4
SEQ ID NO: 394	ttggaagctctttttggga	786	805	SEQ ID NO: 1397	tcccgattcacgcttccaa	10337	10356	1	4
SEQ ID NO: 395	ggaagctctttttgggaag	811	830	SEQ ID NO: 1398	cttcagaaagctaccttcc	12445	12464	1	4
SEQ ID NO: 396	tttttcccagacagtgtca	812	831	SEQ ID NO: 1399	tgaccttctctaagcaaaa	12444	12463	1	4
SEQ ID NO: 397	agacagtgtcaacaaagct	884	903	SEQ ID NO: 1400	agcttggttttgccagtct	3805	3824	1	4
SEQ ID NO: 398	ctttggctataccaaagat	885	904	SEQ ID NO: 1401	atctcgtgtctaggaaaag	3804	3823	1	4
SEQ ID NO: 399	caaagatgataaacatgag	908	927	SEQ ID NO: 1402	ctcaaggataacgtgtttg	9453	9472	1	4
SEQ ID NO: 400	gatatggtaaatggaataa	916	935	SEQ ID NO: 1403	ttatcttataattatatca	13648	13667	1	4
SEQ ID NO: 401	ggaataatgctcagtgttg	989	1008	SEQ ID NO: 1404	caaacacttacttgaattc	1661	1580	1	4
SEQ ID NO: 402	tttgaaatccaaagaagtc	990	1009	SEQ ID NO: 1405	gacttcagagaaatacaaa	1660	1579	1	4
SEQ ID NO: 403	gatcccccagatgattgga	994	1013	SEQ ID NO: 1406	tccaatttccctgtggatc	9667	9686	1	4
SEQ ID NO: 404	cagatattggagaggtcaa	1023	1042	SEQ ID NO: 1407	tgaccacacaaaacagtct	2069	2068	1	4
SEQ ID NO: 405	agaatgacttttttctctc	1082	1101	SEQ ID NO: 1408	tgaagtccggattcattct	5487	5505	1	4
SEQ ID NO: 406	gaactccccactggagctg	1086	1105	SEQ ID NO: 1409	cagctcaaccgtacagttc	13184	13203	1	4
SEQ ID NO: 407	atatcttcatgggagtcaa	1102	1121	SEQ ID NO: 1410	tgacttcagtgcagaatat	14006	14025	1	4
SEQ ID NO: 408	gtcatgctccccggaggca	1104	1123	SEQ ID NO: 1411	tggccccgtttaccatgac	4564	4583	1	4
SEQ ID NO: 409	gctgaagtttatcattcct	1109	1128	SEQ ID NO: 1412	aggaggctttaagttcagc	2570	2589	1	4
SEQ ID NO: 410	attccttccccaaagagac	1124	1143	SEQ ID NO: 1413	gtctcttcctccatggaat	12201	12220	1	4
SEQ ID NO: 411	ctcattgagaacaggcagt	1154	1173	SEQ ID NO: 1414	actgactgcacgctttgag	12265	12284	1	4
SEQ ID NO: 412	ttgagcagtattctgtcag	1166	1185	SEQ ID NO: 1415	cttagagaagtgtcttcaa	10329	10348	1	4
SEQ ID NO: 413	accttgtccagtgaagtcc	1180	1199	SEQ ID NO: 1416	ggacggtactgtcccaggt	12572	12591	1	4
SEQ ID NO: 414	ccagtgaagtccaaattcc	1196	1215	SEQ ID NO: 1417	ggaaggcagagtttactgg	8858	8877	1	4
SEQ ID NO: 415	acattcagaacaagaaaat	1202	1221	SEQ ID NO: 1418	atttcctaaagctggatgc	2161	2180	1	4
SEQ ID NO: 416	gaaaaatcaagggtgttat	1203	1222	SEQ ID NO: 1419	ataaactgcaagatttttc	2160	2179	1	4
SEQ ID NO: 417	aaatcaagggtgttatttc	1210	1229	SEQ ID NO: 1420	gaaacaatgcattagattt	13955	13974	1	4
SEQ ID NO: 418	tggcattatgatgaagaga	1211	1230	SEQ ID NO: 1421	tctcccgtgtataatgcca	11240	11259	1	4
SEQ ID NO: 419	aagagaagattgaatttga	1240	1259	SEQ ID NO: 1422	tcaaaacctactgtctcct	8140	8159	1	4
SEQ ID NO: 420	aaatgacttccaatttccc	1324	1343	SEQ ID NO: 1423	gggaactacaatttcattt	10816	10835	1	4
SEQ ID NO: 421	atgacttccaatttccctg	1341	1390	SEQ ID NO: 1424	caggctgattacgagtcat	3431	3460	1	4
SEQ ID NO: 422	acttccaatttccctgtgg	1432	1451	SEQ ID NO: 1425	ccacgaaaaatatggaagt	5578	5597	1	4
SEQ ID NO: 423	agttgcaatgagctcatgg	1472	1491	SEQ ID NO: 1426	ccatcagttcagataaact	12347	12366	1	4
SEQ ID NO: 424	tttgcaagaccacctcaat	1508	1527	SEQ ID NO: 1427	attgacctgtccattcaaa	9930	9949	1	4
SEQ ID NO: 425	gaaggagttcaacctccag	1538	1557	SEQ ID NO: 1428	ctggaattgtcattccttc	10909	10926	1	4
SEQ ID NO: 426	acttccacatcccagaaaa	1540	1559	SEQ ID NO: 1429	ttttaacaaaaagtggaag	5999	5018	1	4
SEQ ID NO: 427	ctcttcttaaaaagcgatg	1552	1571	SEQ ID NO: 1430	catcactgccaaaggagag	8110	5129	1	4
SEQ ID NO: 428	aaaagcgatggccgggtcc	1602	1621	SEQ ID NO: 1431	tgactcactcattgatttt	9016	9035	1	4
SEQ ID NO: 429	ttcctttgccttttggtgg	1610	1629	SEQ ID NO: 1432	ccacaaaacaatgaaggga	13712	13738	1	4
SEQ ID NO: 430	caagtctgtgggattccat	1621	1640	SEQ ID NO: 1433	atgggaaaaaacaggcttg	1925	1944	1	4
SEQ ID NO: 431	aagtccctacttttaccat	1695	1714	SEQ ID NO: 1434	atgggaagtataagaactt	2477	2496	1	4
SEQ ID NO: 432	tgcctctcctgggtgttct	1730	1749	SEQ ID NO: 1435	agaaaaacaaacaaacgga	5511	5630	1	4
SEQ ID NO: 433	accagcacagaccatttca	1736	1755	SEQ ID NO: 1436	tgaagtgtagtctcctggt	4431	4450	1	4
SEQ ID NO: 434	ccagcacagaccatttcag	1751	1770	SEQ ID NO: 1437	ctgaaatacaatgctctgg	6885	6904	1	4
SEQ ID NO: 435	actatcatgtgatgggtct	1773	1792	SEQ ID NO: 1438	agacacctgattttatagt	9944	9963	1	4
SEQ ID NO: 436	accacagatgtctgcttca	1788	1807	SEQ ID NO: 1439	tgaaggctgactctgtggt	12037	12056	1	4
SEQ ID NO: 437	ccacagagtgtctgcttcg	1882	1901	SEQ ID NO: 1440	ctgagcaacaatttgtgga	10729	10748	1	4
SEQ ID NO: 438	tttggactccaaaaagaaa	1902	1921	SEQ ID NO: 1441	tttctctcatgattacaaa	13992	14011	1	4
SEQ ID NO: 439	tcaaagaagtcaagattga	1905	1924	SEQ ID NO: 1442	tcaaggataactgtttgag	13370	13389	1	4
SEQ ID NO: 440	atgagaactacgagctgac	1916	1935	SEQ ID NO: 1443	gtcagatattgttgctcat	7265	7284	1	4
SEQ ID NO: 441	ttaaactctgacaccaatg	1921	1940	SEQ ID NO: 1444	cattcattgaagatgttaa	13817	13836	1	4
SEQ ID NO: 442	gaagtataagaactttgcc	1922	1941	SEQ ID NO: 1445	ggcaaatttgaaggacttc	13816	13835	1	4
SEQ ID NO: 443	aagtataagaaactttgca	1927	1943	SEQ ID NO: 1446	tggcaaatttgaaggactt	8317	8338	1	4
SEQ ID NO: 444	ttcttcagcctgctttctg	1930	1949	SEQ ID NO: 1447	cagaatccagatacaagaa	10177	10196	1	4
SEQ ID NO: 445	ctggatcactaaattccca	1931	1950	SEQ ID NO: 1448	tgggtctttccagagcccg	10176	10195	1	4
SEQ ID NO: 446	aaattaatagtggtgctca	1935	1954	SEQ ID NO: 1449	tgagaagccccaagaattt	4965	4984	1	4
SEQ ID NO: 447	ggccattccagaagggaag	1936	1955	SEQ ID NO: 1450	cttccgttctgtaatggcc	4964	4983	1	4
SEQ ID NO: 448	tgccatctcgagagttcca	1942	1961	SEQ ID NO: 1451	tggaactctctccatggca	10622	10641	1	4
SEQ ID NO: 449	catgtcaaacactttgtta	1682	2001	SEQ ID NO: 1452	taacaaattccttgacatg	13937	13956	1	4
SEQ ID NO: 450	tttgttataaatcttattg	1999	2018	SEQ ID NO: 1453	caataagatcaatagcaaa	9493	9512	1	4
SEQ ID NO: 451	tctggaaaaagggtcatga	2044	2063	SEQ ID NO: 1454	tccatgtcccatttacaga	8433	8452	1	4
SEQ ID NO: 452	cagctcttgttcaggtcca	2057	2076	SEQ ID NO: 1455	tggacctgcaccaaagctg	7812	7831	1	4
SEQ ID NO: 453	ggaggttccccagctctgc	2060	2079	SEQ ID NO: 1456	gcagccctgggaaaactcc	7814	7833	1	4
SEQ ID NO: 454	ctgttttgaagactctcca	2063	2102	SEQ ID NO: 1457	tggagggtagtcataacag	11813	11832	1	4
SEQ ID NO: 455	agtggctgaaacgtgtgca	2084	213	SEQ ID NO: 1458	tgcagagctttctgccact	14011	14030	1	4
SEQ ID NO: 456	ccaaaatagaagggaatct	2109	2126	SEQ ID NO: 1459	agattcctttctttttcaa	3605	3625	1	4
SEQ ID NO: 457	tgaagagaagattgaattt	2113	2132	SEQ ID NO: 1460	aaattctcttttattttca	5686	5705	1	4
SEQ ID NO: 458	agtggtggcaacaccagca	2114	2133	SEQ ID NO: 1461	tgctagtgaggccaacact	9482	9501	1	4
SEQ ID NO: 459	aaggctccacaagtcatca	2175	2194	SEQ ID NO: 1462	tgatgatatctggaacctt	11701	11720	1	4
SEQ ID NO: 460	gtccgccaggtttctagca	2198	2217	SEQ ID NO: 1463	tgctaagaaccttactgac	11060	11079	1	4
SEQ ID NO: 461	tgatatctggaaccttgga	2245	2264	SEQ ID NO: 1464	ttcactgttcctgaaatca	6134	6153	1	4
SEQ ID NO: 462	gtcaagttgagcaatttct	2249	2268	SEQ ID NO: 1465	agaaaaggcacaccttgac	9849	9868	1	4
SEQ ID NO: 463	atccagatggaaaagggaa	2250	2269	SEQ ID NO: 1466	ttccaatttccctgtggat	4351	4370	1	4
SEQ ID NO: 464	atttgtttgtcaaagaagt	2290	2309	SEQ ID NO: 1467	acttcagagagaaatacat	3325	3344	1	4
SEQ ID NO: 465	ctggaaaatgtcagcctgg	2291	2310	SEQ ID NO: 1468	ccagacttcagttaccagc	8823	6642	1	4
SEQ ID NO: 466	accaggaggttcttcttca	2343	2362	SEQ ID NO: 1469	tgaagtgtgatctcctggt	6788	6807	1	4
SEQ ID NO: 467	aaagaagttctgaaaagat	2387	2406	SEQ ID NO: 1470	attccatcacaaaatcctt	5279	5298	1	4
SEQ ID NO: 468	gctacagcttatggctcca	2397	2416	SEQ ID NO: 1471	tggatctaaatgcagtagc	7606	7625	1	4
SEQ ID NO: 469	atcaatatgatcaatttgt	2510	2529	SEQ ID NO: 1472	caaagaaatcaagattgat	7975	7994	1	4
SEQ ID NO: 470	gaattatctttaaaacatt	2532	2551	SEQ ID NO: 1473	atgtggacaaatataccgg	9075	9094	1	4
SEQ ID NO: 471	cgaggcccgcgctgctggc	2586	2604	SEQ ID NO: 1474	gccagaagtgagatcctcg	10374	10393	1	4
SEQ ID NO: 472	acaactatgaggctgagag	2588	2607	SEQ ID NO: 1475	ctctgagcaacaaattttt	3607	3626	1	4
SEQ ID NO: 473	gctgagagttccagtggag	2595	2614	SEQ ID NO: 1476	ctccatggcaaatgtcagg	9437	9458	1	4
SEQ ID NO: 474	tgaagaaaaaccaagaact	2601	2620	SEQ ID NO: 1477	gagtcattgaggttcttca	6599	6618	1	4
SEQ ID NO: 475	cctacttacatcctgaaca	2602	2621	SEQ ID NO: 1478	tgttcataagggaggtagg	13108	13127	1	4
SEQ ID NO: 476	ctacttacatcctgaacat	2616	2635	SEQ ID NO: 1479	atgttcataagggaggtag	9834	9853	1	4
SEQ ID NO: 477	gagacagaagccaagagcc	2617	2636	SEQ ID NO: 1480	gcttggttttgcccagtct	9833	9852	1	4
SEQ ID NO: 478	cactcactttaccgtcaag	2618	2637	SEQ ID NO: 1481	cttgaacaccaaagtcact	9832	9651	1	4
SEQ ID NO: 479	ctgatcagcagcagccagt	2627	2646	SEQ ID NO: 1482	actgggaagtgcttatcag	9336	9355	1	4
SEQ ID NO: 480	actggacgctaagaggaag	2628	2647	SEQ ID NO: 1483	cttccccaaagagaggagt	9335	9354	1	4
SEQ ID NO: 481	agaggaagcatgtggcaga	2644	2663	SEQ ID NO: 1484	tctggcatttactttctct	6591	6610	1	4
SEQ ID NO: 482	tgaagactctccaggaact	2645	2664	SEQ ID NO: 1485	agttgaaggagactattca	6590	6609	1	4
SEQ ID NO: 483	ctctgagcaaaatatccag	2650	2689	SEQ ID NO: 1486	ctggttactgagctgagag	13996	14015	1	4
SEQ ID NO: 484	atgaagcagtcacatctct	2695	2714	SEQ ID NO: 1487	agagctgccagtcttcatt	7592	7611	1	4
SEQ ID NO: 485	ttgccacagctgattgagg	2720	2739	SEQ ID NO: 1488	cctcctacagtggtggcaa	9220	9239	1	4
SEQ ID NO: 486	agctgattgaggtgtccag	2750	2789	SEQ ID NO: 1489	ctggattccacatgcagct	8530	8549	1	4
SEQ ID NO: 487	tgctccactcacatcctcc	2758	2777	SEQ ID NO: 1490	ggaggctttaagttcagca	9513	9532	1	4
SEQ ID NO: 488	tgaaacgtgtgcatgtcaa	2811	2830	SEQ ID NO: 1491	ttgggagagacaagtttca	2669	2888	1	4
SEQ ID NO: 489	gacattgctaattacctga	2825	2844	SEQ ID NO: 1492	tcagaagctaagcaatgtc	5227	5246	1	4
SEQ ID NO: 490	ttcttcttcagactttcct	2832	2851	SEQ ID NO: 1493	aggagagtccaaatttaga	5132	6151	1	4
SEQ ID NO: 491	ccaatatcttgaaactcag	2858	2877	SEQ ID NO: 1494	tctgaattcattcaattgg	12112	12131	1	4
SEQ ID NO: 492	aaagttagtgaaaagaagt	2664	2883	SEQ ID NO: 1495	aactaccctcactgccttt	5455	5474	1	4
SEQ ID NO: 493	aagttagtgaaagaagttc	3106	3125	SEQ ID NO: 1496	gaacctctggcattttact	12718	12737	1	4
SEQ ID NO: 494	aaagaagttctgaaagaat	3200	3219	SEQ ID NO: 1497	attctctggtaactacttt	8385	8404	1	4
SEQ ID NO: 495	tttggctataccaaagatg	3244	3263	SEQ ID NO: 1498	catcttaggcactgacaaa	3809	3828	1	4
SEQ ID NO: 496	tgttgagaagctgattaaa	3286	3308	SEQ ID NO: 1499	tttagccatcggctcaaca	6349	6968	1	4
SEQ ID NO: 497	caggaagggctcaaagaat	3305	3324	SEQ ID NO: 1500	attcctttaacaattcctg	8930	8949	1	4
SEQ ID NO: 498	aggaagggctcaaagaatg	3307	3326	SEQ ID NO: 1501	cattcctttaacaattcct	13199	13216	1	4
SEQ ID NO: 499	gaagggctcaaagaatgac	3329	3348	SEQ ID NO: 1502	gtcagtcttcaggctcttc	4203	4222	1	4
SEQ ID NO: 500	caaagaatgacttttttct	3337	3356	SEQ ID NO: 1503	agaaggatggcattttttg	7585	7604	1	4
SEQ ID NO: 501	catggagaatgcctttgaa	3384	3403	SEQ ID NO: 1504	ttcagagccaaagtccatg	12431	12450	1	4
SEQ ID NO: 502	ggagccaaggctggagtaa	3395	3414	SEQ ID NO: 1505	ttactccaacgccagctcc	8096	8115	1	4
SEQ ID NO: 503	tcattccttccccaaagag	3414	3433	SEQ ID NO: 1506	ctctctggggcatctatgc	10916	10935	1	4
SEQ ID NO: 504	acctatgagctccagagag	3478	3497	SEQ ID NO: 1507	ctctcaagaccacagaagg	13672	13891	1	4
SEQ ID NO: 505	gggcaaaaacgtcttacag	3493	3512	SEQ ID NO: 1508	tctgaaagacaacgtgccc	5400	5419	1	4
SEQ ID NO: 506	accctggaccttcagaaca	3494	3513	SEQ ID NO: 1509	tgttgctaaggttcagggt	5264	5283	1	4
SEQ ID NO: 507	atgggcgacctaagttgtg	3495	3514	SEQ ID NO: 1510	cacaaattagtttcaccat	5263	5282	1	4
SEQ ID NO: 508	gatgaagagaagattgaat	3546	3565	SEQ ID NO: 1511	atccagcttccccacactt	4559	4578	1	4
SEQ ID NO: 509	caatgtgataccaaaaaaa	3589	3588	SEQ ID NO: 1512	tttttggaaatgccattgt	7601	760	1	4
SEQ ID NO: 510	gtagataccaaaaaaatga	3573	3592	SEQ ID NO: 1513	tcatgtgatgggtctctac	5889	5908	1	4
SEQ ID NO: 511	gcttcagttcatttggact	3592	3611	SEQ ID NO: 1514	agtcaagaaggacttaagc	8869	8888	1	4
SEQ ID NO: 512	tttgtttgtcaaagaagtc	3695	3614	SEQ ID NO: 1515	gacttcagagaaatacaaa	9353	9372	1	4
SEQ ID NO: 513	ttgtttgtcaaagaagtca	3603	3622	SEQ ID NO: 1516	tgacttcagagaaatacaa	8063	8082	1	4
SEQ ID NO: 514	tggcaatgggaaactcgct	3617	3636	SEQ ID NO: 1517	agcgagagtcccctgccat	7863	7682	1	4
SEQ ID NO: 515	aacctctggcatttacttt	3621	3640	SEQ ID NO: 1518	aaaggagatgtcaagggtt	5279	5298	1	4
SEQ ID NO: 516	catttactttctctcatga	3624	3643	SEQ ID NO: 1519	tcatttgaaagaataaatg	8270	8289	1	4
SEQ ID NO: 517	aaagtcagtgccctgctta	3638	3655	SEQ ID NO: 1520	taagaaccttactgacttt	11228	11247	1	4
SEQ ID NO: 518	tcccattttttgagacctt	3850	3559	SEQ ID NO: 1521	aaggacttcaggaatggga	5583	5602	1	4
SEQ ID NO: 519	catcaatattgatcaattt	3668	3667	SEQ ID NO: 1522	aaattaaaaagtcttgatg	8358	8377	1	4
SEQ ID NO: 520	taaagatagttatgattta	3669	3688	SEQ ID NO: 1523	taaaccaaaaacttggtta	8357	8376	1	4
SEQ ID NO: 521	tattgatgaaatcattgaa	3670	3689	SEQ ID NO: 1524	ttcaaagacttaaaaaata	8398	8417	1	4
SEQ ID NO: 522	atgatctacatttgtttat	3752	3771	SEQ ID NO: 1525	ataaagaaattaaagtcat	4082	4101	1	4
SEQ ID NO: 523	agagacacatacagaatat	3785	3814	SEQ ID NO: 1526	atatattgtcagtgcctct	13178	13197	1	4
SEQ ID NO: 524	gacacatacagaatataga	3891	3910	SEQ ID NO: 1527	tctaaattcagttcttgtc	8134	8153	1	4
SEQ ID NO: 525	agcatgtcaaacactttgt	3907	3926	SEQ ID NO: 1528	acaaagtcagtgccctgct	8895	8914	1	4
SEQ ID NO: 526	tttttagaggaaaccaagg	3986	4005	SEQ ID NO: 1529	ccttgtgtacaccaaaaac	9533	9552	1	4
SEQ ID NO: 527	ttttagaggaaaccaaggc	3992	4011	SEQ ID NO: 1530	gcctttgtgtacaccaaaa	13686	13076	1	4
SEQ ID NO: 528	ggaagatagacttcctgaa	4007	4026	SEQ ID NO: 1531	ttcagaaatactgtttttc	6351	6370	1	4
SEQ ID NO: 529	cactgtttctgagtcccag	4028	4047	SEQ ID NO: 1532	ctgggacctaccaagagtg	7586	7605	1	4
SEQ ID NO: 530	cacaaatcctttggctgtg	4037	4056	SEQ ID NO: 1533	cacatttcaaggaattgtg	10025	10044	1	4
SEQ ID NO: 531	ttcctggatacactgttcc	4084	4103	SEQ ID NO: 1534	ggaactgttgactcaggac	9727	9746	1	4
SEQ ID NO: 532	gaaatctcaagctttctct	4096	4115	SEQ ID NO: 1535	agagccaggtcgagctttc	8548	8567	1	4
SEQ ID NO: 533	tttcttcatcttcatctgt	4104	4123	SEQ ID NO: 1536	acagctgaaagagatgaaa	8207	8225	1	4
SEQ ID NO: 534	tctaccgctaaaggagcag	4116	4137	SEQ ID NO: 1537	ctgcacgctttgaggtaga	6076	6097	1	4
SEQ ID NO: 535	ctaccgctaaaggagcagt	4125	4144	SEQ ID NO: 1538	actgcacgctttgaggtcg	8101	8120	1	4
SEQ ID NO: 536	aggggcctcttttcaccaa	4133	4152	SEQ ID NO: 1539	ttggccaggaagtggccct	12001	12020	1	4
SEQ ID NO: 537	tctccatccctgtaaaaag	4276	4295	SEQ ID NO: 1540	ctttttcaccaacggagaa	9016	9035	1	4
SEQ ID NO: 538	gaaaaacaaagcagattat	4309	4326	SEQ ID NO: 1541	ataaactgcaagatttttc	9495	9514	1	4
SEQ ID NO: 539	actcactcattgattttct	4330	4349	SEQ ID NO: 1542	agaaaatcaggatcgtagt	11027	11046	1	4
SEQ ID NO: 540	taaactaatagatgtaatc	4370	4389	SEQ ID NO: 1543	gattaccaccagcagttta	6557	6576	1	4
SEQ ID NO: 541	caaaaacgagcttcggaag	4372	4391	SEQ ID NO: 1544	cttcgtgagaatatttgaa	12125	12144	1	4
SEQ ID NO: 542	tggaataatgctcagtgtt	4399	4418	SEQ ID NO: 1545	aacacttaacttgaattca	7300	7319	1	4
SEQ ID NO: 543	gatttgaaatccaaagaag	4491	4510	SEQ ID NO: 1546	cttcagagaaatacaaatc	8325	8345	1	4
SEQ ID NO: 544	atttgaaatccaaagaagt	4636	4655	SEQ ID NO: 1547	acttcagagaaatacaaat	6976	6995	1	4
SEQ ID NO: 545	atcaacagccgcttctttg	4637	4656	SEQ ID NO: 1548	caaagaagtcaagattgat	6975	6994	1	4
SEQ ID NO: 546	tgttttgaagactctccag	4638	4667	SEQ ID NO: 1549	ctggaaagttaaaacaaac	6974	6993	1	4
SEQ ID NO: 547	cccttctgatagatgtggt	4642	4681	SEQ ID NO: 1550	accaaagctggcaccaggg	12065	12064	1	4
SEQ ID NO: 548	tgagcaagtgaagaacttt	4705	4724	SEQ ID NO: 1551	aaagccattcagtctctca	10202	10221	1	4
SEQ ID NO: 549	atttgaaatccaaagaagt	4737	4756	SEQ ID NO: 1552	actttctaaacttgaaatt	8175	8195	1	4
SEQ ID NO: 550	atccaaagaagtcccggaa	4810	4829	SEQ ID NO: 1553	tccggggaaacctgggatt	7922	7941	1	4
SEQ ID NO: 551	agagcctacctccgcatct	4812	4831	SEQ ID NO: 1554	agatggtacgttagcctct	8732	8751	1	4
SEQ ID NO: 552	aatgcctttgaactcccca	4865	4884	SEQ ID NO: 1555	tgggaactacaattcattt	6836	6854	1	4
SEQ ID NO: 553	gaagtccaaattccggatt	4901	4920	SEQ ID NO: 1556	aatcttcaatttattcttc	9342	9361	1	4
SEQ ID NO: 554	tgcaagcagaagccagaag	4905	4924	SEQ ID NO: 1557	cttcaggttccatcgtgca	8603	8622	1	4
SEQ ID NO: 555	gaagagaagattgaatttg	4968	4987	SEQ ID NO: 1558	caaaacctactgtgcttcc	12264	12283	1	4
SEQ ID NO: 556	atgctaaaggcacatatgg	4976	4995	SEQ ID NO: 1559	ccatatgaaagtcaagcat	10801	10820	1	4
SEQ ID NO: 557	tccctcacctccacctctg	4980	4999	SEQ ID NO: 1560	cagattctcagatgaggga	10034	10053	1	4
SEQ ID NO: 558	atttacagctctgacaagt	4961	5000	SEQ ID NO: 1561	acttttctaaacttgaaat	10033	10052	1	4
SEQ ID NO: 559	aggagcctaccaaaataat	4982	5001	SEQ ID NO: 1562	attatgttgaaaacagtct	4725	4744	1	4
SEQ ID NO: 560	aaagctgaagcacatcaat	5066	5105	SEQ ID NO: 1563	attgttgctcatctccttt	12259	12278	1	4
SEQ ID NO: 561	ctgctggaaacaacgagaa	5092	5111	SEQ ID NO: 1564	ttctgattaccaccagcag	12508	12525	1	4
SEQ ID NO: 562	ttgaaggaattcttgaaaa	5106	5125	SEQ ID NO: 1565	ttttaaaagaaatcttcaa	7726	7745	1	4
SEQ ID NO: 563	gaagtaaaagaaaattttg	5143	5162	SEQ ID NO: 1566	caaaacctactgtctcttc	13571	13590	1	4
SEQ ID NO: 564	tgaagaagatggcaaattt	5170	5189	SEQ ID NO: 1567	aaatgtcagctcttgttca	8641	8660	1	4
SEQ ID NO: 565	aggatctgagttatttttc	5199	5216	SEQ ID NO: 1568	gcaagtcagcccagttcct	9719	9738	1	4
SEQ ID NO: 566	gtgcccttctcggttgctg	5257	5276	SEQ ID NO: 1569	cagccattgacatgagcac	5830	5849	1	4
SEQ ID NO: 567	ggcgcggcctgcgctgctg	5281	5300	SEQ ID NO: 1570	cagctccacagactccgcc	6985	7004	1	4
SEQ ID NO: 568	ctgcgctgctgctgctgct	5316	5335	SEQ ID NO: 1571	agcagaaggtgcgaagcag	7247	7266	1	4
SEQ ID NO: 569	gctggtggcgggcgccagg	5317	5336	SEQ ID NO: 1572	cctggattccacatgcagc	7246	7265	1	4
SEQ ID NO: 570	aagaggaaatgctggaaaa	5333	5352	SEQ ID NO: 1573	tttttcttcactacatctt	7615	7635	1	4
SEQ ID NO: 571	ctggaaaatgtcagcctgg	5338	5357	SEQ ID NO: 1574	ccagacttccacatcccag	12934	12953	1	4
SEQ ID NO: 572	tggagtccctgggactgct	5399	5418	SEQ ID NO: 1575	agcatgcctagtttctcca	8372	8391	1	4
SEQ ID NO: 573	ggagtccctgggactgctg	5404	5423	SEQ ID NO: 1576	cagcatgcctagtttctcc	6496	8515	1	4
SEQ ID NO: 574	tgggactgctgattcaaga	5437	5456	SEQ ID NO: 1577	tcttccatcacttgaccca	9685	9704	1	4
SEQ ID NO: 575	ctgctgattcaagaagtgc	5442	5461	SEQ ID NO: 1578	gcacaccttgacattgcag	4421	4440	1	4
SEQ ID NO: 576	tgccaccaggatcaactgc	5458	5477	SEQ ID NO: 1579	gcaggctgaactggtggca	6380	8399	1	4
SEQ ID NO: 577	gccaccaggatcaactgca	5466	5485	SEQ ID NO: 1580	tgcaggctgaactggtggc	6417	6436	1	4
SEQ ID NO: 578	tgcaaggttgagcgtggag	5486	5506	SEQ ID NO: 1581	cctccatcctctgatctga	11812	11831	1	4
SEQ ID NO: 579	caaggttgagctggaggtt	5498	5517	SEQ ID NO: 1582	aacccctacatgaagcttg	7024	7043	1	4
SEQ ID NO: 580	ctctgcagcttcatcctga	5499	5518	SEQ ID NO: 1583	tcaggaagcttctcaagag	10428	10445	1	4
SEQ ID NO: 581	cagcttcatcctgaagaac	5504	5523	SEQ ID NO: 1584	ggtcttgagttaaatgctg	6123	6142	1	4
SEQ ID NO: 582	gcttcatcctgaagaccag	5576	5595	SEQ ID NO: 1585	ctggacgctaagaggaagc	10767	10776	1	4
SEQ ID NO: 583	tcatcctgaagaccagcca	5649	5668	SEQ ID NO: 1586	tggcatggcattatgatga	12756	12775	1	4
SEQ ID NO: 584	gaaaaccaagaactctgag	5684	5703	SEQ ID NO: 1587	ctcaaccttaatgattttc	9005	9025	1	4
SEQ ID NO: 585	agaactctgaggagtttgc	5767	5785	SEQ ID NO: 1588	gcaagctatatcagtattc	9429	6448	1	4
SEQ ID NO: 586	tctgaggagtttgctgcag	5768	5787	SEQ ID NO: 1589	ctgcagggatcccccagat	13304	13323	1	4
SEQ ID NO: 587	tttgctgcagccatgtcca	5640	5859	SEQ ID NO: 1590	tggaagtgtcagtggcaaa	11168	11187	1	4
SEQ ID NO: 588	caagaggggcatcatttct	5885	5907	SEQ ID NO: 1591	agaataaatgacgttcttg	10911	10930	1	4
SEQ ID NO: 589	tcactttaccgtcaagacg	6035	6054	SEQ ID NO: 1592	cgtctacactatcatgtga	8333	8352	1	4
SEQ ID NO: 590	tttaccgtcaagacgagga	6045	6064	SEQ ID NO: 1593	tccttgacatgttgataaa	11213	11232	1	4
SEQ ID NO: 591	cactggacgctaagaggaa	8080	6099	SEQ ID NO: 1594	ttccagaaagcagccagtg	9372	9391	1	4
SEQ ID NO: 592	aggaagcatgtggcagaag	8081	6100	SEQ ID NO: 1595	cttcatacacattaatcct	10660	10679	1	4
SEQ ID NO: 593	caaggagcaacacctcttc	1639	6158	SEQ ID NO: 1596	gaagtagtactgcatgttg	7931	7950	1	4
SEQ ID NO: 594	acagactttgaaacttgaa	6193	6212	SEQ ID NO: 1597	ttcaattcttcaatgctgt	8670	8889	1	4
SEQ ID NO: 595	tgatgaagcagtcacatct	6233	6252	SEQ ID NO: 1598	agatttgaggattccatca	10201	10220	1	4
SEQ ID NO: 596	agcagtcacatctctcttg	6259	6288	SEQ ID NO: 1599	caaggagaaactgactcgt	9056	9075	1	4
SEQ ID NO: 597	ccagccccatcactttaca	6277	6296	SEQ ID NO: 1600	tgtagtctcctggtgctgg	13310	13329	1	4
SEQ ID NO: 598	ctccactccatcctccagg	6279	6298	SEQ ID NO: 1601	ctggagcttagtaatggag	8413	8432	1	4
SEQ ID NO: 599	catgccaacccccttctga	6312	6331	SEQ ID NO: 1602	tcagatgagggaacacatg	10719	10738	1	4
SEQ ID NO: 600	gagagatcttcaacatggc	6313	6332	SEQ ID NO: 1603	gccaccctggaactctctc	8985	8904	1	4
SEQ ID NO: 601	tcaacatggcgagggatca	6338	6357	SEQ ID NO: 1604	tgatcccacctctcattga	8520	8539	1	4
SEQ ID NO: 602	ccaccttgtatgcgctgag	6344	6363	SEQ ID NO: 1605	ctcagggatctgaaggtgg	11700	11719	1	4
SEQ ID NO: 603	gtcaacaactatcataaga	6372	6391	SEQ ID NO: 1606	tcttgagttaaatgctgac	10427	10446	1	4
SEQ ID NO: 604	tggacattgctaattacct	6407	6426	SEQ ID NO: 1607	aggtatattcgaaagtcca	7991	8010	1	4
SEQ ID NO: 605	ggacattgctaattacctg	6412	6431	SEQ ID NO: 1608	caggtatattcgaaagtcc	11479	11498	1	4
SEQ ID NO: 606	ttctgcgggtcattggaaa	6457	6476	SEQ ID NO: 1609	tttcacatgccaaggagaa	10052	10071	1	4
SEQ ID NO: 607	ccagaactcaagtcttcaa	6486	6505	SEQ ID NO: 1610	ttgaagtgtatgtctcctg	11363	11382	1	4
SEQ ID NO: 608	agtcttcaatcctgaaatg	6487	6506	SEQ ID NO: 1611	catttctgattggtggact	11362	11381	1	4
SEQ ID NO: 609	tgagcaagtgaagaacttt	6532	6551	SEQ ID NO: 1612	aaagtgccacttttactca	7979	7998	1	4
SEQ ID NO: 610	agcaagtgaagaactttgt	6550	6569	SEQ ID NO: 1613	acaaagtcagtgccctgct	9042	9061	1	4
SEQ ID NO: 611	tctgaaagaatctcaactt	6603	6622	SEQ ID NO: 1614	aagtccataatggttcaga	6803	6822	1	4
SEQ ID NO: 612	actgtcatggacttcagaa	6886	6705	SEQ ID NO: 1615	ttctgaatatattgtcagt	6708	6727	1	4
SEQ ID NO: 613	acttgacccagcctcagcc	6688	6707	SEQ ID NO: 1616	ggctcaccctgagagaagt	10433	10452	1	4
SEQ ID NO: 614	tccaaataactaccttcct	6702	6721	SEQ ID NO: 1617	aggaagatatgaagatgga	10151	10170	1	4
SEQ ID NO: 615	actaccctcactgcctttg	6729	6748	SEQ ID NO: 1618	caaatttgtggagggtagt	11153	11172	1	4
SEQ ID NO: 616	ttggatttgcttcagctga	6754	6773	SEQ ID NO: 1619	tcagtataagtacaaccaa	7273	7292	1	4
SEQ ID NO: 617	ttggaagctctttttggga	6815	6834	SEQ ID NO: 1620	tcccgattcacgcttccaa	11396	11415	1	4
SEQ ID NO: 618	ggaagctctttttgggaag	6906	6925	SEQ ID NO: 1621	cttcagaaagctaccttcc	10691	10710	1	4
SEQ ID NO: 619	tttttcccagacagtgtca	6965	6984	SEQ ID NO: 1622	tgaccttctctaagcaaaa	10563	10682	1	4
SEQ ID NO: 620	agacagtgtcaacaaagct	7051	7070	SEQ ID NO: 1623	agcttggttttgccagtct	7096	7115	1	4
SEQ ID NO: 621	ctttggctataccaaagat	7092	7111	SEQ ID NO: 1624	atctcgtgtctaggaaaag	8413	8432	1	4
SEQ ID NO: 622	caaagatgataaacatgag	7191	7210	SEQ ID NO: 1625	ctcaaggataacgtgtttg	7766	7787	1	4
SEQ ID NO: 623	gatatggtaaatggaataa	7219	7238	SEQ ID NO: 1626	ttatcttataattatatca	8518	8537	1	4
SEQ ID NO: 624	ggaataatgctcagtgttg	7224	7243	SEQ ID NO: 1627	caaacacttacttgaattc	10293	10312	1	4
SEQ ID NO: 625	tttgaaatccaaagaagtc	7285	7304	SEQ ID NO: 1628	gacttcagagaaatacaaa	7312	7331	1	4
SEQ ID NO: 626	gatcccccagatgattgga	7319	7335	SEQ ID NO: 1629	tccaatttccctgtggatc	13201	13220	1	4
SEQ ID NO: 627	cagatattggagaggtcaa	7357	7376	SEQ ID NO: 1630	tgaccacacaaaacagtct	7246	7265	1	4
SEQ ID NO: 628	agaatgacttttttctctc	7386	7405	SEQ ID NO: 1631	tgaagtccggattcattct	9715	9734	1	4
SEQ ID NO: 629	gaactccccactggagctg	7456	7475	SEQ ID NO: 1632	cagctcaaccgtacagttc	9068	9087	1	4
SEQ ID NO: 630	atatcttcatgggagtcaa	7467	7485	SEQ ID NO: 1633	tgacttcagtgcagaatat	8517	8536	1	4
SEQ ID NO: 631	gtcatgctccccggaggca	7484	7503	SEQ ID NO: 1634	tggccccgtttaccatgac	10637	1656	1	4
SEQ ID NO: 632	gctgaagtttatcattcct	7570	7569	SEQ ID NO: 1635	aggaggctttaagttcagc	10942	10961	1	4
SEQ ID NO: 633	attccttccccaaagagac	7573	7592	SEQ ID NO: 1636	gtctcttcctccatggaat	10741	10760	1	4
SEQ ID NO: 634	ctcattgagaacaggcagt	7607	7626	SEQ ID NO: 1637	actgactgcacgctttgag	9472	9491	1	4
SEQ ID NO: 635	ttgagcagtattctgtcag	7731	7750	SEQ ID NO: 1638	cttagagaagtgtcttcaa	7857	7876	1	4
SEQ ID NO: 636	accttgtccagtgaagtcc	7862	7881	SEQ ID NO: 1639	ggacggtactgtcccaggt	8914	8933	1	4
SEQ ID NO: 637	ccagtgaagtccaaattcc	7865	7884	SEQ ID NO: 1640	ggaaggcagagtttactgg	6000	6019	1	4
SEQ ID NO: 638	acattcagaacaagaaaat	7666	7885	SEQ ID NO: 1641	atttcctaaagctggatgc	5999	6018	1	4
SEQ ID NO: 639	gaaaaatcaagggtgttat	7901	7920	SEQ ID NO: 1642	ataaactgcaagatttttc	12572	12591	1	4
SEQ ID NO: 640	aaatcaagggtgttatttc	7972	7991	SEQ ID NO: 1643	gaaacaatgcattagattt	13214	13233	1	4
SEQ ID NO: 641	tggcattatgatgaagaga	8042	8061	SEQ ID NO: 1644	tctcccgtgtataatgcca	9410	9429	1	4
SEQ ID NO: 642	aagagaagattgaatttga	8126	8147	SEQ ID NO: 1645	tcaaaacctactgtctcct	12430	12449	1	4
SEQ ID NO: 643	aaatgacttccaatttccc	8218	8237	SEQ ID NO: 1646	gggaactacaatttcattt	12293	12312	1	4
SEQ ID NO: 644	atgacttccaatttccctg	8291	8310	SEQ ID NO: 1647	caggctgattacgagtcat	12571	12590	1	4
SEQ ID NO: 645	acttccaatttccctgtgg	8320	8339	SEQ ID NO: 1648	ccacgaaaaatatggaagt	10018	10037	1	4
SEQ ID NO: 646	agttgcaatgagctcatgg	8430	8449	SEQ ID NO: 1649	ccatcagttcagataaact	9997	10016	1	4
SEQ ID NO: 647	tttgcaagaccacctcaat	8431	8450	SEQ ID NO: 1650	attgacctgtccattcaaa	9237	9256	1	4
SEQ ID NO: 648	gaaggagttcaacctccag	8450	9469	SEQ ID NO: 1651	ctggaattgtcattccttc	9283	9302	1	4
SEQ ID NO: 649	acttccacatcccagaaaa	8468	8487	SEQ ID NO: 1652	ttttaacaaaaagtggaag	10964	10983	1	4
SEQ ID NO: 650	ctcttcttaaaaagcgatg	8543	8562	SEQ ID NO: 1653	catcactgccaaaggagag	13493	13512	1	4
SEQ ID NO: 651	aaaagcgatggccgggtcc	8584	8603	SEQ ID NO: 1654	tgactcactcattgatttt	13780	13799	1	4
SEQ ID NO: 652	ttcctttgccttttggtgg	8644	8863	SEQ ID NO: 1655	ccacaaaacaatgaaggga	13186	13205	1	4
SEQ ID NO: 653	caagtctgtgggattccat	8721	8740	SEQ ID NO: 1656	atgggaaaaaacaggcttg	9223	9242	1	4
SEQ ID NO: 654	aagtccctacttttaccat	8733	8752	SEQ ID NO: 1657	atgggaagtataagaactt	4811	4830	1	4
SEQ ID NO: 655	tgcctctcctgggtgttct	8779	8795	SEQ ID NO: 1658	agaaaaacaaacaaacgga	11164	11183	1	4
SEQ ID NO: 656	accagcacagaccatttca	8787	8808	SEQ ID NO: 1659	tgaagtgtagtctcctggt	12595	12614	1	4
SEQ ID NO: 657	ccagcacagaccatttcag	8791	8810	SEQ ID NO: 1660	ctgaaatacaatgctctgg	9082	9101	1	4
SEQ ID NO: 658	actatcatgtgatgggtct	8806	8825	SEQ ID NO: 1661	agacacctgattttatagt	11002	11021	1	4
SEQ ID NO: 659	accacagatgtctgcttca	8936	8955	SEQ ID NO: 1662	tgaaggctgactctgtggt	12809	12828	1	4
SEQ ID NO: 660	ccacagagtgtctgcttcg	8960	8979	SEQ ID NO: 1663	ctgagcaacaatttgtgga	12284	12303	1	4
SEQ ID NO: 661	tttggactccaaaaagaaa	8980	8999	SEQ ID NO: 1664	tttctctcatgattacaaa	11628	11645	1	4
SEQ ID NO: 662	tcaaagaagtcaagattga	8984	9003	SEQ ID NO: 1665	tcaaggataactgtttgag	13853	13902	1	4
SEQ ID NO: 663	atgagaactacgagctgac	8995	9004	SEQ ID NO: 1666	gtcagatattgttgctcat	13882	13901	1	4
SEQ ID NO: 664	ttaaactctgacaccaatg	9033	9052	SEQ ID NO: 1667	cattcattgaagatgttaa	12247	12266	1	4
SEQ ID NO: 665	gaagtataagaactttgcc	9037	9056	SEQ ID NO: 1668	ggcaaatttgaaggacttc	11659	11676	1	4
SEQ ID NO: 666	aagtataagaaactttgca	9051	9070	SEQ ID NO: 1669	tggcaaatttgaaggactt	11645	11684	1	4
SEQ ID NO: 667	ttcttcagcctgctttctg	9121	9140	SEQ ID NO: 1670	cagaatccagatacaagaa	9741	9750	1	4
SEQ ID NO: 668	ctggatcactaaattccca	9124	9143	SEQ ID NO: 1671	tgggtctttccagagcccg	11069	11088	1	4
SEQ ID NO: 669	aaattaatagtggtgctca	9254	9273	SEQ ID NO: 1672	tgagaagccccaagaattt	7969	7988	1	4
SEQ ID NO: 670	ggccattccagaagggaag	9271	9290	SEQ ID NO: 1673	cttccgttctgtaatggcc	9647	7668	1	4
SEQ ID NO: 671	tgccatctcgagagttcca	9324	9343	SEQ ID NO: 1674	tggaactctctccatggca	12826	12847	1	4
SEQ ID NO: 672	catgtcaaacactttgtta	9424	9443	SEQ ID NO: 1675	taacaaattccttgacatg	13600	13619	1	4
SEQ ID NO: 673	tttgttataaatcttattg	9391	9610	SEQ ID NO: 1676	caataagatcaatagcaaa	11057	11075	1	4
SEQ ID NO: 674	tctggaaaaagggtcatga	9640	9659	SEQ ID NO: 1677	tccatgtcccatttacaga	9682	9701	1	4
SEQ ID NO: 675	cagctcttgttcaggtcca	9646	9665	SEQ ID NO: 1678	tggacctgcaccaaagctg	9272	9291	1	4
SEQ ID NO: 676	ggaggttccccagctctgc	9659	9676	SEQ ID NO: 1679	gcagccctgggaaaactcc	10963	10932	1	4
SEQ ID NO: 677	ctgttttgaagactctcca	9732	9751	SEQ ID NO: 1680	tggagggtagtcataacag	12925	12944	1	4
SEQ ID NO: 678	agtggctgaaacgtgtgca	9749	9768	SEQ ID NO: 1681	tgcagagctttctgccact	10271	10290	1	4
SEQ ID NO: 679	ccaaaatagaagggaatct	9609	9826	SEQ ID NO: 1682	agattcctttctttttcaa	11430	11449	1	4
SEQ ID NO: 680	tgaagagaagattgaattt	9855	9874	SEQ ID NO: 1683	aaattctcttttattttca	8802	8821	1	4
SEQ ID NO: 681	agtggtggcaacaccagca	9882	9901	SEQ ID NO: 1684	tgctagtgaggccaacact	9038	9057	1	4
SEQ ID NO: 682	aaggctccacaagtcatca	9956	9975	SEQ ID NO: 1685	tgatgatatctggaacctt	6885	6904	1	4
SEQ ID NO: 683	gtccgccaggtttctagca	9957	9976	SEQ ID NO: 1686	tgctaagaaccttactgac	6884	5903	1	4
SEQ ID NO: 684	tgatatctggaaccttgga	10011	10030	SEQ ID NO: 1687	ttcactgttcctgaaatca	13297	13316	1	4
SEQ ID NO: 685	gtcaagttgagcaatttct	10169	10188	SEQ ID NO: 1688	agaaaaggcacaccttgac	11176	11197	1	4
SEQ ID NO: 686	atccagatggaaaagggaa	10205	10225	SEQ ID NO: 1689	ttccaatttccctgtggat	13764	13783	1	4
SEQ ID NO: 687	atttgtttgtcaaagaagt	10226	10245	SEQ ID NO: 1690	acttcagagagaaatacat	12072	12091	1	4
SEQ ID NO: 688	ctggaaaatgtcagcctgg	10232	10251	SEQ ID NO: 1691	ccagacttcagttaccagc	11658	11677	1	4
SEQ ID NO: 689	accaggaggttcttcttca	1301	10320	SEQ ID NO: 1692	tgaagtgtgatctcctggt	10539	10558	1	4
SEQ ID NO: 690	aaagaagttctgaaaagat	10400	10419	SEQ ID NO: 1693	attccatcacaaaatcctt	13963	13982	1	4
SEQ ID NO: 691	gctacagcttatggctcca	10416	10435	SEQ ID NO: 1694	tggatctaaatgcagtagc	13208	13227	1	4
SEQ ID NO: 692	atcaatatgatcaatttgt	10451	10480	SEQ ID NO: 1695	caaagaaatcaagattgat	12583	12602	1	4
SEQ ID NO: 693	gaattatctttaaaacatt	10575	10594	SEQ ID NO: 1696	atgtggacaaatataccgg	12355	12374	1	4
SEQ ID NO: 694	cgaggcccgcgctgctggc	10910	10929	SEQ ID NO: 1697	gccagaagtgagatcctcg	4993	5012	1	4
SEQ ID NO: 695	acaactatgaggctgagag	10918	10937	SEQ ID NO: 1698	ctctgagcaacaaattttt	13994	14013	1	4
SEQ ID NO: 696	gctgagagttccagtggag	10975	10994	SEQ ID NO: 1699	ctccatggcaaatgtcagg	8518	8537	1	4
SEQ ID NO: 697	tgaagaaaaaccaagaact	10996	11015	SEQ ID NO: 1700	gagtcattgaggttcttca	12165	12184	1	4
SEQ ID NO: 698	cctacttacatcctgaaca	11256	11275	SEQ ID NO: 1701	tgttcataagggaggtagg	11624	11643	1	4
SEQ ID NO: 699	ctacttacatcctgaacat	11280	11299	SEQ ID NO: 1702	atgttcataagggaggtag	9481	9500	1	4
SEQ ID NO: 700	gagacagaagccaagagcc	11282	11301	SEQ ID NO: 1703	gcttggttttgcccagtct	9039	9058	1	4
SEQ ID NO: 701	cactcactttaccgtcaag	11308	11327	SEQ ID NO: 1704	cttgaacaccaaagtcact	11027	11046	1	4
SEQ ID NO: 702	ctgatcagcagcagccagt	11406	11424	SEQ ID NO: 1705	actgggaagtgcttatcag	8865	8884	1	4
SEQ ID NO: 703	actggacgctaagaggaag	11472	11491	SEQ ID NO: 1706	cttccccaaagagaggagt	12298	12317	1	4
SEQ ID NO: 704	agaggaagcatgtggcaga	11537	11556	SEQ ID NO: 1707	tctggcatttactttctct	13715	13734	1	4
SEQ ID NO: 705	tgaagactctccaggaact	11727	11746	SEQ ID NO: 1708	agttgaaggagactattca	12094	12113	1	4
SEQ ID NO: 706	ctctgagcaaaatatccag	11787	11806	SEQ ID NO: 1709	ctggttactgagctgagag	13096	13115	1	4
SEQ ID NO: 707	atgaagcagtcacatctct	11851	11870	SEQ ID NO: 1710	agagctgccagtcttcatt	6246	6265	1	4
SEQ ID NO: 708	ttgccacagctgattgagg	11964	12003	SEQ ID NO: 1711	cctcctacagtggtggcaa	9212	9231	1	4
SEQ ID NO: 709	agctgattgaggtgtccag	12042	12061	SEQ ID NO: 1712	ctggattccacatgcagct	12661	12680	1	4
SEQ ID NO: 710	tgctccactcacatcctcc	12135	12154	SEQ ID NO: 1713	ggaggctttaagttcagca	8063	8062	1	4
SEQ ID NO: 711	tgaaacgtgtgcatgtcaa	12171	12190	SEQ ID NO: 1714	ttgggagagacaagtttca	10080	10099	1	4
SEQ ID NO: 712	gacattgctaattacctga	12211	12230	SEQ ID NO: 1715	tcagaagctaagcaatgtc	6985	7004	1	4
SEQ ID NO: 713	ttcttcttcagactttcct	12420	12439	SEQ ID NO: 1716	aggagagtccaaatttaga	13439	13458	1	4
SEQ ID NO: 714	ccaatatcttgaaactcag	12439	1258	SEQ ID NO: 1717	tctgaattcattcaattgg	14076	14097	1	4
SEQ ID NO: 715	aaagttagtgaaaagaagt	12440	12459	SEQ ID NO: 1718	aactaccctcactgccttt	14077	14096	1	4
SEQ ID NO: 716	aagttagtgaaagaagttc	12468	12486	SEQ ID NO: 1719	gaacctctggcattttact	13384	13403	1	4
SEQ ID NO: 717	aaagaagttctgaaagaat	12889	12908	SEQ ID NO: 1720	attctctggtaactacttt	13633	13652	1	4
SEQ ID NO: 718	tttggctataccaaagatg	13031	13050	SEQ ID NO: 1721	catcttaggcactgacaaa	14044	14063	1	4
SEQ ID NO: 719	tgttgagaagctgattaaa	13087	13106	SEQ ID NO: 1722	tttagccatcggctcaaca	1392	13811	1	4
SEQ ID NO: 720	caggaagggctcaaagaat	13143	13162	SEQ ID NO: 1723	attcctttaacaattcctg	7024	7043	1	4
SEQ ID NO: 721	aggaagggctcaaagaatg	13182	13201	SEQ ID NO: 1724	cattcctttaacaattcct	10160	10179	1	4
SEQ ID NO: 722	gaagggctcaaagaatgac	13318	13337	SEQ ID NO: 1725	gtcagtcttcaggctcttc	12399	12418	1	4
SEQ ID NO: 723	caaagaatgacttttttct	13369	13388	SEQ ID NO: 1726	agaaggatggcattttttg	10729	10746	1	4
SEQ ID NO: 724	catggagaatgcctttgaa	13443	13462	SEQ ID NO: 1727	ttcagagccaaagtccatg	11480	11499	1	4
SEQ ID NO: 725	ggagccaaggctggagtaa	13552	13571	SEQ ID NO: 1728	ttactccaacgccagctcc	10095	10114	1	4
SEQ ID NO: 726	tcattccttccccaaagag	13599	13618	SEQ ID NO: 1729	ctctctggggcatctatgc	13855	13884	1	4
SEQ ID NO: 727	acctatgagctccagagag	13629	13648	SEQ ID NO: 1730	ctctcaagaccacagaagg	6428	6447	1	4
SEQ ID NO: 728	gggcaaaaacgtcttacag	13718	13737	SEQ ID NO: 1731	tctgaaagacaacgtgccc	9944	9963	1	4
SEQ ID NO: 729	accctggaccttcagaaca	13765	13784	SEQ ID NO: 1732	tgttgctaaggttcagggt	10205	10224	1	4
SEQ ID NO: 730	atgggcgacctaagttgtg	13947	13966	SEQ ID NO: 1733	cacaaattagtttcaccat	7848	7867	1	4
SEQ ID NO: 731	gatgaagagaagattgaat	14050	14069	SEQ ID NO: 1734	atccagcttccccacactt	4222	4241	1	4
SEQ ID NO: 732	caatgtgataccaaaaaaa	4404	4423	SEQ ID NO: 1735	tttttggaaatgccattgt	9595	9614	3	3
SEQ ID NO: 733	gtagataccaaaaaaatga	4543	4562	SEQ ID NO: 1736	tcatgtgatgggtctctac	9055	9074	3	3
SEQ ID NO: 734	gcttcagttcatttggact	25	44	SEQ ID NO: 1737	agtcaagaaggacttaagc	9228	9247	2	3
SEQ ID NO: 735	tttgtttgtcaaagaagtc	39	58	SEQ ID NO: 1738	gacttcagagaaatacaaa	11177	11196	2	3
SEQ ID NO: 736	ttgtttgtcaaagaagtca	219	23	SEQ ID NO: 1739	tgacttcagagaaatacaa	8126	8145	2	3
SEQ ID NO: 737	tggcaatgggaaactcgct	283	302	SEQ ID NO: 1740	agcgagagtcccctgccat	7457	7479	2	3
SEQ ID NO: 738	aacctctggcatttacttt	396	415	SEQ ID NO: 1741	aaaggagatgtcaagggtt	8969	8988	2	3
SEQ ID NO: 739	catttactttctctcatga	464	483	SEQ ID NO: 1742	tcatttgaaagaataaatg	11391	11410	2	3
SEQ ID NO: 740	aaagtcagtgccctgctta	574	593	SEQ ID NO: 1743	taagaaccttactgacttt	7042	7061	2	3
SEQ ID NO: 741	tcccattttttgagacctt	822	841	SEQ ID NO: 1744	aaggacttcaggaatggga	8801	8820	2	3
SEQ ID NO: 742	catcaatattgatcaattt	857	876	SEQ ID NO: 1745	aaattaaaaagtcttgatg	11346	11365	2	3
SEQ ID NO: 743	taaagatagttatgattta	1079	1098	SEQ ID NO: 1746	taaaccaaaaacttggtta	12403	12422	2	3
SEQ ID NO: 744	tattgatgaaatcattgaa	1105	1124	SEQ ID NO: 1747	ttcaaagacttaaaaaata	13104	13123	2	3
SEQ ID NO: 745	atgatctacatttgtttat	1168	1167	SEQ ID NO: 1748	ataaagaaattaaagtcat	3027	3046	2	3
SEQ ID NO: 746	agagacacatacagaatat	1303	1322	SEQ ID NO: 1749	atatattgtcagtgcctct	7848	7867	2	3
SEQ ID NO: 747	gacacatacagaatataga	1432	1451	SEQ ID NO: 1750	tctaaattcagttcttgtc	9130	9149	2	3
SEQ ID NO: 748	agcatgtcaaacactttgt	1492	1511	SEQ ID NO: 1751	acaaagtcagtgccctgct	10920	10939	2	3
SEQ ID NO: 749	tttttagaggaaaccaagg	1567	1586	SEQ ID NO: 1752	ccttgtgtacaccaaaaac	7420	7439	2	3
SEQ ID NO: 750	ttttagaggaaaccaaggc	1619	1638	SEQ ID NO: 1753	gcctttgtgtacaccaaaa	5512	5531	2	3
SEQ ID NO: 751	ggaagatagacttcctgaa	1736	1755	SEQ ID NO: 1754	ttcagaaatactgtttttc	10447	10466	2	3
SEQ ID NO: 752	cactgtttctgagtcccag	1802	1821	SEQ ID NO: 1755	ctgggacctaccaagagtg	13726	13745	2	3
SEQ ID NO: 753	cacaaatcctttggctgtg	1933	1962	SEQ ID NO: 1756	cacatttcaaggaattgtg	3544	3563	2	3
SEQ ID NO: 754	ttcctggatacactgttcc	1948	1967	SEQ ID NO: 1757	ggaactgttgactcaggac	8267	8286	2	3
SEQ ID NO: 755	gaaatctcaagctttctct	2076	2095	SEQ ID NO: 1758	agagccaggtcgagctttc	10459	10476	2	3
SEQ ID NO: 756	tttcttcatcttcatctgt	2213	2232	SEQ ID NO: 1759	acagctgaaagagatgaaa	8316	8335	2	3
SEQ ID NO: 757	tctaccgctaaaggagcag	2366	2385	SEQ ID NO: 1760	ctgcacgctttgaggtaga	9648	9667	2	3
SEQ ID NO: 758	ctaccgctaaaggagcagt	2400	2419	SEQ ID NO: 1761	actgcacgctttgaggtcg	10029	10048	2	3
SEQ ID NO: 759	aggggcctcttttcaccaa	2409	2428	SEQ ID NO: 1762	ttggccaggaagtggccct	4943	4962	2	3
SEQ ID NO: 760	tctccatccctgtaaaaag	2562	2561	SEQ ID NO: 1763	ctttttcaccaacggagaa	10012	10031	2	3
SEQ ID NO: 761	gaaaaacaaagcagattat	2575	2594	SEQ ID NO: 1764	ataaactgcaagatttttc	12152	12171	2	3
SEQ ID NO: 762	actcactcattgattttct	2757	2776	SEQ ID NO: 1765	agaaaatcaggatcgtagt	11807	11626	2	3
SEQ ID NO: 763	taaactaatagatgtaatc	3244	3263	SEQ ID NO: 1766	gattaccaccagcagttta	10895	10914	2	3
SEQ ID NO: 764	caaaaacgagcttcggaag	3660	6379	SEQ ID NO: 1767	cttcgtgagaatatttgaa	11442	11481	2	3
SEQ ID NO: 765	tggaataatgctcagtgtt	6373	3692	SEQ ID NO: 1768	aacacttaacttgaattca	12919	12938	2	3
SEQ ID NO: 766	gatttgaaatccaaagaag	3675	3694	SEQ ID NO: 1769	cttcagagaaatacaaatc	13777	13796	2	3
SEQ ID NO: 767	atttgaaatccaaagaagt	4095	4115	SEQ ID NO: 1770	acttcagagaaatacaaat	9539	9558	2	3
SEQ ID NO: 768	atcaacagccgcttctttg	4543	9583	SEQ ID NO: 1771	caaagaagtcaagattgat	8239	8258	2	3
SEQ ID NO: 769	tgttttgaagactctccag	5127	5146	SEQ ID NO: 1772	ctggaaagttaaaacaaac	13510	13529	2	3
SEQ ID NO: 770	cccttctgatagatgtggt	5345	5364	SEQ ID NO: 1773	accaaagctggcaccaggg	13688	13885	2	3
SEQ ID NO: 771	tgagcaagtgaagaacttt	5412	5431	SEQ ID NO: 1774	aaagccattcagtctctca	8772	8791	2	3
SEQ ID NO: 772	atttgaaatccaaagaagt	5504	5523	SEQ ID NO: 1775	actttctaaacttgaaatt	8786	8805	2	3
SEQ ID NO: 773	atccaaagaagtcccggaa	5840	5859	SEQ ID NO: 1776	tccggggaaacctgggatt	11221	11240	2	3
SEQ ID NO: 774	agagcctacctccgcatct	6277	6295	SEQ ID NO: 1777	agatggtacgttagcctct	13648	13667	2	3
SEQ ID NO: 775	aatgcctttgaactcccca	6280	6299	SEQ ID NO: 1778	tgggaactacaattcattt	8531	8550	2	3
SEQ ID NO: 776	gaagtccaaattccggatt	6312	6331	SEQ ID NO: 1779	aatcttcaatttattcttc	10748	10767	2	3
SEQ ID NO: 777	tgcaagcagaagccagaag	6480	6499	SEQ ID NO: 1780	cttcaggttccatcgtgca	7154	7173	2	3
SEQ ID NO: 778	gaagagaagattgaatttg	6498	6517	SEQ ID NO: 1781	caaaacctactgtgcttcc	11316	11335	2	3
SEQ ID NO: 779	atgctaaaggcacatatgg	6688	6707	SEQ ID NO: 1782	ccatatgaaagtcaagcat	13433	13452	2	3
SEQ ID NO: 780	tccctcacctccacctctg	7052	7071	SEQ ID NO: 1783	cagattctcagatgaggga	12963	12982	2	3
SEQ ID NO: 781	atttacagctctgacaagt	7348	7367	SEQ ID NO: 1784	acttttctaaacttgaaat	12652	12671	2	3
SEQ ID NO: 782	aggagcctaccaaaataat	7745	7764	SEQ ID NO: 1785	attatgttgaaaacagtct	13268	13287	2	3
SEQ ID NO: 783	aaagctgaagcacatcaat	8042	8061	SEQ ID NO: 1786	attgttgctcatctccttt	9473	9492	2	3
SEQ ID NO: 784	ctgctggaaacaacgagaa	9397	9416	SEQ ID NO: 1787	ttctgattaccaccagcag	14075	14094	2	3
SEQ ID NO: 785	ttgaaggaattcttgaaaa	0	19	SEQ ID NO: 1788	ttttaaaagaaatcttcaa	11583	11582	1	3
SEQ ID NO: 786	gaagtaaaagaaaattttg	17	36	SEQ ID NO: 1789	caaaacctactgtctcttc	12670	12689	1	3
SEQ ID NO: 787	tgaagaagatggcaaattt	39	58	SEQ ID NO: 1790	aaatgtcagctcttgttca	1217	1236	1	3
SEQ ID NO: 788	aggatctgagttatttttc	42	61	SEQ ID NO: 1791	gcaagtcagcccagttcct	3744	3763	1	3
SEQ ID NO: 789	gtgcccttctcggttgctg	64	83	SEQ ID NO: 1792	cagccattgacatgagcac	1355	1374	1	3
SEQ ID NO: 790	ggcgcggcctgcgctgctg	81	100	SEQ ID NO: 1793	cagctccacagactccgcc	2674	2693	1	3
SEQ ID NO: 791	ctgcgctgctgctgctgct	95	115	SEQ ID NO: 1794	agcagaaggtgcgaagcag	1080	1099	1	3
SEQ ID NO: 792	gctggtggcgggcgccagg	192	211	SEQ ID NO: 1795	cctggattccacatgcagc	8301	8320	1	3
SEQ ID NO: 793	aagaggaaatgctggaaaa	229	248	SEQ ID NO: 1796	tttttcttcactacatctt	7077	7096	1	3
SEQ ID NO: 794	ctggaaaatgtcagcctgg	245	264	SEQ ID NO: 1797	ccagacttccacatcccag	10059	10078	1	3
SEQ ID NO: 795	tggagtccctgggactgct	289	308	SEQ ID NO: 1798	agcatgcctagtttctcca	8602	8621	1	3
SEQ ID NO: 796	ggagtccctgggactgctg	308	327	SEQ ID NO: 1799	cagcatgcctagtttctcc	13316	13335	1	3
SEQ ID NO: 797	tgggactgctgattcaaga	325	344	SEQ ID NO: 1800	tcttccatcacttgaccca	10696	10715	1	3
SEQ ID NO: 798	ctgctgattcaagaagtgc	335	354	SEQ ID NO: 1801	gcacaccttgacattgcag	4740	4759	1	3
SEQ ID NO: 799	tgccaccaggatcaactgc	340	359	SEQ ID NO: 1802	gcaggctgaactggtggca	1281	1300	1	3
SEQ ID NO: 800	gccaccaggatcaactgca	365	384	SEQ ID NO: 1803	tgcaggctgaactggtggc	1335	1354	1	3
SEQ ID NO: 801	tgcaaggttgagcgtggag	375	394	SEQ ID NO: 1804	cctccatcctctgatctga	5104	5123	1	3
SEQ ID NO: 802	caaggttgagctggaggtt	377	396	SEQ ID NO: 1805	aacccctacatgaagcttg	2688	2707	1	3
SEQ ID NO: 803	ctctgcagcttcatcctga	391	410	SEQ ID NO: 1806	tcaggaagcttctcaagag	1531	1550	1	3
SEQ ID NO: 804	cagcttcatcctgaagaac	396	415	SEQ ID NO: 1807	ggtcttgagttaaatgctg	5222	5241	1	3
SEQ ID NO: 805	gcttcatcctgaagaccag	419	438	SEQ ID NO: 1808	ctggacgctaagaggaagc	3525	3544	1	3
SEQ ID NO: 806	tcatcctgaagaccagcca	422	441	SEQ ID NO: 1809	tggcatggcattatgatga	2199	2218	1	3
SEQ ID NO: 807	gaaaaccaagaactctgag	423	442	SEQ ID NO: 1810	ctcaaccttaatgattttc	9719	9738	1	3
SEQ ID NO: 808	agaactctgaggagtttgc	443	462	SEQ ID NO: 1811	gcaagctatatcagtattc	9066	9085	1	3
SEQ ID NO: 809	tctgaggagtttgctgcag	445	464	SEQ ID NO: 1812	ctgcagggatcccccagat	5639	5658	1	3
SEQ ID NO: 810	tttgctgcagccatgtcca	475	494	SEQ ID NO: 1813	tggaagtgtcagtggcaaa	2996	3015	1	3
SEQ ID NO: 811	caagaggggcatcatttct	476	495	SEQ ID NO: 1814	agaataaatgacgttcttg	2995	3014	1	3
SEQ ID NO: 812	tcactttaccgtcaagacg	482	501	SEQ ID NO: 1815	cgtctacactatcatgtga	1285	1304	1	3
SEQ ID NO: 813	tttaccgtcaagacgagga	499	518	SEQ ID NO: 1816	tccttgacatgttgataaa	7481	7500	1	3
SEQ ID NO: 814	cactggacgctaagaggaa	518	537	SEQ ID NO: 1817	ttccagaaagcagccagtg	13813	13832	1	3
SEQ ID NO: 815	aggaagcatgtggcagaag	520	539	SEQ ID NO: 1818	cttcatacacattaatcct	7564	7583	1	3
SEQ ID NO: 816	caaggagcaacacctcttc	547	566	SEQ ID NO: 1819	gaagtagtactgcatgttg	4844	4853	1	3
SEQ ID NO: 817	acagactttgaaacttgaa	567	586	SEQ ID NO: 1820	ttcaattcttcaatgctgt	7969	7958	1	3
SEQ ID NO: 818	tgatgaagcagtcacatct	568	587	SEQ ID NO: 1821	agatttgaggattccatca	7965	7987	1	3
SEQ ID NO: 819	agcagtcacatctctcttg	570	589	SEQ ID NO: 1822	caaggagaaactgactcgt	7900	7919	1	3
SEQ ID NO: 820	ccagccccatcactttaca	573	592	SEQ ID NO: 1823	tgtagtctcctggtgctgg	6290	6309	1	3
SEQ ID NO: 821	ctccactccatcctccagg	574	593	SEQ ID NO: 1824	ctggagcttagtaatggag	6289	6308	1	3
SEQ ID NO: 822	catgccaacccccttctga	589	608	SEQ ID NO: 1825	tcagatgagggaacacatg	7408	7124	1	3
SEQ ID NO: 823	gagagatcttcaacatggc	607	626	SEQ ID NO: 1826	gccaccctggaactctctc	10058	10077	1	3
SEQ ID NO: 824	tcaacatggcgagggatca	621	640	SEQ ID NO: 1827	tgatcccacctctcattga	8071	8090	1	3
SEQ ID NO: 825	ccaccttgtatgcgctgag	639	658	SEQ ID NO: 1828	ctcagggatctgaaggtgg	8767	8786	1	3
SEQ ID NO: 826	gtcaacaactatcataaga	655	674	SEQ ID NO: 1829	tcttgagttaaatgctgac	2920	2939	1	3
SEQ ID NO: 827	tggacattgctaattacct	662	681	SEQ ID NO: 1830	aggtatattcgaaagtcca	4532	4551	1	3
SEQ ID NO: 828	ggacattgctaattacctg	672	691	SEQ ID NO: 1831	caggtatattcgaaagtcc	10572	10591	1	3
SEQ ID NO: 829	ttctgcgggtcattggaaa	677	696	SEQ ID NO: 1832	tttcacatgccaaggagaa	2483	2502	1	3
SEQ ID NO: 830	ccagaactcaagtcttcaa	679	698	SEQ ID NO: 1833	ttgaagtgtatgtctcctg	11169	11185	1	3
SEQ ID NO: 831	agtcttcaatcctgaaatg	690	709	SEQ ID NO: 1834	catttctgattggtggact	5956	5975	1	3
SEQ ID NO: 832	tgagcaagtgaagaacttt	691	710	SEQ ID NO: 1835	aaagtgccacttttactca	3507	3528	1	3
SEQ ID NO: 833	agcaagtgaagaactttgt	692	711	SEQ ID NO: 1836	acaaagtcagtgccctgct	3468	2487	1	3
SEQ ID NO: 834	tctgaaagaatctcaactt	694	713	SEQ ID NO: 1837	aagtccataatggttcaga	13366	13375	1	3
SEQ ID NO: 835	actgtcatggacttcagaa	695	714	SEQ ID NO: 1838	ttctgaatatattgtcagt	1801	1820	1	3
SEQ ID NO: 836	acttgacccagcctcagcc	769	788	SEQ ID NO: 1839	ggctcaccctgagagaagt	3011	3030	1	3
SEQ ID NO: 837	tccaaataactaccttcct	770	789	SEQ ID NO: 1840	aggaagatatgaagatgga	3010	3029	1	3
SEQ ID NO: 838	actaccctcactgcctttg	775	794	SEQ ID NO: 1841	caaatttgtggagggtagt	12660	12679	1	3
SEQ ID NO: 839	ttggatttgcttcagctga	815	834	SEQ ID NO: 1842	tcagtataagtacaaccaa	4811	4830	1	3
SEQ ID NO: 840	ttggaagctctttttggga	857	876	SEQ ID NO: 1843	tcccgattcacgcttccaa	9331	9360	1	3
SEQ ID NO: 841	ggaagctctttttgggaag	894	913	SEQ ID NO: 1844	cttcagaaagctaccttcc	13457	13476	1	3
SEQ ID NO: 842	tttttcccagacagtgtca	805	914	SEQ ID NO: 1845	tgaccttctctaagcaaaa	13456	13475	1	3
SEQ ID NO: 843	agacagtgtcaacaaagct	900	919	SEQ ID NO: 1846	agcttggttttgccagtct	4284	4303	1	3
SEQ ID NO: 844	ctttggctataccaaagat	901	920	SEQ ID NO: 1847	atctcgtgtctaggaaaag	1067	1085	1	3
SEQ ID NO: 845	caaagatgataaacatgag	925	944	SEQ ID NO: 1848	ctcaaggataacgtgtttg	6786	6805	1	3
SEQ ID NO: 846	gatatggtaaatggaataa	926	945	SEQ ID NO: 1849	ttatcttataattatatca	1236	1257	1	3
SEQ ID NO: 847	ggaataatgctcagtgttg	946	965	SEQ ID NO: 1850	caaacacttacttgaattc	4584	4603	1	3
SEQ ID NO: 848	tttgaaatccaaagaagtc	947	966	SEQ ID NO: 1851	gacttcagagaaatacaaa	4583	4602	1	3
SEQ ID NO: 849	gatcccccagatgattgga	948	967	SEQ ID NO: 1852	tccaatttccctgtggatc	5091	5110	1	3
SEQ ID NO: 850	cagatattggagaggtcaa	970	989	SEQ ID NO: 1853	tgaccacacaaaacagtct	7979	7998	1	3
SEQ ID NO: 851	agaatgacttttttctctc	1000	1019	SEQ ID NO: 1854	tgaagtccggattcattct	8364	8383	1	3
SEQ ID NO: 852	gaactccccactggagctg	1004	1023	SEQ ID NO: 1855	cagctcaaccgtacagttc	11159	11176	1	3
SEQ ID NO: 853	atatcttcatgggagtcaa	1015	1035	SEQ ID NO: 1856	tgacttcagtgcagaatat	10329	10345	1	3
SEQ ID NO: 854	gtcatgctccccggaggca	1038	1057	SEQ ID NO: 1857	tggccccgtttaccatgac	10372	10391	1	3
SEQ ID NO: 855	gctgaagtttatcattcct	1042	1061	SEQ ID NO: 1858	aggaggctttaagttcagc	4868	4887	1	3
SEQ ID NO: 856	attccttccccaaagagac	1079	1098	SEQ ID NO: 1859	gtctcttcctccatggaat	5872	5891	1	3
SEQ ID NO: 857	ctcattgagaacaggcagt	1105	1124	SEQ ID NO: 1860	actgactgcacgctttgag	7104	7123	1	3
SEQ ID NO: 858	ttgagcagtattctgtcag	1106	1125	SEQ ID NO: 1861	cttagagaagtgtcttcaa	11044	11063	1	3
SEQ ID NO: 859	accttgtccagtgaagtcc	1122	1141	SEQ ID NO: 1862	ggacggtactgtcccaggt	12252	12271	1	3
SEQ ID NO: 860	ccagtgaagtccaaattcc	1146	1167	SEQ ID NO: 1863	ggaaggcagagtttactgg	6318	6337	1	3
SEQ ID NO: 861	acattcagaacaagaaaat	1168	1187	SEQ ID NO: 1864	atttcctaaagctggatgc	1359	1378	1	3
SEQ ID NO: 862	gaaaaatcaagggtgttat	1190	1209	SEQ ID NO: 1865	ataaactgcaagatttttc	7104	7123	1	3
SEQ ID NO: 863	aaatcaagggtgttatttc	1192	1211	SEQ ID NO: 1866	gaaacaatgcattagattt	12289	12308	1	3
SEQ ID NO: 864	tggcattatgatgaagaga	1204	1223	SEQ ID NO: 1867	tctcccgtgtataatgcca	13938	12957	1	3
SEQ ID NO: 865	aagagaagattgaatttga	1207	1228	SEQ ID NO: 1868	tcaaaacctactgtctcct	12271	12290	1	3
SEQ ID NO: 866	aaatgacttccaatttccc	1208	1227	SEQ ID NO: 1869	gggaactacaatttcattt	8520	8539	1	3
SEQ ID NO: 867	atgacttccaatttccctg	1223	1242	SEQ ID NO: 1870	caggctgattacgagtcat	4725	4744	1	3
SEQ ID NO: 868	acttccaatttccctgtgg	1269	1278	SEQ ID NO: 1871	ccacgaaaaatatggaagt	4993	5012	1	3
SEQ ID NO: 869	agttgcaatgagctcatgg	1288	1307	SEQ ID NO: 1872	ccatcagttcagataaact	7231	7250	1	3
SEQ ID NO: 870	tttgcaagaccacctcaat	1377	1396	SEQ ID NO: 1873	attgacctgtccattcaaa	12315	12334	1	3
SEQ ID NO: 871	gaaggagttcaacctccag	1380	1399	SEQ ID NO: 1874	ctggaattgtcattccttc	13043	13062	1	3
SEQ ID NO: 872	acttccacatcccagaaaa	1407	1426	SEQ ID NO: 1875	ttttaacaaaaagtggaag	5710	5729	1	3
SEQ ID NO: 873	ctcttcttaaaaagcgatg	1470	1480	SEQ ID NO: 1876	catcactgccaaaggagag	3928	3947	1	3
SEQ ID NO: 874	aaaagcgatggccgggtcc	1491	1510	SEQ ID NO: 1877	tgactcactcattgatttt	4652	4671	1	3
SEQ ID NO: 875	ttcctttgccttttggtgg	1492	1511	SEQ ID NO: 1878	ccacaaaacaatgaaggga	7856	7875	1	3
SEQ ID NO: 876	caagtctgtgggattccat	1497	1515	SEQ ID NO: 1879	atgggaaaaaacaggcttg	12279	12298	1	3
SEQ ID NO: 877	aagtccctacttttaccat	1557	1576	SEQ ID NO: 1880	atgggaagtataagaactt	6345	6364	1	3
SEQ ID NO: 878	tgcctctcctgggtgttct	1567	1586	SEQ ID NO: 1881	agaaaaacaaacaaacgga	7153	7172	1	3
SEQ ID NO: 879	accagcacagaccatttca	1574	1593	SEQ ID NO: 1882	tgaagtgtagtctcctggt	6428	6447	1	3
SEQ ID NO: 880	ccagcacagaccatttcag	1601	1620	SEQ ID NO: 1883	ctgaaatacaatgctctgg	9954	9973	1	3
SEQ ID NO: 881	actatcatgtgatgggtct	1607	1628	SEQ ID NO: 1884	agacacctgattttatagt	8392	8411	1	3
SEQ ID NO: 882	accacagatgtctgcttca	1617	1636	SEQ ID NO: 1885	tgaaggctgactctgtggt	11648	11665	1	3
SEQ ID NO: 883	ccacagagtgtctgcttcg	1619	1638	SEQ ID NO: 1886	ctgagcaacaatttgtgga	13912	1931	1	3
SEQ ID NO: 884	tttggactccaaaaagaaa	1655	1674	SEQ ID NO: 1887	tttctctcatgattacaaa	4421	4440	1	3
SEQ ID NO: 885	tcaaagaagtcaagattga	1664	1683	SEQ ID NO: 1888	tcaaggataactgtttgag	10875	10894	1	3
SEQ ID NO: 886	atgagaactacgagctgac	1665	1664	SEQ ID NO: 1889	gtcagatattgttgctcat	13991	14010	1	3
SEQ ID NO: 887	ttaaactctgacaccaatg	1677	1696	SEQ ID NO: 1890	cattcattgaagatgttaa	11165	11184	1	3
SEQ ID NO: 888	gaagtataagaactttgcc	1680	1699	SEQ ID NO: 1891	ggcaaatttgaaggacttc	5510	5529	1	3
SEQ ID NO: 889	aagtataagaaactttgca	1723	1742	SEQ ID NO: 1892	tggcaaatttgaaggactt	4534	4553	1	3
SEQ ID NO: 890	ttcttcagcctgctttctg	1726	1745	SEQ ID NO: 1893	cagaatccagatacaagaa	7234	7253	1	3
SEQ ID NO: 891	ctggatcactaaattccca	1729	1748	SEQ ID NO: 1894	tgggtctttccagagcccg	4282	4301	1	3
SEQ ID NO: 892	aaattaatagtggtgctca	1742	1761	SEQ ID NO: 1895	tgagaagccccaagaattt	2559	1578	1	3
SEQ ID NO: 893	ggccattccagaagggaag	1744	1763	SEQ ID NO: 1896	cttccgttctgtaatggcc	11299	11818	1	3
SEQ ID NO: 894	tgccatctcgagagttcca	1802	1821	SEQ ID NO: 1897	tggaactctctccatggca	9038	9057	1	3
SEQ ID NO: 895	catgtcaaacactttgtta	1816	1835	SEQ ID NO: 1898	taacaaattccttgacatg	7140	7159	1	3
SEQ ID NO: 896	tttgttataaatcttattg	1817	1836	SEQ ID NO: 1899	caataagatcaatagcaaa	7139	7158	1	3
SEQ ID NO: 897	tctggaaaaagggtcatga	1823	1842	SEQ ID NO: 1900	tccatgtcccatttacaga	6561	6600	1	3
SEQ ID NO: 898	cagctcttgttcaggtcca	1828	1847	SEQ ID NO: 1901	tggacctgcaccaaagctg	2752	2771	1	3
SEQ ID NO: 899	ggaggttccccagctctgc	1834	1853	SEQ ID NO: 1902	gcagccctgggaaaactcc	4534	4553	1	3
SEQ ID NO: 900	ctgttttgaagactctcca	1869	1888	SEQ ID NO: 1903	tggagggtagtcataacag	5326	5345	1	3
SEQ ID NO: 901	agtggctgaaacgtgtgca	1875	1894	SEQ ID NO: 1904	tgcagagctttctgccact	12669	12688	1	3
SEQ ID NO: 902	ccaaaatagaagggaatct	1879	1898	SEQ ID NO: 1905	agattcctttctttttcaa	8901	8920	1	3
SEQ ID NO: 903	tgaagagaagattgaattt	1884	1903	SEQ ID NO: 1906	aaattctcttttattttca	8990	9009	1	3
SEQ ID NO: 904	agtggtggcaacaccagca	1888	1907	SEQ ID NO: 1907	tgctagtgaggccaacact	7623	7642	1	3
SEQ ID NO: 905	aaggctccacaagtcatca	1892	1911	SEQ ID NO: 1908	tgatgatatctggaacctt	12737	12756	1	3
SEQ ID NO: 906	gtccgccaggtttctagca	1893	1912	SEQ ID NO: 1909	tgctaagaaccttactgac	12268	12287	1	3
SEQ ID NO: 907	tgatatctggaaccttgga	1500	1919	SEQ ID NO: 1910	ttcactgttcctgaaatca	9344	9363	1	3
SEQ ID NO: 908	gtcaagttgagcaatttct	1926	1945	SEQ ID NO: 1911	agaaaaggcacaccttgac	9392	9411	1	3
SEQ ID NO: 909	atccagatggaaaagggaa	1933	1952	SEQ ID NO: 1912	ttccaatttccctgtggat	1978	1997	1	3
SEQ ID NO: 910	atttgtttgtcaaagaagt	1941	1960	SEQ ID NO: 1913	acttcagagagaaatacat	7907	7926	1	3
SEQ ID NO: 911	ctggaaaatgtcagcctgg	1946	1967	SEQ ID NO: 1914	ccagacttcagttaccagc	1970	1989	1	3
SEQ ID NO: 912	accaggaggttcttcttca	1972	1991	SEQ ID NO: 1915	tgaagtgtgatctcctggt	12342	12361	1	3
SEQ ID NO: 913	aaagaagttctgaaaagat	1989	2008	SEQ ID NO: 1916	attccatcacaaaatcctt	7028	7047	1	3
SEQ ID NO: 914	gctacagcttatggctcca	2021	2040	SEQ ID NO: 1917	tggatctaaatgcagtagc	6970	6989	1	3
SEQ ID NO: 915	atcaatatgatcaatttgt	2023	2042	SEQ ID NO: 1918	caaagaaatcaagattgat	10276	10295	1	3
SEQ ID NO: 916	gaattatctttaaaacatt	2071	2090	SEQ ID NO: 1919	atgtggacaaatataccgg	5449	5468	1	3
SEQ ID NO: 917	cgaggcccgcgctgctggc	2075	2094	SEQ ID NO: 1920	gccagaagtgagatcctcg	4892	4911	1	3
SEQ ID NO: 918	acaactatgaggctgagag	2076	2095	SEQ ID NO: 1921	ctctgagcaacaaattttt	5279	5298	1	3
SEQ ID NO: 919	gctgagagttccagtggag	2093	2112	SEQ ID NO: 1922	ctccatggcaaatgtcagg	3453	3472	1	3
SEQ ID NO: 920	tgaagaaaaaccaagaact	2150	2169	SEQ ID NO: 1923	gagtcattgaggttcttca	10924	10943	1	3
SEQ ID NO: 921	cctacttacatcctgaaca	2154	2173	SEQ ID NO: 1924	tgttcataagggaggtagg	12257	12276	1	3
SEQ ID NO: 922	ctacttacatcctgaacat	2183	2202	SEQ ID NO: 1925	atgttcataagggaggtag	7842	7861	1	3
SEQ ID NO: 923	gagacagaagccaagagcc	2185	2204	SEQ ID NO: 1926	gcttggttttgcccagtct	4076	4097	1	3
SEQ ID NO: 924	cactcactttaccgtcaag	2196	2215	SEQ ID NO: 1927	cttgaacaccaaagtcact	12950	12969	1	3
SEQ ID NO: 925	ctgatcagcagcagccagt	2201	2220	SEQ ID NO: 1928	actgggaagtgcttatcag	13513	13532	1	3
SEQ ID NO: 926	actggacgctaagaggaag	2202	2221	SEQ ID NO: 1929	cttccccaaagagaggagt	13512	13531	1	3
SEQ ID NO: 927	agaggaagcatgtggcaga	2207	2226	SEQ ID NO: 1930	tctggcatttactttctct	4531	4550	1	3
SEQ ID NO: 928	tgaagactctccaggaact	2212	2231	SEQ ID NO: 1931	agttgaaggagactattca	3777	3795	1	3
SEQ ID NO: 929	ctctgagcaaaatatccag	2218	2237	SEQ ID NO: 1932	ctggttactgagctgagag	7790	7809	1	3
SEQ ID NO: 930	atgaagcagtcacatctct	2221	2240	SEQ ID NO: 1933	agagctgccagtcttcatt	6722	6741	1	3
SEQ ID NO: 931	ttgccacagctgattgagg	2239	2258	SEQ ID NO: 1934	cctcctacagtggtggcaa	6715	6734	1	3
SEQ ID NO: 932	agctgattgaggtgtccag	2323	2342	SEQ ID NO: 1935	ctggattccacatgcagct	2668	2687	1	3
SEQ ID NO: 933	tgctccactcacatcctcc	2329	2348	SEQ ID NO: 1936	ggaggctttaagttcagca	6272	8291	1	3
SEQ ID NO: 934	tgaaacgtgtgcatgtcaa	2349	2368	SEQ ID NO: 1937	ttgggagagacaagtttca	4254	4273	1	3
SEQ ID NO: 935	gacattgctaattacctga	2368	2377	SEQ ID NO: 1938	tcagaagctaagcaatgtc	9946	9985	1	3
SEQ ID NO: 936	ttcttcttcagactttcct	2369	2378	SEQ ID NO: 1939	aggagagtccaaatttaga	10801	10820	1	3
SEQ ID NO: 937	ccaatatcttgaaactcag	2362	2381	SEQ ID NO: 1940	tctgaattcattcaattgg	10359	13078	1	3
SEQ ID NO: 938	aaagttagtgaaaagaagt	2366	2385	SEQ ID NO: 1941	aactaccctcactgccttt	7973	7992	1	3
SEQ ID NO: 939	aagttagtgaaagaagttc	2377	2396	SEQ ID NO: 1942	gaacctctggcattttact	9534	9553	1	3
SEQ ID NO: 940	aaagaagttctgaaagaat	2376	2397	SEQ ID NO: 1943	attctctggtaactacttt	9533	9552	1	3
SEQ ID NO: 941	tttggctataccaaagatg	2379	2398	SEQ ID NO: 1944	catcttaggcactgacaaa	9532	9551	1	3
SEQ ID NO: 942	tgttgagaagctgattaaa	2393	2412	SEQ ID NO: 1945	tttagccatcggctcaaca	6866	6885	1	3
SEQ ID NO: 943	caggaagggctcaaagaat	2400	3419	SEQ ID NO: 1946	attcctttaacaattcctg	8240	8259	1	3
SEQ ID NO: 944	aggaagggctcaaagaatg	2401	2420	SEQ ID NO: 1947	cattcctttaacaattcct	8239	8258	1	3
SEQ ID NO: 945	gaagggctcaaagaatgac	2408	2427	SEQ ID NO: 1948	gtcagtcttcaggctcttc	3680	3899	1	3
SEQ ID NO: 946	caaagaatgacttttttct	2409	2428	SEQ ID NO: 1949	agaaggatggcattttttg	3679	3698	1	3
SEQ ID NO: 947	catggagaatgcctttgaa	2430	2449	SEQ ID NO: 1950	ttcagagccaaagtccatg	8563	8582	1	3
SEQ ID NO: 948	ggagccaaggctggagtaa	241	2450	SEQ ID NO: 1951	ttactccaacgccagctcc	8562	8581	1	3
SEQ ID NO: 949	tcattccttccccaaagag	2447	2466	SEQ ID NO: 1952	ctctctggggcatctatgc	12519	12536	1	3
SEQ ID NO: 950	acctatgagctccagagag	2458	2475	SEQ ID NO: 1953	ctctcaagaccacagaagg	2726	2745	1	3
SEQ ID NO: 951	gggcaaaaacgtcttacag	2461	2480	SEQ ID NO: 1954	tctgaaagacaacgtgccc	10763	10782	1	3
SEQ ID NO: 952	accctggaccttcagaaca	2471	2490	SEQ ID NO: 1955	tgttgctaaggttcagggt	3530	3549	1	3
SEQ ID NO: 953	atgggcgacctaagttgtg	2475	2494	SEQ ID NO: 1956	cacaaattagtttcaccat	2664	2683	1	3
SEQ ID NO: 954	gatgaagagaagattgaat	2493	2512	SEQ ID NO: 1957	atccagcttccccacactt	7931	7950	1	3
SEQ ID NO: 955	caatgtgataccaaaaaaa	2553	2572	SEQ ID NO: 1958	tttttggaaatgccattgt	3506	3525	1	3
SEQ ID NO: 956	gtagataccaaaaaaatga	2574	2593	SEQ ID NO: 1959	tcatgtgatgggtctctac	3827	3846	1	3
SEQ ID NO: 957	gcttcagttcatttggact	2582	2601	SEQ ID NO: 1960	agtcaagaaggacttaagc	6099	6118	1	3
SEQ ID NO: 958	tttgtttgtcaaagaagtc	2597	2618	SEQ ID NO: 1961	gacttcagagaaatacaaa	10710	10729	1	3
SEQ ID NO: 959	ttgtttgtcaaagaagtca	2600	2619	SEQ ID NO: 1962	tgacttcagagaaatacaa	12273	12292	1	3
SEQ ID NO: 960	tggcaatgggaaactcgct	2610	2629	SEQ ID NO: 1963	agcgagagtcccctgccat	5729	5748	1	3
SEQ ID NO: 961	aacctctggcatttacttt	2613	2632	SEQ ID NO: 1964	aaaggagatgtcaagggtt	5376	5394	1	3
SEQ ID NO: 962	catttactttctctcatga	2664	2703	SEQ ID NO: 1965	tcatttgaaagaataaatg	5072	5091	1	3
SEQ ID NO: 963	aaagtcagtgccctgctta	2690	2709	SEQ ID NO: 1966	taagaaccttactgacttt	3051	3070	1	3
SEQ ID NO: 964	tcccattttttgagacctt	2702	2721	SEQ ID NO: 1967	aaggacttcaggaatggga	4098	4117	1	3
SEQ ID NO: 965	catcaatattgatcaattt	2757	2776	SEQ ID NO: 1968	aaattaaaaagtcttgatg	11228	11247	1	3
SEQ ID NO: 966	taaagatagttatgattta	2759	2778	SEQ ID NO: 1969	taaaccaaaaacttggtta	4377	4396	1	3
SEQ ID NO: 967	tattgatgaaatcattgaa	2786	2605	SEQ ID NO: 1970	ttcaaagacttaaaaaata	12333	12352	1	3
SEQ ID NO: 968	atgatctacatttgtttat	2800	2819	SEQ ID NO: 1971	ataaagaaattaaagtcat	4171	4190	1	3
SEQ ID NO: 969	agagacacatacagaatat	2826	2845	SEQ ID NO: 1972	atatattgtcagtgcctct	12554	12573	1	3
SEQ ID NO: 970	gacacatacagaatataga	2833	2852	SEQ ID NO: 1973	tctaaattcagttcttgtc	4799	4818	1	3
SEQ ID NO: 971	agcatgtcaaacactttgt	2835	2554	SEQ ID NO: 1974	acaaagtcagtgccctgct	7631	7650	1	3
SEQ ID NO: 972	tttttagaggaaaccaagg	2861	2860	SEQ ID NO: 1975	ccttgtgtacaccaaaaac	7943	7952	1	3
SEQ ID NO: 973	ttttagaggaaaccaaggc	2871	2690	SEQ ID NO: 1976	gcctttgtgtacaccaaaa	6261	6230	1	3
SEQ ID NO: 974	ggaagatagacttcctgaa	2900	2919	SEQ ID NO: 1977	ttcagaaatactgtttttc	13206	13225	1	3
SEQ ID NO: 975	cactgtttctgagtcccag	2926	2945	SEQ ID NO: 1978	ctgggacctaccaagagtg	10183	10202	1	3
SEQ ID NO: 976	cacaaatcctttggctgtg	2931	2950	SEQ ID NO: 1979	cacatttcaaggaattgtg	11881	11900	1	3
SEQ ID NO: 977	ttcctggatacactgttcc	2932	2951	SEQ ID NO: 1980	ggaactgttgactcaggac	7098	7117	1	3
SEQ ID NO: 978	gaaatctcaagctttctct	2956	2975	SEQ ID NO: 1981	agagccaggtcgagctttc	3197	3216	1	3
SEQ ID NO: 979	tttcttcatcttcatctgt	2979	2998	SEQ ID NO: 1982	acagctgaaagagatgaaa	12023	12042	1	3
SEQ ID NO: 980	tctaccgctaaaggagcag	2961	3000	SEQ ID NO: 1983	ctgcacgctttgaggtaga	8333	8352	1	3
SEQ ID NO: 981	ctaccgctaaaggagcagt	3035	3054	SEQ ID NO: 1984	actgcacgctttgaggtcg	6432	6451	1	3
SEQ ID NO: 982	aggggcctcttttcaccaa	3066	3085	SEQ ID NO: 1985	ttggccaggaagtggccct	3184	3203	1	3
SEQ ID NO: 983	tctccatccctgtaaaaag	3093	3112	SEQ ID NO: 1986	ctttttcaccaacggagaa	12251	12270	1	3
SEQ ID NO: 984	gaaaaacaaagcagattat	3112	3131	SEQ ID NO: 1987	ataaactgcaagatttttc	5136	5155	1	3
SEQ ID NO: 985	actcactcattgattttct	3167	3186	SEQ ID NO: 1988	agaaaatcaggatcgtagt	6541	6560	1	3
SEQ ID NO: 986	taaactaatagatgtaatc	3194	3213	SEQ ID NO: 1989	gattaccaccagcagttta	13209	13226	1	3
SEQ ID NO: 987	caaaaacgagcttcggaag	3214	3233	SEQ ID NO: 1990	cttcgtgagaatatttgaa	9522	9541	1	3
SEQ ID NO: 988	tggaataatgctcagtgtt	3217	3236	SEQ ID NO: 1991	aacacttaacttgaattca	6947	6956	1	3
SEQ ID NO: 989	gatttgaaatccaaagaag	3225	3244	SEQ ID NO: 1992	cttcagagaaatacaaatc	4083	4102	1	3
SEQ ID NO: 990	atttgaaatccaaagaagt	3226	3245	SEQ ID NO: 1993	acttcagagaaatacaaat	4082	4101	1	3
SEQ ID NO: 991	atcaacagccgcttctttg	3280	3299	SEQ ID NO: 1994	caaagaagtcaagattgat	10543	10862	1	3
SEQ ID NO: 992	tgttttgaagactctccag	3261	3300	SEQ ID NO: 1995	ctggaaagttaaaacaaac	10842	10861	1	3
SEQ ID NO: 993	cccttctgatagatgtggt	3297	3316	SEQ ID NO: 1996	accaaagctggcaccaggg	10044	10063	1	3
SEQ ID NO: 994	tgagcaagtgaagaacttt	3363	3382	SEQ ID NO: 1997	aaagccattcagtctctca	4207	4226	1	3
SEQ ID NO: 995	atttgaaatccaaagaagt	3364	3383	SEQ ID NO: 1998	actttctaaacttgaaatt	4054	4073	1	3
SEQ ID NO: 996	atccaaagaagtcccggaa	3382	3401	SEQ ID NO: 1999	tccggggaaacctgggatt	9068	9087	1	3
SEQ ID NO: 997	agagcctacctccgcatct	3390	3409	SEQ ID NO: 2000	agatggtacgttagcctct	9218	9237	1	3
SEQ ID NO: 998	aatgcctttgaactcccca	3427	3446	SEQ ID NO: 2001	tgggaactacaattcattt	6627	6646	1	3
SEQ ID NO: 999	gaagtccaaattccggatt	3430	3449	SEQ ID NO: 2002	aatcttcaatttattcttc	6308	6327	1	3
SEQ ID NO: 1000	tgcaagcagaagccagaag	3441	3460	SEQ ID NO: 2003	cttcaggttccatcgtgca	13830	13849	1	3
SEQ ID NO: 1001	gaagagaagattgaatttg	3446	3465	SEQ ID NO: 2004	caaaacctactgtgcttcc	10212	10231	1	3
SEQ ID NO: 1002	atgctaaaggcacatatgg	3447	3486	SEQ ID NO: 2005	ccatatgaaagtcaagcat	10211	10230	1	3
SEQ ID NO: 1003	tccctcacctccacctctg	3465	3474	SEQ ID NO: 2006	cagattctcagatgaggga	11340	11359	1	3
SEQ ID NO: 2007	atttacagctctgacaagt	3947	3966	SEQ ID NO: 2313	acttttctaaacttgaaat	6335	6354	1	3
SEQ ID NO: 2008	aggagcctaccaaaataat	3963	3982	SEQ ID NO: 2314	attatgttgaaaacagtct	7356	7374	1	3
SEQ ID NO: 2009	aaagctgaagcacatcaat	3976	3995	SEQ ID NO: 2315	attgttgctcatctccttt	8302	8321	1	3
SEQ ID NO: 2010	ctgctggaaacaacgagaa	3967	4006	SEQ ID NO: 2316	ttctgattaccaccagcag	9039	9058	1	3
SEQ ID NO: 2011	ttgaaggaattcttgaaaa	3988	4007	SEQ ID NO: 2317	ttttaaaagaaatcttcaa	10446	10485	1	3
SEQ ID NO: 2012	gaagtaaaagaaaattttg	3990	4009	SEQ ID NO: 2318	caaaacctactgtctcttc	12022	12041	1	3
SEQ ID NO: 2013	tgaagaagatggcaaattt	4038	4057	SEQ ID NO: 2319	aaatgtcagctcttgttca	10537	10556	1	3
SEQ ID NO: 2014	aggatctgagttatttttc	4044	4063	SEQ ID NO: 2320	gcaagtcagcccagttcct	8065	8084	1	3
SEQ ID NO: 2015	gtgcccttctcggttgctg	4066	4085	SEQ ID NO: 2321	cagccattgacatgagcac	8805	8824	1	3
SEQ ID NO: 2016	ggcgcggcctgcgctgctg	4067	4086	SEQ ID NO: 2322	cagctccacagactccgcc	5720	5739	1	3
SEQ ID NO: 2017	ctgcgctgctgctgctgct	4094	4113	SEQ ID NO: 2323	agcagaaggtgcgaagcag	5322	5341	1	3
SEQ ID NO: 2018	gctggtggcgggcgccagg	4134	4153	SEQ ID NO: 2324	cctggattccacatgcagc	6246	5265	1	3
SEQ ID NO: 2019	aagaggaaatgctggaaaa	4148	4167	SEQ ID NO: 2325	tttttcttcactacatctt	8835	5854	1	3
SEQ ID NO: 2020	ctggaaaatgtcagcctgg	4170	4189	SEQ ID NO: 2326	ccagacttccacatcccag	4727	4746	1	3
SEQ ID NO: 2021	tggagtccctgggactgct	4184	4203	SEQ ID NO: 2327	agcatgcctagtttctcca	5580	5599	1	3
SEQ ID NO: 2022	ggagtccctgggactgctg	4167	4206	SEQ ID NO: 2328	cagcatgcctagtttctcc	11663	11682	1	3
SEQ ID NO: 2023	tgggactgctgattcaaga	4188	4207	SEQ ID NO: 2329	tcttccatcacttgaccca	11662	11681	1	3
SEQ ID NO: 2024	ctgctgattcaagaagtgc	4224	4243	SEQ ID NO: 2330	gcacaccttgacattgcag	13351	13370	1	3
SEQ ID NO: 2025	tgccaccaggatcaactgc	4272	4291	SEQ ID NO: 2331	gcaggctgaactggtggca	8603	8622	1	3
SEQ ID NO: 2026	gccaccaggatcaactgca	4283	4302	SEQ ID NO: 2332	tgcaggctgaactggtggc	13161	13160	1	3
SEQ ID NO: 2027	tgcaaggttgagcgtggag	4295	4314	SEQ ID NO: 2333	cctccatcctctgatctga	6957	6976	1	3
SEQ ID NO: 2028	caaggttgagctggaggtt	4304	4323	SEQ ID NO: 2334	aacccctacatgaagcttg	11080	11099	1	3
SEQ ID NO: 2029	ctctgcagcttcatcctga	4305	4324	SEQ ID NO: 2335	tcaggaagcttctcaagag	11079	11098	1	3
SEQ ID NO: 2030	cagcttcatcctgaagaac	4311	4330	SEQ ID NO: 2336	ggtcttgagttaaatgctg	8569	8588	1	3
SEQ ID NO: 2031	gcttcatcctgaagaccag	4344	4363	SEQ ID NO: 2337	ctggacgctaagaggaagc	9976	9995	1	3
SEQ ID NO: 2032	tcatcctgaagaccagcca	4345	4364	SEQ ID NO: 2338	tggcatggcattatgatga	9975	9994	1	3
SEQ ID NO: 2033	gaaaaccaagaactctgag	4355	4374	SEQ ID NO: 2339	ctcaaccttaatgattttc	6479	6498	1	3
SEQ ID NO: 2034	agaactctgaggagtttgc	4398	4417	SEQ ID NO: 2340	gcaagctatatcagtattc	8677	6698	1	3
SEQ ID NO: 2035	tctgaggagtttgctgcag	4404	4423	SEQ ID NO: 2341	ctgcagggatcccccagat	7094	7113	1	3
SEQ ID NO: 2036	tttgctgcagccatgtcca	4441	4460	SEQ ID NO: 2342	tggaagtgtcagtggcaaa	9206	9225	1	3
SEQ ID NO: 2037	caagaggggcatcatttct	4448	4467	SEQ ID NO: 2343	agaataaatgacgttcttg	11294	11313	1	3
SEQ ID NO: 2038	tcactttaccgtcaagacg	4454	4473	SEQ ID NO: 2344	cgtctacactatcatgtga	12223	12242	1	3
SEQ ID NO: 2039	tttaccgtcaagacgagga	4455	4474	SEQ ID NO: 2345	tccttgacatgttgataaa	12222	12241	1	3
SEQ ID NO: 2040	cactggacgctaagaggaa	4457	4476	SEQ ID NO: 2346	ttccagaaagcagccagtg	9272	9291	1	3
SEQ ID NO: 2041	aggaagcatgtggcagaag	4535	4554	SEQ ID NO: 2347	cttcatacacattaatcct	112061	11225	1	3
SEQ ID NO: 2042	caaggagcaacacctcttc	4543	4562	SEQ ID NO: 2348	gaagtagtactgcatgttg	8014	8033	1	3
SEQ ID NO: 2043	acagactttgaaacttgaa	4561	4580	SEQ ID NO: 2349	ttcaattcttcaatgctgt	13414	13433	1	3
SEQ ID NO: 2044	tgatgaagcagtcacatct	4576	4597	SEQ ID NO: 2350	agatttgaggattccatca	11987	12006	1	3
SEQ ID NO: 2045	agcagtcacatctctcttg	4580	4599	SEQ ID NO: 2351	caaggagaaactgactcgt	8885	8884	1	3
SEQ ID NO: 2046	ccagccccatcactttaca	4597	4616	SEQ ID NO: 2352	tgtagtctcctggtgctgg	9237	9256	1	3
SEQ ID NO: 2047	ctccactccatcctccagg	4606	4625	SEQ ID NO: 2353	ctggagcttagtaatggag	9364	9383	1	3
SEQ ID NO: 2048	catgccaacccccttctga	4659	4678	SEQ ID NO: 2354	tcagatgagggaacacatg	6182	6201	1	3
SEQ ID NO: 2049	gagagatcttcaacatggc	4660	4679	SEQ ID NO: 2355	gccaccctggaactctctc	9300	9319	1	3
SEQ ID NO: 2050	tcaacatggcgagggatca	4684	4703	SEQ ID NO: 2356	tgatcccacctctcattga	9368	9387	1	3
SEQ ID NO: 2051	ccaccttgtatgcgctgag	4722	4741	SEQ ID NO: 2357	ctcagggatctgaaggtgg	12283	12302	1	3
SEQ ID NO: 2052	gtcaacaactatcataaga	4754	4773	SEQ ID NO: 2358	tcttgagttaaatgctgac	12255	12274	1	3
SEQ ID NO: 2053	tggacattgctaattacct	4755	4774	SEQ ID NO: 2359	aggtatattcgaaagtcca	12254	12273	1	3
SEQ ID NO: 2054	ggacattgctaattacctg	4785	4804	SEQ ID NO: 2360	caggtatattcgaaagtcc	5969	5988	1	3
SEQ ID NO: 2055	ttctgcgggtcattggaaa	4796	4815	SEQ ID NO: 2361	tttcacatgccaaggagaa	6912	6931	1	3
SEQ ID NO: 2056	ccagaactcaagtcttcaa	4860	4879	SEQ ID NO: 2362	ttgaagtgtatgtctcctg	13024	13043	1	3
SEQ ID NO: 2057	agtcttcaatcctgaaatg	4899	4918	SEQ ID NO: 2363	catttctgattggtggact	6893	6912	1	3
SEQ ID NO: 2058	tgagcaagtgaagaacttt	4932	4951	SEQ ID NO: 2364	aaagtgccacttttactca	5340	5359	1	3
SEQ ID NO: 2059	agcaagtgaagaactttgt	4955	4974	SEQ ID NO: 2365	acaaagtcagtgccctgct	12541	12560	1	3
SEQ ID NO: 2060	tctgaaagaatctcaactt	4973	4992	SEQ ID NO: 2366	aagtccataatggttcaga	10095	10114	1	3
SEQ ID NO: 2061	actgtcatggacttcagaa	4999	5016	SEQ ID NO: 2367	ttctgaatatattgtcagt	14011	14030	1	3
SEQ ID NO: 2062	acttgacccagcctcagcc	5032	5051	SEQ ID NO: 2368	ggctcaccctgagagaagt	6786	6805	1	3
SEQ ID NO: 2063	tccaaataactaccttcct	5075	5094	SEQ ID NO: 2369	aggaagatatgaagatgga	6177	5196	1	3
SEQ ID NO: 2064	actaccctcactgcctttg	5084	5103	SEQ ID NO: 2370	caaatttgtggagggtagt	8234	6520	1	3
SEQ ID NO: 2065	ttggatttgcttcagctga	5108	5127	SEQ ID NO: 2371	tcagtataagtacaaccaa	9214	6233	1	3
SEQ ID NO: 2066	ttggaagctctttttggga	5127	5146	SEQ ID NO: 2372	tcccgattcacgcttccaa	6891	6910	1	3
SEQ ID NO: 2067	ggaagctctttttgggaag	5140	5159	SEQ ID NO: 2373	cttcagaaagctaccttcc	6491	6510	1	3
SEQ ID NO: 2068	tttttcccagacagtgtca	5142	5161	SEQ ID NO: 2374	tgaccttctctaagcaaaa	6428	6447	1	3
SEQ ID NO: 2069	agacagtgtcaacaaagct	5185	5204	SEQ ID NO: 2375	agcttggttttgccagtct	9498	7517	1	3
SEQ ID NO: 2070	ctttggctataccaaagat	5223	5242	SEQ ID NO: 2376	atctcgtgtctaggaaaag	8141	8160	1	3
SEQ ID NO: 2071	caaagatgataaacatgag	5239	5258	SEQ ID NO: 2377	ctcaaggataacgtgtttg	11348	11367	1	3
SEQ ID NO: 2072	gatatggtaaatggaataa	6295	5314	SEQ ID NO: 2378	ttatcttataattatatca	11969	11988	1	3
SEQ ID NO: 2073	ggaataatgctcagtgttg	5328	5347	SEQ ID NO: 2379	caaacacttacttgaattc	8478	6497	1	3
SEQ ID NO: 2074	tttgaaatccaaagaagtc	5341	5360	SEQ ID NO: 2380	gacttcagagaaatacaaa	5738	5757	1	3
SEQ ID NO: 2075	gatcccccagatgattgga	5345	5364	SEQ ID NO: 2381	tccaatttccctgtggatc	6694	6713	1	3
SEQ ID NO: 2076	cagatattggagaggtcaa	5378	5397	SEQ ID NO: 2382	tgaccacacaaaacagtct	10988	11007	1	3
SEQ ID NO: 2077	agaatgacttttttctctc	5381	5400	SEQ ID NO: 2383	tgaagtccggattcattct	12482	12501	1	3
SEQ ID NO: 2078	gaactccccactggagctg	5387	5406	SEQ ID NO: 2384	cagctcaaccgtacagttc	11844	11863	1	3
SEQ ID NO: 2079	atatcttcatgggagtcaa	5412	5431	SEQ ID NO: 2385	tgacttcagtgcagaatat	7362	7381	1	3
SEQ ID NO: 2080	gtcatgctccccggaggca	5427	5446	SEQ ID NO: 2386	tggccccgtttaccatgac	8014	8033	1	3
SEQ ID NO: 2081	gctgaagtttatcattcct	5435	5454	SEQ ID NO: 2387	aggaggctttaagttcagc	10666	10685	1	3
SEQ ID NO: 2082	attccttccccaaagagac	5480	5479	SEQ ID NO: 2388	gtctcttcctccatggaat	6570	5589	1	3
SEQ ID NO: 2083	ctcattgagaacaggcagt	5433	5502	SEQ ID NO: 2389	actgactgcacgctttgag	7267	7286	1	3
SEQ ID NO: 2084	ttgagcagtattctgtcag	5585	5607	SEQ ID NO: 2390	cttagagaagtgtcttcaa	9368	9387	1	3
SEQ ID NO: 2085	accttgtccagtgaagtcc	5591	5610	SEQ ID NO: 2391	ggacggtactgtcccaggt	6263	6262	1	3
SEQ ID NO: 2086	ccagtgaagtccaaattcc	5594	5513	SEQ ID NO: 2392	ggaaggcagagtttactgg	10119	10138	1	3
SEQ ID NO: 2087	acattcagaacaagaaaat	5608	5627	SEQ ID NO: 2393	atttcctaaagctggatgc	7057	7076	1	3
SEQ ID NO: 2088	gaaaaatcaagggtgttat	5618	5537	SEQ ID NO: 2394	ataaactgcaagatttttc	12150	12169	1	3
SEQ ID NO: 2089	aaatcaagggtgttatttc	5678	5597	SEQ ID NO: 2395	gaaacaatgcattagattt	9079	9098	1	3
SEQ ID NO: 2090	tggcattatgatgaagaga	5697	5716	SEQ ID NO: 2396	tctcccgtgtataatgcca	7442	7461	1	3
SEQ ID NO: 2091	aagagaagattgaatttga	5732	5751	SEQ ID NO: 2397	tcaaaacctactgtctcct	8588	8607	1	3
SEQ ID NO: 2092	aaatgacttccaatttccc	5782	5601	SEQ ID NO: 2398	gggaactacaatttcattt	8620	8639	1	3
SEQ ID NO: 2093	atgacttccaatttccctg	5763	5802	SEQ ID NO: 2399	caggctgattacgagtcat	8619	8638	1	3
SEQ ID NO: 2094	acttccaatttccctgtgg	5784	5803	SEQ ID NO: 2400	ccacgaaaaatatggaagt	8618	8367	1	3
SEQ ID NO: 2095	agttgcaatgagctcatgg	5786	5805	SEQ ID NO: 2401	ccatcagttcagataaact	12404	12423	1	3
SEQ ID NO: 2096	tttgcaagaccacctcaat	5792	5811	SEQ ID NO: 2402	attgacctgtccattcaaa	6493	6512	1	3
SEQ ID NO: 2097	gaaggagttcaacctccag	5793	5812	SEQ ID NO: 2403	ctggaattgtcattccttc	12967	12986	1	3
SEQ ID NO: 2098	acttccacatcccagaaaa	5851	5870	SEQ ID NO: 2404	ttttaacaaaaagtggaag	12205	12224	1	3
SEQ ID NO: 2099	ctcttcttaaaaagcgatg	5871	5890	SEQ ID NO: 2405	catcactgccaaaggagag	1080	1099	1	3
SEQ ID NO: 2100	aaaagcgatggccgggtcc	5906	5925	SEQ ID NO: 2406	tgactcactcattgatttt	8268	8285	1	3
SEQ ID NO: 2101	ttcctttgccttttggtgg	8975	6994	SEQ ID NO: 2407	ccacaaaacaatgaaggga	13604	13623	1	3
SEQ ID NO: 2102	caagtctgtgggattccat	5985	6004	SEQ ID NO: 2408	atgggaaaaaacaggcttg	13343	13362	1	3
SEQ ID NO: 2103	aagtccctacttttaccat	6001	6020	SEQ ID NO: 2409	atgggaagtataagaactt	6410	6429	1	3
SEQ ID NO: 2104	tgcctctcctgggtgttct	6038	6057	SEQ ID NO: 2410	agaaaaacaaacaaacgga	11602	11621	1	3
SEQ ID NO: 2105	accagcacagaccatttca	6053	6072	SEQ ID NO: 2411	tgaagtgtagtctcctggt	8029	8046	1	3
SEQ ID NO: 2106	ccagcacagaccatttcag	6076	6095	SEQ ID NO: 2412	ctgaaatacaatgctctgg	9674	9693	1	3
SEQ ID NO: 2107	actatcatgtgatgggtct	6095	6114	SEQ ID NO: 2413	agacacctgattttatagt	8591	8610	1	3
SEQ ID NO: 2108	accacagatgtctgcttca	6124	6143	SEQ ID NO: 2414	tgaaggctgactctgtggt	8265	8264	1	3
SEQ ID NO: 2109	ccacagagtgtctgcttcg	6190	6209	SEQ ID NO: 2415	ctgagcaacaatttgtgga	6980	6999	1	3
SEQ ID NO: 2110	tttggactccaaaaagaaa	6227	6246	SEQ ID NO: 2416	tttctctcatgattacaaa	9189	9188	1	3
SEQ ID NO: 2111	tcaaagaagtcaagattga	6249	6268	SEQ ID NO: 2417	tcaaggataactgtttgag	12962	12981	1	3
SEQ ID NO: 2112	atgagaactacgagctgac	6268	6267	SEQ ID NO: 2418	gtcagatattgttgctcat	101779	10198	1	3
SEQ ID NO: 2113	ttaaactctgacaccaatg	8275	6297	SEQ ID NO: 2419	cattcattgaagatgttaa	13647	13666	1	3
SEQ ID NO: 2114	gaagtataagaactttgcc	6280	6299	SEQ ID NO: 2420	ggcaaatttgaaggacttc	6350	6369	1	3
SEQ ID NO: 2115	aagtataagaaactttgca	6307	6326	SEQ ID NO: 2421	tggcaaatttgaaggactt	9568	9587	1	3
SEQ ID NO: 2116	ttcttcagcctgctttctg	6329	6348	SEQ ID NO: 2422	cagaatccagatacaagaa	9558	9577	1	3
SEQ ID NO: 2117	ctggatcactaaattccca	6336	6355	SEQ ID NO: 2423	tgggtctttccagagcccg	12940	12959	1	3
SEQ ID NO: 2118	aaattaatagtggtgctca	6415	6434	SEQ ID NO: 2424	tgagaagccccaagaattt	11821	11840	1	3
SEQ ID NO: 2119	ggccattccagaagggaag	6443	6462	SEQ ID NO: 2425	cttccgttctgtaatggcc	13614	13633	1	3
SEQ ID NO: 2120	tgccatctcgagagttcca	6462	6471	SEQ ID NO: 2426	tggaactctctccatggca	7716	7735	1	3
SEQ ID NO: 2121	catgtcaaacactttgtta	6461	6480	SEQ ID NO: 2427	taacaaattccttgacatg	12881	12880	1	3
SEQ ID NO: 2122	tttgttataaatcttattg	6469	6508	SEQ ID NO: 2428	caataagatcaatagcaaa	10471	10490	1	3
SEQ ID NO: 2123	tctggaaaaagggtcatga	6495	6514	SEQ ID NO: 2429	tccatgtcccatttacaga	11800	11819	1	3
SEQ ID NO: 2124	cagctcttgttcaggtcca	6526	6545	SEQ ID NO: 2430	tggacctgcaccaaagctg	11155	11174	1	3
SEQ ID NO: 2125	ggaggttccccagctctgc	6533	6552	SEQ ID NO: 2431	gcagccctgggaaaactcc	8372	8391	1	3
SEQ ID NO: 2126	ctgttttgaagactctcca	6536	6556	SEQ ID NO: 2432	tggagggtagtcataacag	9687	9706	1	3
SEQ ID NO: 2127	agtggctgaaacgtgtgca	6633	6652	SEQ ID NO: 2433	tgcagagctttctgccact	1925	1944	1	3
SEQ ID NO: 2128	ccaaaatagaagggaatct	6649	6668	SEQ ID NO: 2434	agattcctttctttttcaa	13807	13826	1	3
SEQ ID NO: 2129	tgaagagaagattgaattt	6675	6894	SEQ ID NO: 2435	aaattctcttttattttca	13120	13139	1	3
SEQ ID NO: 2130	agtggtggcaacaccagca	6689	6708	SEQ ID NO: 2436	tgctagtgaggccaacact	13856	13875	1	3
SEQ ID NO: 2131	aaggctccacaagtcatca	6690	6709	SEQ ID NO: 2437	tgatgatatctggaacctt	13855	13874	1	3
SEQ ID NO: 2132	gtccgccaggtttctagca	6695	6714	SEQ ID NO: 2438	tgctaagaaccttactgac	14076	14095	1	3
SEQ ID NO: 2133	tgatatctggaaccttgga	6711	6730	SEQ ID NO: 2439	ttcactgttcctgaaatca	8260	8279	1	3
SEQ ID NO: 2134	gtcaagttgagcaatttct	6739	6758	SEQ ID NO: 2440	agaaaaggcacaccttgac	12858	12875	1	3
SEQ ID NO: 2135	atccagatggaaaagggaa	6740	6759	SEQ ID NO: 2441	ttccaatttccctgtggat	8781	8780	1	3
SEQ ID NO: 2136	atttgtttgtcaaagaagt	6745	6764	SEQ ID NO: 2442	acttcagagagaaatacat	13093	13112	1	3
SEQ ID NO: 2137	ctggaaaatgtcagcctgg	6769	6788	SEQ ID NO: 2443	ccagacttcagttaccagc	11694	11713	1	3
SEQ ID NO: 2138	accaggaggttcttcttca	6772	6791	SEQ ID NO: 2444	tgaagtgtgatctcctggt	11802	11821	1	3
SEQ ID NO: 2139	aaagaagttctgaaaagat	6797	6816	SEQ ID NO: 2445	attccatcacaaaatcctt	11167	11186	1	3
SEQ ID NO: 2140	gctacagcttatggctcca	6816	6835	SEQ ID NO: 2446	tggatctaaatgcagtagc	9863	9882	1	3
SEQ ID NO: 2141	atcaatatgatcaatttgt	6820	6839	SEQ ID NO: 2447	caaagaaatcaagattgat	8005	8025	1	3
SEQ ID NO: 2142	gaattatctttaaaacatt	6880	6899	SEQ ID NO: 2448	atgtggacaaatataccgg	10080	10099	1	3
SEQ ID NO: 2143	cgaggcccgcgctgctggc	6886	6905	SEQ ID NO: 2449	gccagaagtgagatcctcg	9057	9076	1	3
SEQ ID NO: 2144	acaactatgaggctgagag	6916	6935	SEQ ID NO: 2450	ctctgagcaacaaattttt	9483	9502	1	3
SEQ ID NO: 2145	gctgagagttccagtggag	6942	6961	SEQ ID NO: 2451	ctccatggcaaatgtcagg	3680	3699	1	3
SEQ ID NO: 2146	tgaagaaaaaccaagaact	7052	7071	SEQ ID NO: 2452	gagtcattgaggttcttca	6183	6202	1	3
SEQ ID NO: 2147	cctacttacatcctgaaca	7053	7072	SEQ ID NO: 2453	tgttcataagggaggtagg	5326	5345	1	3
SEQ ID NO: 2148	ctacttacatcctgaacat	7062	7061	SEQ ID NO: 2454	atgttcataagggaggtag	13125	13144	1	3
SEQ ID NO: 2149	gagacagaagccaagagcc	7103	7122	SEQ ID NO: 2455	gcttggttttgcccagtct	12021	12040	1	3
SEQ ID NO: 2150	cactcactttaccgtcaag	7152	7171	SEQ ID NO: 2456	cttgaacaccaaagtcact	10323	10342	1	3
SEQ ID NO: 2151	ctgatcagcagcagccagt	7156	7176	SEQ ID NO: 2457	actgggaagtgcttatcag	12148	12167	1	3
SEQ ID NO: 2152	actggacgctaagaggaag	7215	7234	SEQ ID NO: 2458	cttccccaaagagaggagt	10160	10179	1	3
SEQ ID NO: 2153	agaggaagcatgtggcaga	7256	7275	SEQ ID NO: 2459	tctggcatttactttctct	11326	11345	1	3
SEQ ID NO: 2154	tgaagactctccaggaact	7283	7282	SEQ ID NO: 2460	agttgaaggagactattca	2069	2068	1	3
SEQ ID NO: 2155	ctctgagcaaaatatccag	7272	7291	SEQ ID NO: 2461	ctggttactgagctgagag	9061	9060	1	3
SEQ ID NO: 2156	atgaagcagtcacatctct	7280	7299	SEQ ID NO: 2462	agagctgccagtcttcatt	7435	7454	1	3
SEQ ID NO: 2157	ttgccacagctgattgagg	7284	7303	SEQ ID NO: 2463	cctcctacagtggtggcaa	10411	10430	1	3
SEQ ID NO: 2158	agctgattgaggtgtccag	7292	7311	SEQ ID NO: 2464	ctggattccacatgcagct	9945	9964	1	3
SEQ ID NO: 2159	tgctccactcacatcctcc	7345	7364	SEQ ID NO: 2465	ggaggctttaagttcagca	7414	7433	1	3
SEQ ID NO: 2160	tgaaacgtgtgcatgtcaa	7346	7355	SEQ ID NO: 2466	ttgggagagacaagtttca	7413	7432	1	3
SEQ ID NO: 2161	gacattgctaattacctga	7347	7366	SEQ ID NO: 2467	tcagaagctaagcaatgtc	10487	10506	1	3
SEQ ID NO: 2162	ttcttcttcagactttcct	7348	7367	SEQ ID NO: 2468	aggagagtccaaatttaga	10486	10505	1	3
SEQ ID NO: 2163	ccaatatcttgaaactcag	7349	7368	SEQ ID NO: 2469	tctgaattcattcaattgg	10485	10504	1	3
SEQ ID NO: 2164	aaagttagtgaaaagaagt	7372	7391	SEQ ID NO: 2470	aactaccctcactgccttt	11479	11498	1	3
SEQ ID NO: 2165	aagttagtgaaagaagttc	7398	7417	SEQ ID NO: 2471	gaacctctggcattttact	8783	8802	1	3
SEQ ID NO: 2166	aaagaagttctgaaagaat	7433	7452	SEQ ID NO: 2472	attctctggtaactacttt	7954	7983	1	3
SEQ ID NO: 2167	tttggctataccaaagatg	7434	7453	SEQ ID NO: 2473	catcttaggcactgacaaa	8630	8649	1	3
SEQ ID NO: 2168	tgttgagaagctgattaaa	7444	7463	SEQ ID NO: 2474	tttagccatcggctcaaca	10723	10742	1	3
SEQ ID NO: 2169	caggaagggctcaaagaat	7465	7464	SEQ ID NO: 2475	attcctttaacaattcctg	8401	8420	1	3
SEQ ID NO: 2170	aggaagggctcaaagaatg	7539	7558	SEQ ID NO: 2476	cattcctttaacaattcct	8532	8551	1	3
SEQ ID NO: 2171	gaagggctcaaagaatgac	7608	7627	SEQ ID NO: 2477	gtcagtcttcaggctcttc	10165	10184	1	3
SEQ ID NO: 2172	caaagaatgacttttttct	7633	7652	SEQ ID NO: 2478	agaaggatggcattttttg	10205	10224	1	3
SEQ ID NO: 2173	catggagaatgcctttgaa	7676	7695	SEQ ID NO: 2479	ttcagagccaaagtccatg	11013	11032	1	3
SEQ ID NO: 2174	ggagccaaggctggagtaa	7892	7711	SEQ ID NO: 2480	ttactccaacgccagctcc	6246	6266	1	3
SEQ ID NO: 2175	tcattccttccccaaagag	7714	7733	SEQ ID NO: 2481	ctctctggggcatctatgc	9369	9386	1	3
SEQ ID NO: 2176	acctatgagctccagagag	7715	7734	SEQ ID NO: 2482	ctctcaagaccacagaagg	8802	8821	1	3
SEQ ID NO: 2177	gggcaaaaacgtcttacag	7724	7743	SEQ ID NO: 2483	tctgaaagacaacgtgccc	10527	10546	1	3
SEQ ID NO: 2178	accctggaccttcagaaca	7730	7749	SEQ ID NO: 2484	tgttgctaaggttcagggt	11017	11036	1	3
SEQ ID NO: 2179	atgggcgacctaagttgtg	7734	7753	SEQ ID NO: 2485	cacaaattagtttcaccat	13673	13692	1	3
SEQ ID NO: 2180	gatgaagagaagattgaat	7745	7764	SEQ ID NO: 2486	atccagcttccccacactt	10275	10294	1	3
SEQ ID NO: 2181	caatgtgataccaaaaaaa	7762	7781	SEQ ID NO: 2487	tttttggaaatgccattgt	14018	14037	1	3
SEQ ID NO: 2182	gtagataccaaaaaaatga	7839	7858	SEQ ID NO: 2488	tcatgtgatgggtctctac	10200	10219	1	3
SEQ ID NO: 2183	gcttcagttcatttggact	7840	7859	SEQ ID NO: 2489	agtcaagaaggacttaagc	10199	10216	1	3
SEQ ID NO: 2184	tttgtttgtcaaagaagtc	7860	7879	SEQ ID NO: 2490	gacttcagagaaatacaaa	8951	8970	1	3
SEQ ID NO: 2185	ttgtttgtcaaagaagtca	7879	7898	SEQ ID NO: 2491	tgacttcagagaaatacaa	7965	7984	1	3
SEQ ID NO: 2186	tggcaatgggaaactcgct	7889	7908	SEQ ID NO: 2492	agcgagagtcccctgccat	73970	13989	1	3
SEQ ID NO: 2187	aacctctggcatttacttt	7921	7940	SEQ ID NO: 2493	aaaggagatgtcaagggtt	9580	8599	1	3
SEQ ID NO: 2188	catttactttctctcatga	7996	8015	SEQ ID NO: 2494	tcatttgaaagaataaatg	13175	13194	1	3
SEQ ID NO: 2189	aaagtcagtgccctgctta	8005	8024	SEQ ID NO: 2495	taagaaccttactgacttt	6821	6840	1	3
SEQ ID NO: 2190	tcccattttttgagacctt	8031	8080	SEQ ID NO: 2496	aaggacttcaggaatggga	9464	9483	1	3
SEQ ID NO: 2191	catcaatattgatcaattt	8055	8074	SEQ ID NO: 2497	aaattaaaaagtcttgatg	9881	9900	1	3
SEQ ID NO: 2192	taaagatagttatgattta	8081	8100	SEQ ID NO: 2498	taaaccaaaaacttggtta	10406	10425	1	3
SEQ ID NO: 2193	tattgatgaaatcattgaa	8137	8156	SEQ ID NO: 2499	ttcaaagacttaaaaaata	12924	12943	1	3
SEQ ID NO: 2194	atgatctacatttgtttat	8225	8244	SEQ ID NO: 2500	ataaagaaattaaagtcat	6514	8533	1	3
SEQ ID NO: 2195	agagacacatacagaatat	8312	8331	SEQ ID NO: 2501	atatattgtcagtgcctct	8876	8895	1	3
SEQ ID NO: 2196	gacacatacagaatataga	8331	8350	SEQ ID NO: 2502	tctaaattcagttcttgtc	8913	8932	1	3
SEQ ID NO: 2197	agcatgtcaaacactttgt	8379	8396	SEQ ID NO: 2503	acaaagtcagtgccctgct	11604	11623	1	3
SEQ ID NO: 2198	tttttagaggaaaccaagg	8391	8410	SEQ ID NO: 2504	ccttgtgtacaccaaaaac	11544	11583	1	3
SEQ ID NO: 2199	ttttagaggaaaccaaggc	8414	8433	SEQ ID NO: 2505	gcctttgtgtacaccaaaa	9730	9749	1	3
SEQ ID NO: 2200	ggaagatagacttcctgaa	8428	8447	SEQ ID NO: 2506	ttcagaaatactgtttttc	9662	9681	1	3
SEQ ID NO: 2201	cactgtttctgagtcccag	8500	8519	SEQ ID NO: 2507	ctgggacctaccaagagtg	13402	13421	1	3
SEQ ID NO: 2202	cacaaatcctttggctgtg	8501	8520	SEQ ID NO: 2508	cacatttcaaggaattgtg	13401	13420	1	3
SEQ ID NO: 2203	ttcctggatacactgttcc	8519	8538	SEQ ID NO: 2509	ggaactgttgactcaggac	11529	11548	1	3
SEQ ID NO: 2204	gaaatctcaagctttctct	8522	8541	SEQ ID NO: 2510	agagccaggtcgagctttc	13652	13671	1	3
SEQ ID NO: 2205	tttcttcatcttcatctgt	8541	8560	SEQ ID NO: 2511	acagctgaaagagatgaaa	12452	12471	1	3
SEQ ID NO: 2206	tctaccgctaaaggagcag	8596	8615	SEQ ID NO: 2512	ctgcacgctttgaggtaga	113801	11399	1	3
SEQ ID NO: 2207	ctaccgctaaaggagcagt	8608	8627	SEQ ID NO: 2513	actgcacgctttgaggtcg	13772	13791	1	3
SEQ ID NO: 2208	aggggcctcttttcaccaa	8670	8689	SEQ ID NO: 2514	ttggccaggaagtggccct	13414	1343	1	3
SEQ ID NO: 2209	tctccatccctgtaaaaag	8743	8762	SEQ ID NO: 2515	ctttttcaccaacggagaa	13372	13391	1	3
SEQ ID NO: 2210	gaaaaacaaagcagattat	8757	8776	SEQ ID NO: 2516	ataaactgcaagatttttc	12694	12713	1	3
SEQ ID NO: 2211	actcactcattgattttct	8821	8640	SEQ ID NO: 2517	agaaaatcaggatcgtagt	12251	12270	1	3
SEQ ID NO: 2212	taaactaatagatgtaatc	8921	8940	SEQ ID NO: 2518	gattaccaccagcagttta	10030	10049	1	3
SEQ ID NO: 2213	caaaaacgagcttcggaag	9052	9071	SEQ ID NO: 2519	cttcgtgagaatatttgaa	12610	12629	1	3
SEQ ID NO: 2214	tggaataatgctcagtgtt	9059	9078	SEQ ID NO: 2520	aacacttaacttgaattca	7300	7919	1	3
SEQ ID NO: 2215	gatttgaaatccaaagaag	9069	9088	SEQ ID NO: 2521	cttcagagaaatacaaatc	13558	13577	1	3
SEQ ID NO: 2216	atttgaaatccaaagaagt	9133	9152	SEQ ID NO: 2522	acttcagagaaatacaaat	12496	12517	1	3
SEQ ID NO: 2217	atcaacagccgcttctttg	9134	9153	SEQ ID NO: 2523	caaagaagtcaagattgat	2890	2908	1	3
SEQ ID NO: 2218	tgttttgaagactctccag	9213	9232	SEQ ID NO: 2524	ctggaaagttaaaacaaac	13630	13649	1	3
SEQ ID NO: 2219	cccttctgatagatgtggt	9222	9241	SEQ ID NO: 2525	accaaagctggcaccaggg	13486	13505	1	3
SEQ ID NO: 2220	tgagcaagtgaagaacttt	9275	9294	SEQ ID NO: 2526	aaagccattcagtctctca	10372	10391	1	3
SEQ ID NO: 2221	atttgaaatccaaagaagt	9304	9323	SEQ ID NO: 2527	actttctaaacttgaaatt	9840	9850	1	3
SEQ ID NO: 2222	atccaaagaagtcccggaa	9397	9416	SEQ ID NO: 2528	tccggggaaacctgggatt	14030	14048	1	3
SEQ ID NO: 2223	agagcctacctccgcatct	9427	9446	SEQ ID NO: 2529	agatggtacgttagcctct	13372	13391	1	3
SEQ ID NO: 2224	aatgcctttgaactcccca	9455	9474	SEQ ID NO: 2530	tgggaactacaattcattt	11726	11745	1	3
SEQ ID NO: 2225	gaagtccaaattccggatt	9470	9489	SEQ ID NO: 2531	aatcttcaatttattcttc	11711	11730	1	3
SEQ ID NO: 2226	tgcaagcagaagccagaag	9494	9513	SEQ ID NO: 2532	cttcaggttccatcgtgca	13198	13217	1	3
SEQ ID NO: 2227	gaagagaagattgaatttg	9497	9516	SEQ ID NO: 2533	caaaacctactgtgcttcc	9613	9632	1	3
SEQ ID NO: 2228	atgctaaaggcacatatgg	9526	9545	SEQ ID NO: 2534	ccatatgaaagtcaagcat	11203	11222	1	3
SEQ ID NO: 2229	tccctcacctccacctctg	9553	9572	SEQ ID NO: 2535	cagattctcagatgaggga	3926	3947	1	3
SEQ ID NO: 2230	atttacagctctgacaagt	9570	9589	SEQ ID NO: 2536	acttttctaaacttgaaat	13013	13032	1	3
SEQ ID NO: 2231	aggagcctaccaaaataat	9582	9601	SEQ ID NO: 2537	attatgttgaaaacagtct	10414	10433	1	3
SEQ ID NO: 2232	aaagctgaagcacatcaat	9583	9602	SEQ ID NO: 2538	attgttgctcatctccttt	11283	11302	1	3
SEQ ID NO: 2233	ctgctggaaacaacgagaa	9632	9651	SEQ ID NO: 2539	ttctgattaccaccagcag	9797	9816	1	3
SEQ ID NO: 2234	ttgaaggaattcttgaaaa	9712	9731	SEQ ID NO: 2540	ttttaaaagaaatcttcaa	11680	11699	1	3
SEQ ID NO: 2235	gaagtaaaagaaaattttg	9781	9800	SEQ ID NO: 2541	caaaacctactgtctcttc	12754	1273	1	3
SEQ ID NO: 2236	tgaagaagatggcaaattt	9851	9870	SEQ ID NO: 2542	aaatgtcagctcttgttca	8930	8949	1	3
SEQ ID NO: 2237	aggatctgagttatttttc	9863	9887	SEQ ID NO: 2543	gcaagtcagcccagttcct	13199	13218	1	3
SEQ ID NO: 2238	gtgcccttctcggttgctg	9886	9905	SEQ ID NO: 2544	cagccattgacatgagcac	4834	4853	1	3
SEQ ID NO: 2239	ggcgcggcctgcgctgctg	9942	9961	SEQ ID NO: 2545	cagctccacagactccgcc	11072	11091	1	3
SEQ ID NO: 2240	ctgcgctgctgctgctgct	10105	10124	SEQ ID NO: 2546	agcagaaggtgcgaagcag	6432	6451	1	3
SEQ ID NO: 2241	gctggtggcgggcgccagg	10159	10178	SEQ ID NO: 2547	cctggattccacatgcagc	7216	7235	1	3
SEQ ID NO: 2242	aagaggaaatgctggaaaa	10193	10212	SEQ ID NO: 2548	tttttcttcactacatctt	12881	12900	1	3
SEQ ID NO: 2243	ctggaaaatgtcagcctgg	10196	10215	SEQ ID NO: 2549	ccagacttccacatcccag	12423	12442	1	3
SEQ ID NO: 2244	tggagtccctgggactgct	10224	10243	SEQ ID NO: 2550	agcatgcctagtttctcca	13920	13939	1	3
SEQ ID NO: 2245	ggagtccctgggactgctg	10297	10316	SEQ ID NO: 2551	cagcatgcctagtttctcc	8187	8206	1	3
SEQ ID NO: 2246	tgggactgctgattcaaga	10322	10341	SEQ ID NO: 2552	tcttccatcacttgaccca	7153	7172	1	3
SEQ ID NO: 2247	ctgctgattcaagaagtgc	10369	10388	SEQ ID NO: 2553	gcacaccttgacattgcag	10770	10789	1	3
SEQ ID NO: 2248	tgccaccaggatcaactgc	10445	10454	SEQ ID NO: 2554	gcaggctgaactggtggca	13015	13034	1	3
SEQ ID NO: 2249	gccaccaggatcaactgca	10465	10474	SEQ ID NO: 2555	tgcaggctgaactggtggc	12851	12870	1	3
SEQ ID NO: 2250	tgcaaggttgagcgtggag	10468	10485	SEQ ID NO: 2556	cctccatcctctgatctga	10916	10935	1	3
SEQ ID NO: 2251	caaggttgagctggaggtt	10474	10493	SEQ ID NO: 2557	aacccctacatgaagcttg	7140	7159	1	3
SEQ ID NO: 2252	ctctgcagcttcatcctga	10504	10523	SEQ ID NO: 2558	tcaggaagcttctcaagag	12234	12253	1	3
SEQ ID NO: 2253	cagcttcatcctgaagaac	10540	10559	SEQ ID NO: 2559	ggtcttgagttaaatgctg	12025	12047	1	3
SEQ ID NO: 2254	gcttcatcctgaagaccag	10585	10584	SEQ ID NO: 2560	ctggacgctaagaggaagc	10641	10660	1	3
SEQ ID NO: 2255	tcatcctgaagaccagcca	10702	10721	SEQ ID NO: 2561	tggcatggcattatgatga	12281	12300	1	3
SEQ ID NO: 2256	gaaaaccaagaactctgag	10715	10734	SEQ ID NO: 2562	ctcaaccttaatgattttc	12737	12756	1	3
SEQ ID NO: 2257	agaactctgaggagtttgc	10852	10871	SEQ ID NO: 2563	gcaagctatatcagtattc	2459	2478	1	3
SEQ ID NO: 2258	tctgaggagtttgctgcag	10889	10908	SEQ ID NO: 2564	ctgcagggatcccccagat	12952	12971	1	3
SEQ ID NO: 2259	tttgctgcagccatgtcca	10890	10909	SEQ ID NO: 2565	tggaagtgtcagtggcaaa	12951	12970	1	3
SEQ ID NO: 2260	caagaggggcatcatttct	10906	10925	SEQ ID NO: 2566	agaataaatgacgttcttg	14058	14077	1	3
SEQ ID NO: 2261	tcactttaccgtcaagacg	10907	10926	SEQ ID NO: 2567	cgtctacactatcatgtga	14057	14076	1	3
SEQ ID NO: 2262	tttaccgtcaagacgagga	10932	10951	SEQ ID NO: 2568	tccttgacatgttgataaa	12248	12267	1	3
SEQ ID NO: 2263	cactggacgctaagaggaa	10979	10998	SEQ ID NO: 2569	ttccagaaagcagccagtg	11178	11197	1	3
SEQ ID NO: 2264	aggaagcatgtggcagaag	10986	11005	SEQ ID NO: 2570	cttcatacacattaatcct	13339	13358	1	3
SEQ ID NO: 2265	caaggagcaacacctcttc	10987	11006	SEQ ID NO: 2571	gaagtagtactgcatgttg	13338	13357	1	3
SEQ ID NO: 2266	acagactttgaaacttgaa	10995	11014	SEQ ID NO: 2572	ttcaattcttcaatgctgt	13814	13633	1	3
SEQ ID NO: 2267	tgatgaagcagtcacatct	11010	11029	SEQ ID NO: 2573	agatttgaggattccatca	9272	9291	1	3
SEQ ID NO: 2268	agcagtcacatctctcttg	11024	11043	SEQ ID NO: 2574	caaggagaaactgactcgt	13158	13177	1	3
SEQ ID NO: 2269	ccagccccatcactttaca	11071	11090	SEQ ID NO: 2575	tgtagtctcctggtgctgg	10458	10477	1	3
SEQ ID NO: 2270	ctccactccatcctccagg	11107	11126	SEQ ID NO: 2576	ctggagcttagtaatggag	7564	7583	1	3
SEQ ID NO: 2271	catgccaacccccttctga	11191	11210	SEQ ID NO: 2577	tcagatgagggaacacatg	12362	12381	1	3
SEQ ID NO: 2272	gagagatcttcaacatggc	11231	11250	SEQ ID NO: 2578	gccaccctggaactctctc	13140	13159	1	3
SEQ ID NO: 2273	tcaacatggcgagggatca	11269	11268	SEQ ID NO: 2579	tgatcccacctctcattga	8585	8904	1	3
SEQ ID NO: 2274	ccaccttgtatgcgctgag	11324	11343	SEQ ID NO: 2580	ctcagggatctgaaggtgg	13326	13345	1	3
SEQ ID NO: 2275	gtcaacaactatcataaga	11424	11443	SEQ ID NO: 2581	tcttgagttaaatgctgac	13102	13121	1	3
SEQ ID NO: 2276	tggacattgctaattacct	11445	11464	SEQ ID NO: 2582	aggtatattcgaaagtcca	12633	12652	1	3
SEQ ID NO: 2277	ggacattgctaattacctg	11528	11547	SEQ ID NO: 2583	caggtatattcgaaagtcc	8520	8539	1	3
SEQ ID NO: 2278	ttctgcgggtcattggaaa	11583	11602	SEQ ID NO: 2584	tttcacatgccaaggagaa	12685	12704	1	3
SEQ ID NO: 2279	ccagaactcaagtcttcaa	11600	11619	SEQ ID NO: 2585	ttgaagtgtatgtctcctg	11825	11844	1	3
SEQ ID NO: 2280	agtcttcaatcctgaaatg	11631	11650	SEQ ID NO: 2586	catttctgattggtggact	9223	9242	1	3
SEQ ID NO: 2281	tgagcaagtgaagaacttt	11683	11702	SEQ ID NO: 2587	aaagtgccacttttactca	12251	12270	1	3
SEQ ID NO: 2282	agcaagtgaagaactttgt	11684	11703	SEQ ID NO: 2588	acaaagtcagtgccctgct	12250	12269	1	3
SEQ ID NO: 2283	tctgaaagaatctcaactt	11695	11714	SEQ ID NO: 2589	aagtccataatggttcaga	13171	13190	1	3
SEQ ID NO: 2284	actgtcatggacttcagaa	11798	11818	SEQ ID NO: 2590	ttctgaatatattgtcagt	6496	6515	1	3
SEQ ID NO: 2285	acttgacccagcctcagcc	11995	12015	SEQ ID NO: 2591	ggctcaccctgagagaagt	11430	11449	1	3
SEQ ID NO: 2286	tccaaataactaccttcct	12048	12067	SEQ ID NO: 2592	aggaagatatgaagatgga	13278	13297	1	3
SEQ ID NO: 2287	actaccctcactgcctttg	12052	12071	SEQ ID NO: 2593	caaatttgtggagggtagt	13952	13971	1	3
SEQ ID NO: 2288	ttggatttgcttcagctga	12056	12085	SEQ ID NO: 2594	tcagtataagtacaaccaa	13703	13722	1	3
SEQ ID NO: 2289	ttggaagctctttttggga	12256	12275	SEQ ID NO: 2595	tcccgattcacgcttccaa	11659	11678	1	3
SEQ ID NO: 2290	ggaagctctttttgggaag	12292	12311	SEQ ID NO: 2596	cttcagaaagctaccttcc	3010	3029	1	3
SEQ ID NO: 2291	tttttcccagacagtgtca	12319	12338	SEQ ID NO: 2597	tgaccttctctaagcaaaa	8500	6519	1	3
SEQ ID NO: 2292	agacagtgtcaacaaagct	12354	12373	SEQ ID NO: 2597	agcttggttttgccagtct	12755	12774	1	3
SEQ ID NO: 2293	ctttggctataccaaagat	12457	12486	SEQ ID NO: 2598	atctcgtgtctaggaaaag	13107	13126	1	3
SEQ ID NO: 2294	caaagatgataaacatgag	12576	12595	SEQ ID NO: 2599	ctcaaggataacgtgtttg	12632	12651	1	3
SEQ ID NO: 2295	gatatggtaaatggaataa	12728	12747	SEQ ID NO: 2600	ttatcttataattatatca	12857	12875	1	3
SEQ ID NO: 2296	ggaataatgctcagtgttg	12869	12888	SEQ ID NO: 2601	caaacacttacttgaattc	13516	13535	1	3
SEQ ID NO: 2297	tttgaaatccaaagaagtc	12966	12985	SEQ ID NO: 2602	gacttcagagaaatacaaa	5794	5813	1	3
SEQ ID NO: 2298	gatcccccagatgattgga	12003	13012	SEQ ID NO: 2603	tccaatttccctgtggatc	13956	13975	1	3
SEQ ID NO: 2299	cagatattggagaggtcaa	13057	13076	SEQ ID NO: 2604	tgaccacacaaaacagtct	13192	13211	1	3
SEQ ID NO: 2300	agaatgacttttttctctc	13072	13091	SEQ ID NO: 2605	tgaagtccggattcattct	13807	13826	1	3
SEQ ID NO: 2301	gaactccccactggagctg	13142	13161	SEQ ID NO: 2606	cagctcaaccgtacagttc	9558	9577	1	3
SEQ ID NO: 2302	atatcttcatgggagtcaa	13172	13191	SEQ ID NO: 2607	tgacttcagtgcagaatat	11694	11713	1	3
SEQ ID NO: 2303	gtcatgctccccggaggca	13271	13290	SEQ ID NO: 2608	tggccccgtttaccatgac	13929	13948	1	3
SEQ ID NO: 2304	gctgaagtttatcattcct	13303	13322	SEQ ID NO: 2609	aggaggctttaagttcagc	8287	8306	1	3
SEQ ID NO: 2305	attccttccccaaagagac	13434	13453	SEQ ID NO: 2610	gtctcttcctccatggaat	13826	13845	1	3
SEQ ID NO: 2306	ctcattgagaacaggcagt	13501	13520	SEQ ID NO: 2611	actgactgcacgctttgag	13613	13832	1	3
SEQ ID NO: 2307	ttgagcagtattctgtcag	13562	13581	SEQ ID NO: 2612	cttagagaagtgtcttcaa	7991	8010	1	3
SEQ ID NO: 2308	accttgtccagtgaagtcc	13672	13691	SEQ ID NO: 2613	ggacggtactgtcccaggt	10414	10435	1	3
SEQ ID NO: 2309	ccagtgaagtccaaattcc	13685	13704	SEQ ID NO: 2614	ggaaggcagagtttactgg	7362	7381	1	3
SEQ ID NO: 2310	acattcagaacaagaaaat	13803	13822	SEQ ID NO: 2615	atttcctaaagctggatgc	10374	10393	1	3
SEQ ID NO: 2311	gaaaaatcaagggtgttat	14035	14054	SEQ ID NO: 2616	ataaactgcaagatttttc	7856	7875	1	3
SEQ ID NO: 2312	aaatcaagggtgttatttc	14049	14068	SEQ ID NO: 2617	gaaacaatgcattagattt	9834	9853	1	3
# = Match Number
B = Middle Matching Bases

TABLE 9 — Selected palindromic sequences from human ApoB

		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 2619	ggccattccagaagggaag	517	536	SEQ ID NO: 3948	cttccgttctgtaatggcc	5803	6322	1	9
SEQ ID NO: 2620	tgccatctcgagagttcca	4107	4126	SEQ ID NO: 3949	tggaactctctccatggca	10884	10903	1	8
SEQ ID NO: 2621	catgtcaaacactttgtta	7064	7083	SEQ ID NO: 3950	taacaaattccttgacatg	7368	7385	1	8
SEQ ID NO: 2622	ttgttataaatcttaattg	7076	7095	SEQ ID NO: 3951	caataagatcaatagcaaa	8999	9017	1	8
SEQ ID NO: 2623	tctggaaaagggtcatgga	8888	8907	SEQ ID NO: 3959	tccatgtcccatttacaga	11384	11383	1	8
SEQ ID NO: 2624	cagctcttgttcaggtcca	10908	10927	SEQ ID NO: 3960	tggacctgcaccaaagctg	13980	13979	1	8
SEQ ID NO: 2625	ggaggttccccagactcgc	354	363	SEQ ID NO: 3961	gcagccctgggaaaactcc	6465	5474	1	7
SEQ ID NO: 2626	ctgttttgaagactctcca	1088	1108	SEQ ID NO: 3962	tggagggtagtcataacag	10335	10354	1	7
SEQ ID NO: 2627	agtggctgaaacgtgtgca	1305	1324	SEQ ID NO: 3963	tgcagagctttctgcccac	13516	13635	1	7
SEQ ID NO: 2628	ccaaaatagaagggaatct	2076	2095	SEQ ID NO: 3964	agattcctttgccttttgg	4008	4027	1	7
SEQ ID NO: 2629	tgaagagaagattgaattt	3820	3647	SEQ ID NO: 3965	aaattctcttttcttttca	8220	9239	1	7
SEQ ID NO: 2630	agtggtgccaataccagca	4238	4257	SEQ ID NO: 3966	tgctagtgaggccaacact	10857	10676	1	7
SEQ ID NO: 2631	aaggctccacaagtcatca	5956	5977	SEQ ID NO: 3967	tgatgatatctggaacctt	10732	10751	1	7
SEQ ID NO: 2632	gtcagccaggtttatagca	7733	7752	SEQ ID NO: 3968	tgctaagaaccttactgac	7789	7808	1	7
SEQ ID NO: 2633	tgatatctggaaccttgaa	10735	10754	SEQ ID NO: 3969	ttcactgttcctgaaatca	7871	7890	1	7
SEQ ID NO: 2634	gtcaagttgagcaatttct	13431	13450	SEQ ID NO: 3970	agaaaaggcacaccttgac	11080	11099	1	7
SEQ ID NO: 2635	atccagatggaaaagggaa	13488	13507	SEQ ID NO: 3971	ttccccatttccctgtggt	3666	3707	1	7
SEQ ID NO: 2636	atttgtttgtcaaagaagt	4551	4570	SEQ ID NO: 3972	acttcagagaaatacaaat	11409	11427	4	6
SEQ ID NO: 2637	ctggaaaatgtcagcctgg	212	231	SEQ ID NO: 3973	ccagacttccgtttcccag	8243	6262	2	6
SEQ ID NO: 2638	accaggaggttcttcttca	1737	1756	SEQ ID NO: 3974	tgaagtgtagtctcctggt	5097	5118	2	6
SEQ ID NO: 2639	aaagaagttctgaaagaat	1964	1983	SEQ ID NO: 3975	attccatcacaatcctttt	9669	9688	2	6
SEQ ID NO: 2640	gctacagcttatggctcca	3578	3597	SEQ ID NO: 3976	tggatctaaatgcagtagc	11631	11650	2	6
SEQ ID NO: 2641	atccatcttgatcaatttg	6422	6441	SEQ ID NO: 3977	caaagaagtcaagattgat	4561	4580	2	6
SEQ ID NO: 2642	gaattatcttttaaaacat	7334	7353	SEQ ID NO: 3978	atgtgttaaccaaatattc	11502	11521	2	6
SEQ ID NO: 2643	cgaggcccgcgctgctggc	138	157	SEQ ID NO: 3979	gccagaagtgagatcctcg	3515	3534	1	6
SEQ ID NO: 2644	acaactatgaggctgagag	279	293	SEQ ID NO: 3980	ctctgagcaacaaatttgt	10317	10338	1	6
SEQ ID NO: 2645	gctgagagttccagtggcg	290	309	SEQ ID NO: 3981	ctccatggcaaatgtcagc	10593	10912	1	6
SEQ ID NO: 2646	tgaagaaaaccaagaactc	468	475	SEQ ID NO: 3982	gagtcattgcggttcttca	4937	4956	1	6
SEQ ID NO: 2647	cctcttacatcctgaacaa	506	586	SEQ ID NO: 3983	tgttcataagggaggtcgg	12774	12703	1	6
SEQ ID NO: 2648	ctacttacatcctgaacat	567	588	SEQ ID NO: 3984	atgttcataagggcggtag	12773	12782	1	6
SEQ ID NO: 2649	gagacagaagaagccaagc	623	642	SEQ ID NO: 3985	gcttggttttgccagtctc	2467	2488	1	6
SEQ ID NO: 2650	cactcactttaccgtcaag	679	698	SEQ ID NO: 3986	cttgaacacaaagtcagtg	8008	5027	1	6
SEQ ID NO: 2651	ctgatcagcagcagccagt	830	849	SEQ ID NO: 3987	actgggaagtgcttatcag	5245	5264	1	6
SEQ ID NO: 2652	actggccgctaagaggaag	862	961	SEQ ID NO: 3988	cttccccaaagagaccagt	2869	2917	1	6
SEQ ID NO: 2653	agaggaagcatgtggcaga	873	892	SEQ ID NO: 3989	tctggcatttactttctct	6929	5048	1	6
SEQ ID NO: 2654	tgaagactctccaggaact	1086	1114	SEQ ID NO: 3990	agttgaaggagactattca	7224	7243	1	6
SEQ ID NO: 2655	ctctgagcaaaatatccag	1129	1148	SEQ ID NO: 3991	ctggttactgagctgagag	1169	1188	1	6
SEQ ID NO: 2656	atgaagcagtcacatctct	1197	1216	SEQ ID NO: 3992	agagctgccagtccttcat	10024	10043	1	6
SEQ ID NO: 2657	ttgccacagctgattgagg	1217	1236	SEQ ID NO: 3993	cctcctacagtggtggcaa	4230	4249	1	6
SEQ ID NO: 2659	agctgattgaggtgtccag	1224	1243	SEQ ID NO: 3994	ctggattccacatgcagct	11856	11874	1	6
SEQ ID NO: 2650	tgctccactcacatcctcc	1286	1305	SEQ ID NO: 3995	ggaggctttaagttcagca	7609	7628	1	6
SEQ ID NO: 2660	tgaagcgtgtgcatgccaa	1311	1330	SEQ ID NO: 3996	ttgggagagacaagtttca	6508	6527	1	6
SEQ ID NO: 2661	gacattgctaattacctga	1511	1630	SEQ ID NO: 3997	tcagaagctaagcaatgtc	7240	7259	1	6
SEQ ID NO: 2662	ttcttcttcagactttcct	1746	1765	SEQ ID NO: 3998	aggagagtccaaattagaa	6506	8526	1	6
SEQ ID NO: 2663	ccaatatcttgaactcaga	1911	1930	SEQ ID NO: 3999	tctgaattcattcaattgg	6493	6512	1	6
SEQ ID NO: 2664	aaagttggtgaaagaagtt	1954	1973	SEQ ID NO: 4000	aactaccctcactgccttt	2140	2159	1	6
SEQ ID NO: 2665	aagtagtgaaagaaagttc	1955	1974	SEQ ID NO: 4001	gaacctctggcatttactt	5924	5943	1	6
SEQ ID NO: 2666	aaagaagttctgaaagaat	1964	1983	SEQ ID NO: 4002	attctctggtaactacttt	5490	5509	1	6
SEQ ID NO: 2667	tttggctataccaaagatg	2330	2349	SEQ ID NO: 4003	catcttaggcactgacaaa	5005	5024	1	6
SEQ ID NO: 2668	tgttgagaagctgattaaa	2389	2408	SEQ ID NO: 4004	tttagccatcggctcaaca	5708	5727	1	6
SEQ ID NO: 2669	caggaagggctcaaagaat	3569	2589	SEQ ID NO: 4005	attcctttaacaggttcag	9500	9519	1	6
SEQ ID NO: 2670	aggaagggctcaaagaatg	2570	2589	SEQ ID NO: 4006	cattcctttaacaattcct	9499	9518	1	6
SEQ ID NO: 2671	gaagggctcaaagaatgac	2572	2591	SEQ ID NO: 4007	gtcagtcttcaggctcttc	7922	7941	1	6
SEQ ID NO: 2672	caaagaatgacttttttct	2580	2599	SEQ ID NO: 4008	agaaggatggcattttttg	14008	14027	1	6
SEQ ID NO: 2673	catggagaatgcctttgaa	2611	2630	SEQ ID NO: 4009	ttcagagccaaagtccatg	7127	7146	1	6
SEQ ID NO: 2674	ggagccacggatggagtaa	2687	2706	SEQ ID NO: 4010	ttatccaacgccagctccc	3058	3077	1	6
SEQ ID NO: 2675	tcattccttccccaaagag	2892	2911	SEQ ID NO: 4011	ctctcggggcatctatgaa	5147	5166	1	6
SEQ ID NO: 2676	acctatgagctccagagag	3173	3192	SEQ ID NO: 4012	ctctcaagaccacagaggt	12984	13003	1	6
SEQ ID NO: 2677	gggcaaacgtcttacagaa	3373	3392	SEQ ID NO: 4013	tctgaaagaaacgtgtgca	12325	12344	1	6
SEQ ID NO: 2678	accctggacattcagaaca	3395	3414	SEQ ID NO: 4014	tgttgctaaggttcagggt	5683	5702	1	6
SEQ ID NO: 2679	atgggcgacctaagttgtg	3437	3456	SEQ ID NO: 4015	cacaaattagttcaccatt	8949	8968	1	6
SEQ ID NO: 2680	gatgaagagagagttgagt	3626	3645	SEQ ID NO: 4016	attccagcttccccacatc	8338	8357	1	6
SEQ ID NO: 2681	caatgtagataccaaaaaa	3664	3683	SEQ ID NO: 4017	tttttggaaatgccattgc	8651	6670	1	6
SEQ ID NO: 2682	gtagataccaaaaaaatga	3868	3687	SEQ ID NO: 4018	tcatgtgatgggtctcacc	4379	4398	1	6
SEQ ID NO: 2683	gcttcagttcatttggact	4517	4536	SEQ ID NO: 4019	agtcaagaaggacttaagc	5312	5331	1	6
SEQ ID NO: 2684	tttgtttgttcaaagaagt	4552	4571	SEQ ID NO: 4020	gactcagagaaatacaaac	11406	11427	1	6
SEQ ID NO: 2685	ttgtttgtcaaagaagtca	4553	4572	SEQ ID NO: 4021	tgactcagagaaatacaac	11407	11428	1	6
SEQ ID NO: 2686	tggcaatgggaaactcgct	5854	5873	SEQ ID NO: 4022	agcgagaatcaccctgcca	8227	8248	1	6
SEQ ID NO: 2687	aacctctggcatttacttt	5925	5944	SEQ ID NO: 4023	aaaggagatgtcaagggtt	10607	10626	1	6
SEQ ID NO: 2688	catttactttctctcatga	5934	5953	SEQ ID NO: 4024	tcatttgaaagaataaatg	7034	7053	1	6
SEQ ID NO: 2689	aaagtcagtgccctgctta	6017	6036	SEQ ID NO: 4025	taagaacctactgaacttt	7792	7811	1	6
SEQ ID NO: 2690	tcccattttttgagacctt	6330	6349	SEQ ID NO: 4026	aaggacttcaggaatggga	12012	12031	1	6
SEQ ID NO: 2691	catcaatattgatcaattt	6421	6440	SEQ ID NO: 4027	aatattaaaagtcttgatg	6740	5759	1	6
SEQ ID NO: 2692	taaagatagttatgattta	6673	6692	SEQ ID NO: 4028	taaaccaaaacttggttta	9027	9046	1	6
SEQ ID NO: 2693	tattgatgaaatcattgaa	6721	6740	SEQ ID NO: 4029	ttcaaagacttaaaacaat	8015	8034	1	6
SEQ ID NO: 2694	atgatctacatttgtttat	6798	6817	SEQ ID NO: 4030	ataaagaaattgaatcaat	7388	7407	1	6
SEQ ID NO: 2695	agagacacatacagaatat	6927	6946	SEQ ID NO: 4031	atatattgtcagtgcctct	13390	13409	1	6
SEQ ID NO: 2696	gacacatacagaatataga	6930	6949	SEQ ID NO: 4032	tctaaattcagttcttgtc	11335	11354	1	6
SEQ ID NO: 2697	agcatgtcaaacatttgtc	7062	7081	SEQ ID NO: 4033	acaaagtcagtgccctgct	6015	8034	1	6
SEQ ID NO: 2698	ttttagaggaaaccaaggc	7523	7542	SEQ ID NO: 4034	cctttgtgtacaccccaaa	11238	11257	1	6
SEQ ID NO: 2699	ttttagaggaaaccaaggc	7524	7543	SEQ ID NO: 4035	gcctttgtgtacaccaaaa	11237	11258	1	6
SEQ ID NO: 2700	ggaagatagacttcctgaa	9315	9334	SEQ ID NO: 4036	ttcagaaatactgtttccg	12832	12851	1	6
SEQ ID NO: 2701	cactgtttctgagtcccag	9342	9361	SEQ ID NO: 4037	ctgggacctaccaagagtg	12531	12550	1	6
SEQ ID NO: 2702	cacaaatcctttggctgtg	9676	9695	SEQ ID NO: 4038	cacatttcaggattgtggg	10071	10090	1	6
SEQ ID NO: 2703	ttcctggatacactgttcc	9801	9880	SEQ ID NO: 4039	ggaactgttgactcaggaa	12577	12596	1	6
SEQ ID NO: 2704	gaaatctcaagctttctat	10050	10069	SEQ ID NO: 4040	agagccaggtccgagcttc	11052	11071	1	6
SEQ ID NO: 2705	tttcttcatcttcatctgt	10218	10234	SEQ ID NO: 4041	acagctgaaagagatgaaa	13083	13082	1	6
SEQ ID NO: 2706	tctaccgctaaaggagcag	10529	10548	SEQ ID NO: 4042	ctgcacgcttgaggtagaa	11769	11788	1	6
SEQ ID NO: 2707	ctaccgtcaaaggagcagt	10530	10549	SEQ ID NO: 4043	actgcacgctttgaggtag	11768	11787	1	6
SEQ ID NO: 2708	agggcctctttttcaccaa	10839	10868	SEQ ID NO: 4044	ttggccaggaagtggccct	10965	10984	1	6
SEQ ID NO: 2709	ttctccatccctgtaaaag	11273	11292	SEQ ID NO: 4045	cttttcaccaaacgggaga	10846	10605	1	6
SEQ ID NO: 2710	gaaaaccaaagcagattct	11824	11343	SEQ ID NO: 4046	ataaactgcaagattttct	13608	13627	1	6
SEQ ID NO: 2711	actcactcattgatttcct	12690	12709	SEQ ID NO: 4047	agaaaatcaggatctgagt	14035	14054	1	6
SEQ ID NO: 2712	taaactaatagatgtaatc	12898	12917	SEQ ID NO: 4048	gattaccaccagcggttta	13586	13605	1	6
SEQ ID NO: 2713	caaaagagcttcaggaagc	13208	13227	SEQ ID NO: 4049	cttcgtgaagaatattttg	13268	13287	1	6
SEQ ID NO: 2714	tggaataatgctcagtgtt	2374	2393	SEQ ID NO: 4050	aacacttcattgaattcca	10670	10889	3	5
SEQ ID NO: 2715	gatttgaaatccaaaggag	2406	2427	SEQ ID NO: 4051	cttcagagaatacaaaatc	11410	11429	3	5
SEQ ID NO: 2716	atttgaaatccaaagaagt	2409	2428	SEQ ID NO: 4052	acttcagcgaaatacaaat	11409	11428	3	5
SEQ ID NO: 2717	atcaacagccgcttctttg	998	1017	SEQ ID NO: 4053	caaagaagtcaagattgat	4561	4580	2	5
SEQ ID NO: 2718	tgttttgaagactctccag	1090	1109	SEQ ID NO: 4054	ctggaaagttaaaacaaca	6963	6932	2	5
SEQ ID NO: 1719	cccttctgatagatgtggt	1332	1351	SEQ ID NO: 4055	accaaagctggcaccaggg	13989	13958	2	5
SEQ ID NO: 2720	tgagcaagtgaagaacttt	1876	1895	SEQ ID NO: 4056	aaagccattcagtctctca	12971	12990	2	5
SEQ ID NO: 2721	atttgaaatccaaagaagt	2409	2426	SEQ ID NO: 4057	acttttctaaacttgaaat	9063	9082	2	5
SEQ ID NO: 2722	atccaaagaagtcccggaa	2416	2435	SEQ ID NO: 4058	ttccggggaaacctgggat	12729	12748	2	5
SEQ ID NO: 2723	agagcctacctccgcatct	2438	2457	SEQ ID NO: 4059	agatggtacgttagcctct	11929	11948	2	5
SEQ ID NO: 2724	aatgcctttgaaactcccc	2615	2837	SEQ ID NO: 4060	tgggaactacaatttcatt	7020	7039	2	5
SEQ ID NO: 2725	gaagtccaaattccggatt	3305	3324	SEQ ID NO: 4061	aatcttcaatttattcttc	13823	13842	2	5
SEQ ID NO: 2726	tgcaagcagaagccagaag	3304	3625	SEQ ID NO: 4062	cttcaggtttccatcgtga	11384	11403	2	5
SEQ ID NO: 2727	gaagagaagattgaatttg	3629	3848	SEQ ID NO: 4063	caaaacctactgtctcctc	10467	10486	2	5
SEQ ID NO: 2728	atgctaaaggcacatatgg	4605	4524	SEQ ID NO: 4064	ccatatgaaagtcaagcat	12554	12683	2	5
SEQ ID NO: 2729	tccctcacctccacctctg	4745	4764	SEQ ID NO: 4065	cagattctcgatgagggat	8920	8939	2	5
SEQ ID NO: 2730	atttacagctctgacaagt	5435	5454	SEQ ID NO: 4066	acttttctaaacttgaatt	9353	9062	2	5
SEQ ID NO: 2731	aggagcctaccaaaattat	5602	5621	SEQ ID NO: 4067	attatgttgaaaacagttc	11838	11857	2	5
SEQ ID NO: 2732	aaagctgaagcacatcaat	8409	5428	SEQ ID NO: 4068	attgttgctcatctccttt	10202	10221	2	5
SEQ ID NO: 2733	ctgctggaaacaacgagaa	9426	9448	SEQ ID NO: 4069	tttctgataccacccacag	13682	13801	2	5
SEQ ID NO: 2734	ttgaaggaattcttgaaaa	9590	9509	SEQ ID NO: 4070	ttttaaaagaaaatctcaa	13813	12832	2	5
SEQ ID NO: 2735	gaagtaaaagaaatttggg	10751	10770	SEQ ID NO: 4071	caaaaacctactgtctggg	10467	10486	2	5
SEQ ID NO: 2736	tgaagaagatggcaaattt	11992	12011	SEQ ID NO: 4072	aaatgtcagctcttgttca	10902	10921	2	5
SEQ ID NO: 2737	aggatctgagttattttgc	14043	14062	SEQ ID NO: 4073	gcaagtcagcccagttcct	10928	10947	2	5
SEQ ID NO: 2738	gtgcccttctcggttgctg	26	45	SEQ ID NO: 4074	cagccattgacatgagcac	5748	5767	1	5
SEQ ID NO: 2739	ggcgctgcctgcgctgctg	154	173	SEQ ID NO: 4075	cagctccaccgactccgcc	3070	3089	1	5
SEQ ID NO: 2740	ctgcgctgctgctgctgct	162	181	SEQ ID NO: 4076	agcagaaggtgcaagcagg	3232	3251	1	5
SEQ ID NO: 2741	gctggtggcgggcgccagg	178	197	SEQ ID NO: 4077	cctggattccacatgcagc	11854	11673	1	5
SEQ ID NO: 2742	aagaggaaatgctggaaaa	201	220	SEQ ID NO: 4078	ttttcttcactactacttc	2592	2611	1	5
SEQ ID NO: 2743	ctggaaaatgtcagcctgg	212	231	SEQ ID NO: 4079	ccagacttccacatcccag	3923	3942	1	5
SEQ ID NO: 2744	tggagtccctgggactgct	305	323	SEQ ID NO: 4080	agcatgcctagtttctcca	9953	9972	1	5
SEQ ID NO: 2745	ggagctcccgggacttgtg	305	324	SEQ ID NO: 4081	cagtacgcctagtttctcc	9952	9971	1	5
SEQ ID NO: 2746	tgggactgctgattcaaga	313	332	SEQ ID NO: 4082	tcttccatcacttggaaac	2050	2039	1	5
SEQ ID NO: 2747	ctgctgattcaagaagtgc	316	337	SEQ ID NO: 4083	gcacaccttgacattgaca	11087	11106	1	5
SEQ ID NO: 2748	tgccaccaggatcaactgc	334	363	SEQ ID NO: 4084	gcaggctgaactggtggca	2725	2744	1	5
SEQ ID NO: 2749	gccaccaggatcaactgca	335	364	SEQ ID NO: 4085	tgcaggctgaactggttgc	2724	2743	1	5
SEQ ID NO: 2750	tgcaaggttgagctggagg	350	389	SEQ ID NO: 4086	cctccacctctgatctgca	4762	4771	1	5
SEQ ID NO: 2751	cataggttgagctggaggt	352	371	SEQ ID NO: 4089	aaccctacatggaaagctt	13783	13782	1	5
SEQ ID NO: 2752	ctctgcagtccatccctga	377	395	SEQ ID NO: 4090	tcaggaagcttctcaagag	13210	13238	1	5
SEQ ID NO: 2753	cagcttcatcctgaagacc	382	401	SEQ ID NO: 4091	ggtcttgagttaaatgctg	4985	5004	1	5
SEQ ID NO: 2754	gcttcatcctgaatgacag	384	403	SEQ ID NO: 4092	ctggacgctaagaggaagc	863	882	1	5
SEQ ID NO: 2755	tcatcctgaagaccagcca	387	406	SEQ ID NO: 4093	tggcatggcattatgatcc	3612	3631	1	5
SEQ ID NO: 2756	gaaaaccaagaactctgag	460	479	SEQ ID NO: 4094	ctcaaccttaatgattttc	8294	8313	1	5
SEQ ID NO: 2757	agaactctgaggagtttgc	468	437	SEQ ID NO: 4095	gcaagctctacagtattct	8385	8404	1	5
SEQ ID NO: 2758	tctgaggagtttgccgggt	473	492	SEQ ID NO: 4096	ctgcaggggatcccccaga	2534	2553	1	5
SEQ ID NO: 2759	tttgctgcagccatgtcca	482	801	SEQ ID NO: 4097	tggaagtgcagtggcaaat	10360	10399	1	5
SEQ ID NO: 2760	caagaggggcatcatttct	550	805	SEQ ID NO: 4098	agaataaatgacgttcttg	7043	7062	1	5
SEQ ID NO: 2761	tcactttaccgtcaagacg	662	701	SEQ ID NO: 4099	cgtctacactatagaaatt	4368	4387	1	5
SEQ ID NO: 2762	tttaccgtcaagacgaagg	686	705	SEQ ID NO: 4100	tccttgaccatgttgataa	7374	7393	1	5
SEQ ID NO: 2763	cactggacgctaagaaaga	861	880	SEQ ID NO: 4101	ttccagaaagcagccagtg	12506	12525	1	5
SEQ ID NO: 2764	aggaagcatgtggcagaag	675	894	SEQ ID NO: 4102	cttcatacacattatcctt	9996	10015	1	5
SEQ ID NO: 2765	caaggagcaacacctcttc	901	920	SEQ ID NO: 4103	gaagtagtactgcatcttg	6643	6882	1	5
SEQ ID NO: 2766	acagactttgaaacttgaa	957	988	SEQ ID NO: 4104	ttcaatcttcaatgctgtt	10508	10527	1	5
SEQ ID NO: 2767	tgatgaagcagtcacatct	1195	1214	SEQ ID NO: 4105	agatttgaggattccatca	7984	8003	1	5
SEQ ID NO: 2768	agcagtcacatctctcttg	1201	1220	SEQ ID NO: 4106	caaggagaaaactgactgc	6532	8551	1	5
SEQ ID NO: 2769	cagccccatcactttacat	1239	1258	SEQ ID NO: 4107	tgtagtctcctggtgctgg	5102	5121	1	5
SEQ ID NO: 2770	ctccactcatccttcagtg	1388	1307	SEQ ID NO: 4108	caggagcttagtaatggag	8717	8736	1	5
SEQ ID NO: 2771	catgcccaccccctcctgg	1322	1341	SEQ ID NO: 4109	tcagatgagggaacacatg	8927	8946	1	5
SEQ ID NO: 2772	gagagatcttcaacatggc	1398	1417	SEQ ID NO: 4110	gccaccctggaaactctct	10877	10896	1	5
SEQ ID NO: 2773	tcaaacatggcgacgcatc	1407	1428	SEQ ID NO: 4111	tgatcccacctctcattga	2973	2992	1	5
SEQ ID NO: 2774	ccaccttgtatgcgctgag	1437	1456	SEQ ID NO: 4112	ctcagggatctgaggggtg	8195	8214	1	5
SEQ ID NO: 2775	gtcaaacaactatatcata	1463	1482	SEQ ID NO: 4113	tcttgagttaaatgctgac	4987	5006	1	5
SEQ ID NO: 2776	tggacattgctaattacct	1509	1525	SEQ ID NO: 4114	aggtatattcgaaagtcca	12807	12826	1	5
SEQ ID NO: 2777	ggacattgctaattacctg	1510	1629	SEQ ID NO: 4115	caggtatattcgaaagtcc	12806	12825	1	5
SEQ ID NO: 2778	ttctgcgggtcattggaaa	1551	1600	SEQ ID NO: 4116	tttcacactgcccaagaaa	5522	6541	1	5
SEQ ID NO: 2779	ccagaactcaagtcttcaa	1628	1647	SEQ ID NO: 4117	ttgaagtgtgtctcctggc	5096	5115	1	5
SEQ ID NO: 2780	agtcttcaattcctgaagt	1638	1657	SEQ ID NO: 4118	catttctgattggtggact	7765	7784	1	5
SEQ ID NO: 2781	tgagcaagtgaagaaactt	1876	1895	SEQ ID NO: 4119	aaagtgccactttttacta	6191	6210	1	5
SEQ ID NO: 2782	agcaagtgaaagaacttgt	1878	1897	SEQ ID NO: 4120	acaaagtcagttgcctggt	6015	6034	1	5
SEQ ID NO: 2783	tctgaaaagaatctcaact	1972	1991	SEQ ID NO: 4121	aagtccataatggttcaga	12819	12833	1	5
SEQ ID NO: 2784	actgtcatggacttcagaa	1994	2013	SEQ ID NO: 4122	ttctgaatatattgtcagt	13384	13703	1	5
SEQ ID NO: 2785	acttgacccagcctcagcc	2059	2078	SEQ ID NO: 4123	ggctccccctgagagaagt	12399	1241	1	5
SEQ ID NO: 2786	tccaaataactaccttcct	2104	2123	SEQ ID NO: 4124	aggaagatagaagatggat	4720	4739	1	5
SEQ ID NO: 2787	actaccctcactgcctttg	2141	2160	SEQ ID NO: 4125	caaatttgtggagggtagt	10327	10346	1	5
SEQ ID NO: 2788	ttggatttgcttcagctga	2159	2176	SEQ ID NO: 4126	tcagtataaatacaccaaa	9400	9419	1	5
SEQ ID NO: 2789	ttggaagctcttgggaaat	2219	2238	SEQ ID NO: 4127	tcccgattcacgcttccaa	11585	11604	1	5
SEQ ID NO: 2790	ggaagctctttttgggaag	2221	2240	SEQ ID NO: 4128	cttcagaaagctaccttcc	7837	7955	1	5
SEQ ID NO: 2791	tttttcccagacagtgtca	2246	2285	SEQ ID NO: 4129	tgacctctctaagccaaat	4884	4903	1	5
SEQ ID NO: 2792	agacagtgtcaacaagctt	2254	2273	SEQ ID NO: 4130	agctggttttgcccagttt	2466	2405	1	5
SEQ ID NO: 2793	cttggctataccaaagatt	2329	2348	SEQ ID NO: 4131	atctcgtgtctaggaaaag	2976	5995	1	5
SEQ ID NO: 2794	caaagatgataaaacatgg	2341	2300	SEQ ID NO: 4132	ctcaaggataacgtgtttg	12617	12636	1	5
SEQ ID NO: 2795	gatatggtaaatggaataa	2363	2382	SEQ ID NO: 4133	tttacttagggttaatttg	13087	13106	1	5
SEQ ID NO: 2796	ggaataatgctcagtgttg	2375	2394	SEQ ID NO: 4134	caacacttacttgaattcc	10689	10688	1	5
SEQ ID NO: 2797	tttgaaatccaaagaaatc	2410	2429	SEQ ID NO: 4135	gacttcagagaaatacaat	11408	11427	1	5
SEQ ID NO: 2798	cagatgattggagaggtca	2542	2591	SEQ ID NO: 4136	tccaatttcctgtgggtgg	3689	3708	1	5
SEQ ID NO: 2799	agaatgacttttttcttca	2549	2588	SEQ ID NO: 4137	tgaccacacaaacagtgct	5371	6390	1	5
SEQ ID NO: 2800	agaatgactttttcttcac	2583	2505	SEQ ID NO: 4138	tgaagtccggcttctttct	11023	11042	1	5
SEQ ID NO: 2801	gaactcccactggagctgc	2627	2846	SEQ ID NO: 4139	cagctcaaccgtacagttc	11809	11856	1	5
SEQ ID NO: 2802	atatctaaggtggtccaca	2660	2879	SEQ ID NO: 4140	tgacttcagtgcagaatat	11974	11993	1	5
SEQ ID NO: 2803	gctgaagtttatcattcct	2675	2694	SEQ ID NO: 4141	tggccccgtttaccataga	5817	5836	1	5
SEQ ID NO: 2804	attccttcccccaaagaga	2881	2900	SEQ ID NO: 4142	aggaggctttaagttcagc	7608	627	1	5
SEQ ID NO: 2805	ctcattgagaaacaggcag	2894	2913	SEQ ID NO: 4143	gtctcttcctccatggaat	10478	10497	1	5
SEQ ID NO: 2806	ctcattgagaacaggcagt	2984	3003	SEQ ID NO: 4144	actgactgcacgctttgag	11764	11783	1	5
SEQ ID NO: 2807	ttgagcagtattctgtcag	3150	3169	SEQ ID NO: 4145	gctgagaaagtgcttcctt	12407	142428	1	5
SEQ ID NO: 2808	accttgtccagtgaagtcc	3293	3312	SEQ ID NO: 4146	ggacggtactgtcccaggt	12792	12811	1	5
SEQ ID NO: 2809	cacgtgaagtccaaattcc	3300	3319	SEQ ID NO: 4147	ggaaggcagagtttactgg	9150	9176	1	5
SEQ ID NO: 2810	acattcagaaaagaaagcg	3402	3421	SEQ ID NO: 4148	atttcctaaagctggattg	11175	11194	1	5
SEQ ID NO: 2811	gaaaaatcaagggtgttat	3471	3490	SEQ ID NO: 4149	ataaactgcaaagattttt	13609	13627	1	5
SEQ ID NO: 2812	aaatcaagggtgttatttc	3474	3493	SEQ ID NO: 4150	gaaaacaatgcattagatt	9753	9772	1	5
SEQ ID NO: 2813	tggcattatgatgaagaga	3817	3636	SEQ ID NO: 4151	tctcccgtgtatatgccaa	11789	11808	1	5
SEQ ID NO: 2814	aagagaagattgaatttga	3630	3649	SEQ ID NO: 4152	tcaaaacctactgtctctt	10466	10435	1	5
SEQ ID NO: 2815	aaatgacttccaatttccc	3681	3700	SEQ ID NO: 4153	gggaactacaatttcattt	7021	7040	1	5
SEQ ID NO: 2816	atgacttccaattttccct	3383	3702	SEQ ID NO: 4154	caggctgattacgagtcat	4925	4944	1	5
SEQ ID NO: 2817	acttccaattttccctgtg	3686	3705	SEQ ID NO: 4155	ccacgaaaaatatggaagt	10388	10387	1	5
SEQ ID NO: 2818	agttgcaatgagctcatgg	3811	3830	SEQ ID NO: 4156	ccatcagttcagataaact	7997	8016	1	5
SEQ ID NO: 2819	tttgcaagaccacctcaat	3868	3887	SEQ ID NO: 4157	attgacctgtccattcaaa	13679	136696	1	5
SEQ ID NO: 2820	gaaggagttcaacctccag	2892	3911	SEQ ID NO: 4158	ctggaaatttgtcattccc	11735	11756	1	5
SEQ ID NO: 2821	acttccacatcccagaaaa	3927	3946	SEQ ID NO: 4159	ttttaacaaaaagtggtgg	6629	6848	1	5
SEQ ID NO: 2822	ctcttcttaaaaagcgatg	3947	3966	SEQ ID NO: 4160	catcactgccaaaggagag	8494	8513	1	5
SEQ ID NO: 2823	aaaagagagggcgggtcaa	3956	3975	SEQ ID NO: 4161	tgactactcagggaaattt	12555	12707	1	5
SEQ ID NO: 2824	ttcctttgccttttggtgg	4011	4030	SEQ ID NO: 4162	ccacaaacaatgaagggaa	9264	9283	1	5
SEQ ID NO: 2825	caagtctcggggattccat	4087	4106	SEQ ID NO: 4163	atggaaaaaaaacagggct	9574	9693	1	5
SEQ ID NO: 2826	aagtccctactttttacat	4125	4144	SEQ ID NO: 4164	atgggaagtataagaactt	4842	4891	1	5
SEQ ID NO: 2827	tgcctctcctgggtgtttc	4157	4186	SEQ ID NO: 4165	agaaaacaaccaaggcaaa	9651	9670	1	5
SEQ ID NO: 2828	accagcacagaccacccca	4250	4269	SEQ ID NO: 4166	tgaagtgtagtctcctggt	5097	5118	1	5
SEQ ID NO: 2829	ccagcacagaccatttcag	4251	4270	SEQ ID NO: 4167	ctgaaatacaagcttctgg	6519	5538	1	5
SEQ ID NO: 2830	actatcatgtgatgggtct	4375	4394	SEQ ID NO: 4168	agacacctgatttttatat	7956	7976	1	5
SEQ ID NO: 2831	accacagatgtctgcttca	4304	4523	SEQ ID NO: 4169	tgaaggctgatttcttgtt	4290	4309	1	5
SEQ ID NO: 2832	ccacagatgtctgcttcag	4505	4524	SEQ ID NO: 4170	ctgagcaactttttttgtt	10319	10338	1	5
SEQ ID NO: 2833	tttggactccaaaaaagaa	4528	4547	SEQ ID NO: 4171	tttctctcatgatacaaaa	5941	5960	1	5
SEQ ID NO: 2834	tcaaagaagtcaagattga	4560	4579	SEQ ID NO: 4172	tcaaggataacgtgtttga	12618	12637	1	5
SEQ ID NO: 2835	atgagaactacgagctgac	4808	4625	SEQ ID NO: 4173	gtcagatattggtgctcat	10196	10214	1	5
SEQ ID NO: 2836	ttaaatctgacaacattgg	4826	4845	SEQ ID NO: 4174	cattcatttgaaggggaac	7350	7369	1	5
SEQ ID NO: 2837	gaagtataagaactttgcc	4846	4665	SEQ ID NO: 4175	ggcaaatttgaaggacttc	12002	12021	1	5
SEQ ID NO: 2838	aagtataagaactttgcca	4847	4866	SEQ ID NO: 4176	tggcaaatttgaaggactt	12001	12020	1	5
SEQ ID NO: 2839	ttcttcagcctgtttctgg	4949	4968	SEQ ID NO: 4177	cagaatccagatacaagaa	6892	6914	1	5
SEQ ID NO: 2840	ctggatcactaaattccca	4965	4984	SEQ ID NO: 4178	tgggtcttccagaaggccg	11041	11060	1	5
SEQ ID NO: 2841	aaattaatagtggtgctca	5022	5041	SEQ ID NO: 4179	tgagaagccccagaatttt	6266	6275	1	5
SEQ ID NO: 2842	agtgcaacgaccaacttga	5081	5100	SEQ ID NO: 4180	tcaaattcctggattacac	9866	9875	1	5
SEQ ID NO: 2843	ctgggaagtgcttatcagg	5246	5265	SEQ ID NO: 4181	cctgaccttcacataccag	8316	8337	1	5
SEQ ID NO: 2844	gcaaaaacaaatttcaggg	5286	5305	SEQ ID NO: 4182	aagtcaaagaaattttgct	10752	10771	1	5
SEQ ID NO: 2845	aaaaaacatttcaacttca	5288	5307	SEQ ID NO: 4183	tgaagtaaaagaaattttt	10750	10760	1	5
SEQ ID NO: 2846	tcagtcaagaagaacttaa	5310	5329	SEQ ID NO: 4184	ttaaggaacttccattcta	13371	13380	1	5
SEQ ID NO: 2847	tcaaatgacatgatgggct	5333	5352	SEQ ID NO: 4185	agcccatcaatatattgaa	6213	6232	1	5
SEQ ID NO: 2848	cacaaaacagtctgaacaa	5375	5394	SEQ ID NO: 4186	tgttttcaactgccttgta	11227	11246	1	5
SEQ ID NO: 2849	tcttcaaaacttgaccaca	5417	5436	SEQ ID NO: 4187	tgttttcctattttcaaga	12843	12862	1	5
SEQ ID NO: 2850	caagtttttattaggcact	5449	6468	SEQ ID NO: 4188	agttatttgctaaagcttg	14061	14070	1	5
SEQ ID NO: 2851	tggtaactactttaccagg	5496	5515	SEQ ID NO: 4189	ctgttttagaggaaagccg	7520	7539	1	5
SEQ ID NO: 2852	aacagtgacctgaaataca	5510	5529	SEQ ID NO: 4190	tgtatagcaaattcctgtt	5898	5917	1	5
SEQ ID NO: 2853	gggaaactacggctagaac	5552	5571	SEQ ID NO: 4191	gttccttccagatttccgc	10941	10960	1	5
SEQ ID NO: 2854	aacaaacttgccatcctcc	5028	5647	SEQ ID NO: 4192	gagaacagctttcgtttgg	11212	11231	1	5
SEQ ID NO: 2855	tcagcaagctataaagcag	5660	5679	SEQ ID NO: 4193	ctgctaagaaccttactga	7788	7807	1	5
SEQ ID NO: 2856	gcagacactgttgctaagg	5675	5694	SEQ ID NO: 4194	cctttcaagcactgactgc	11764	11773	1	5
SEQ ID NO: 2857	tctggggagaacatactgg	5674	5893	SEQ ID NO: 4195	ccaggttttccacaaccga	8046	8084	1	5
SEQ ID NO: 2858	ttctctcatgattacaaag	5942	5961	SEQ ID NO: 4196	ctttttcaaccaacgggaa	10846	10885	1	5
SEQ ID NO: 2859	ctgagcagacaggcacctg	6042	6661	SEQ ID NO: 4197	caggaggctttaagttgtc	7607	7526	1	5
SEQ ID NO: 2860	caatttaaacaaacaagat	6074	6093	SEQ ID NO: 4198	attccttcctttacaattg	8090	8109	1	5
SEQ ID NO: 2861	tggacgaaactctggctac	6146	6167	SEQ ID NO: 4199	gtcagcccaggttcctcaa	10932	10951	1	5
SEQ ID NO: 2862	ctttttactcagtgaccca	6200	6219	SEQ ID NO: 4200	tgggctaaacgtttaattg	7835	7854	1	5
SEQ ID NO: 2863	tcattgtggctttagaaga	6226	6244	SEQ ID NO: 4201	atcttcataagttcaatga	13182	13201	1	5
SEQ ID NO: 2864	aaaaccaagatgttcactc	6303	6322	SEQ ID NO: 4202	gagtgaaatgctgtttttt	8638	8867	1	5
SEQ ID NO: 2865	aggaatcgacaaccattat	6365	6384	SEQ ID NO: 4203	taatgatttcaatgttcct	6302	8321	1	5
SEQ ID NO: 2866	tagttgtactggaaaaacg	6384	6403	SEQ ID NO: 4204	acgttagcctctatagact	11935	11955	1	5
SEQ ID NO: 2867	ggaaaactgatcagagaag	6394	6413	SEQ ID NO: 4205	cttttacaattcatttccc	13022	13041	1	5
SEQ ID NO: 2868	gaaaacgtacaggaccccc	9695	6414	SEQ ID NO: 4206	gctttctctttcacaaatg	10080	10079	1	5
SEQ ID NO: 2869	aaagctgaagcccatcaat	6409	6425	SEQ ID NO: 4207	atttgattagttggcaagg	6992	7011	1	5
SEQ ID NO: 2870	aagtctaagacctaggaga	6410	6429	SEQ ID NO: 4208	tattgatgttagagtgctt	6991	7010	1	5
SEQ ID NO: 2871	tgaagcacatcaatattga	6414	6433	SEQ ID NO: 4209	tcaaccttaatgattttaa	8295	6314	1	5
SEQ ID NO: 2872	atcaatattgatcaatttg	6422	6441	SEQ ID NO: 4210	caaagccatacatgatggg	1658	1687	1	5
SEQ ID NO: 2873	taatgattatctgaattca	6484	8503	SEQ ID NO: 4211	tgaaatcattgaaaattta	6727	5746	1	5
SEQ ID NO: 2874	gattatcgaattcattcat	6488	6507	SEQ ID NO: 4212	tgaaagtacgctgaagttc	7102	7121	1	5
SEQ ID NO: 2875	aattgggagagaagccttt	6506	6625	SEQ ID NO: 4213	aaacattcctttaccactt	9496	9515	1	5
SEQ ID NO: 2876	aaaatagctattgctaata	3701	6720	SEQ ID NO: 4214	tattgaaatatgatttttt	6814	6839	1	5
SEQ ID NO: 2877	aaaattaacaagtccgtat	6739	6758	SEQ ID NO: 4215	atcataggttgaggttttt	6785	6784	1	5
SEQ ID NO: 2878	ttgaaaaatatttgattta	6818	6835	SEQ ID NO: 4216	ttaatctttcataagtcaa	13176	13198	1	5
SEQ ID NO: 2879	agacatccagcacctagct	6946	6965	SEQ ID NO: 4217	tgggctaaacgtttaattg	2468	2485	1	5
SEQ ID NO: 2880	caattcatttgaaagaaat	7029	7048	SEQ ID NO: 4218	tattgaaatatgatttttt	8090	8109	1	5
SEQ ID NO: 2881	aggtttaaatggataattt	7182	7201	SEQ ID NO: 4219	aattgttgaaagaaaaact	13155	13174	1	5
SEQ ID NO: 2882	cagaagctaagcaatgtcc	7241	7250	SEQ ID NO: 4220	ggacaaggcccagaatctg	12553	12572	1	5
SEQ ID NO: 2883	taagattaaatatttactt	7270	7289	SEQ ID NO: 4221	aaagaaacctatggcctta	13183	13182	1	5
SEQ ID NO: 2884	aaagattactttgagaaat	7277	7298	SEQ ID NO: 4222	attcttaacgggattcctt	9489	9508	1	5
SEQ ID NO: 2885	aggaaatagttggattacc	7280	7308	SEQ ID NO: 4223	taaagccattcaggtctct	12970	12969	1	5
SEQ ID NO: 2886	attttgatgatgctggccc	7303	7322	SEQ ID NO: 4224	gacatgtttgaagggtgga	7372	7398	1	5
SEQ ID NO: 2887	gaattatcttttaaacatt	734	7353	SEQ ID NO: 4225	tgggctaaacgtttaattg	7685	7704	1	5
SEQ ID NO: 2888	ttaccaccagtttgaagtt	7411	7430	SEQ ID NO: 4226	tattgaaatatgatttttt	10739	10758	1	5
SEQ ID NO: 2889	ttgcagtgtatcggacaga	7548	7567	SEQ ID NO: 4227	agcttgtttgccagtgggc	8420	8439	1	5
SEQ ID NO: 2890	cattcagaaccagggaaga	7699	7718	SEQ ID NO: 4228	tgaaatcattgaaaattta	12009	12020	1	5
SEQ ID NO: 2891	acaaactgtttatagtccc	7958	7977	SEQ ID NO: 4229	caggaggctttaagttgtc	12614	12633	1	5
SEQ ID NO: 2892	ggatttcaatacatttcac	7992	8011	SEQ ID NO: 4230	agcttgtttgccagtgggc	13124	13143	1	5
SEQ ID NO: 2893	ttgtagaaatgaaagtaaa	8112	8131	SEQ ID NO: 4231	taaagccattcaggtctct	12360	12379	1	5
SEQ ID NO: 2894	ctgaacagtgagctgcagt	8166	8175	SEQ ID NO: 4232	aattgttgaaagaaaaact	8809	8828	1	5
SEQ ID NO: 2895	aatccaatctcctctttgc	8407	3420	SEQ ID NO: 4233	taaagccattcaggtctct	11017	11036	1	5
SEQ ID NO: 2896	atttgattttcaagcaaag	8532	8554	SEQ ID NO: 4234	ttaatctttcataagtcaa	14023	14042	1	5
SEQ ID NO: 2897	ttttgattttcaagcaaat	8535	8552	SEQ ID NO: 4235	ggacaaggcccagaatctg	9622	9641	1	5
SEQ ID NO: 2898	tgatttcaagcaaatgcag	8536	8555	SEQ ID NO: 4236	attcttaacgggattcctt	14025	14044	1	5
SEQ ID NO: 2899	atgctgtttgggaaattgg	8645	8664	SEQ ID NO: 4237	aattgttgaaagaaaaact	11203	11222	1	5
SEQ ID NO: 2900	tgctgttttttggaaatgc	8646	8855	SEQ ID NO: 4238	agcttgtttgccagtgggc	11202	11221	1	5
SEQ ID NO: 2901	aaaaaaatacactggacgt	8706	8725	SEQ ID NO: 4239	ttaatctttcataagtcaa	10533	10652	1	5
SEQ ID NO: 2902	actggagcttagtaagggc	8716	8735	SEQ ID NO: 4240	agcttgtttgccagtgggc	1289	1308	1	5
SEQ ID NO: 2903	cttctggaaagagggtcat	8886	8905	SEQ ID NO: 4241	agcttgtttgccagtgggc	13759	13778	1	5
SEQ ID NO: 2904	ggaaaagggtcatggaaat	8891	8910	SEQ ID NO: 4242	attccttccttacaattgg	8282	9304	1	5
SEQ ID NO: 2905	gggcctgccccagatttct	8910	8929	SEQ ID NO: 4243	gagaacatttatggaggcc	9440	9450	1	5
SEQ ID NO: 2906	ttctcagataggggaacac	8924	8943	SEQ ID NO: 4244	gtgtcctcaagctgagggg	12416	12435	1	5
SEQ ID NO: 2907	gatgaaagaattaagggga	8930	8949	SEQ ID NO: 4245	attccagcttcccaactac	8338	8357	1	5
SEQ ID NO: 2908	cttggactgtcaaataagt	8996	9005	SEQ ID NO: 4246	cttatgggatttcctaagg	11167	11186	1	5
SEQ ID NO: 2909	gcatccacaaacaatggag	9200	9279	SEQ ID NO: 4247	cttcatcgttcatttgagt	10227	10246	1	5
SEQ ID NO: 2910	cacaaacaatgaagggaat	9265	8284	SEQ ID NO: 4248	cttatgggatttcctaagg	11456	11507	1	5
SEQ ID NO: 2911	ccaaaatttctctcggctt	9415	9434	SEQ ID NO: 4249	cttcatcgttcatttgagt	9671	9690	1	5
SEQ ID NO: 2912	caaattttctctgctaaaa	9416	9435	SEQ ID NO: 4250	ttccatcacaaatcctttg	9670	9689	1	5
SEQ ID NO: 2913	tctgctggaaacaacaacg	9425	9444	SEQ ID NO: 4251	tctcaaagagttacaacag	13328	13248	1	5
SEQ ID NO: 2914	cagaagctaagcaatgtcc	9426	6445	SEQ ID NO: 4252	tattgaaatatgatttttt	13228	13247	1	5
SEQ ID NO: 2915	taagattaaatatttactt	9441	9460	SEQ ID NO: 4253	aattgttgaaagaaaaact	8909	8928	1	5
SEQ ID NO: 2916	aaagattactttgagaaat	9475	9494	SEQ ID NO: 4254	ggacaaggcccagaatctg	13821	13840	1	5
SEQ ID NO: 2917	aggaaatagttggattacc	9565	9584	SEQ ID NO: 4255	aaagaaacctatggcctta	14021	14040	1	5
SEQ ID NO: 2918	attttgatgatgctggccc	9712	9731	SEQ ID NO: 4256	attcttaacgggattcctt	14033	14052	1	5
SEQ ID NO: 2919	gaattatcttttaaacatt	9751	9770	SEQ ID NO: 4257	taaagccattcaggtctct	5633	8662	1	5
SEQ ID NO: 2920	ttaccaccagtttgaagtt	10001	10020	SEQ ID NO: 4258	gacatgtttgaagggtgga	14089	14108	1	5
SEQ ID NO: 2921	ttgcagtgtatcggacaga	10194	10213	SEQ ID NO: 4259	tgggctaaacgtttaattg	4807	4826	1	5
SEQ ID NO: 2922	cattcagaaccagggaaga	10338	10355	SEQ ID NO: 4260	tattgaaatatgatttttt	27347	2753	1	5
SEQ ID NO: 2923	acaaactgtttatagtccc	10404	10423	SEQ ID NO: 4261	agcttgtttgccagtgggc	8368	8385	1	5
SEQ ID NO: 2924	ggatttcaatacatttcac	10405	10424	SEQ ID NO: 4262	tgaaatcattgaaaattta	8365	8384	1	5
SEQ ID NO: 2925	ttgtagaaatgaaagtaaa	10438	10455	SEQ ID NO: 4263	caggaggctttaagttgtc	13258	13277	1	5
SEQ ID NO: 2926	ttaccaccagtttgaagtt	10578	10597	SEQ ID NO: 4264	agcttgtttgccagtgggc	12974	12966	1	5
SEQ ID NO: 2927	ttgcagtgtatcggacaga	10863	10882	SEQ ID NO: 4265	taaagccattcaggtctct	12676	12695	1	5
SEQ ID NO: 2928	cattcagaaccagggaaga	10672	10691	SEQ ID NO: 4266	aattgttgaaagaaaaact	5008	6027	1	5
SEQ ID NO: 2929	acaaactgtttatagtccc	10751	10770	SEQ ID NO: 4267	taaagccattcaggtctct	5287	5306	1	5
SEQ ID NO: 2930	ggatttcaatacatttcac	10882	10901	SEQ ID NO: 4268	ttaatctttcataagtcaa	11372	11391	1	5
SEQ ID NO: 2931	ttgtagaaatgaaagtaaa	11164	11203	SEQ ID NO: 4269	ggacaaggcccagaatctg	11856	11874	1	5
SEQ ID NO: 2932	ctgaacagtgagctgcagt	11485	11504	SEQ ID NO: 4270	attcttaacgggattcctt	6705	5784	1	5
SEQ ID NO: 2933	aatccaatctcctctttgc	11613	11632	SEQ ID NO: 4271	aattgttgaaagaaaaact	12424	12443	1	5
SEQ ID NO: 2934	atttgattttcaagcaaag	11614	11633	SEQ ID NO: 4272	agcttgtttgccagtgggc	12423	12442	1	5
SEQ ID NO: 2935	ttttgattttcaagcaaat	11642	11861	SEQ ID NO: 4273	ttaatctttcataagtcaa	13103	13122	1	5
SEQ ID NO: 2936	acaaactgtttatagtccc	12229	12248	SEQ ID NO: 4274	agcttgtttgccagtgggc	13631	13650	1	5
SEQ ID NO: 2937	ggatttcaatacatttcac	12936	12957	SEQ ID NO: 4275	agcttgtttgccagtgggc	13826	13845	1	5
SEQ ID NO: 2938	ttgtagaaatgaaagtaaa	13034	13053	SEQ ID NO: 4276	attccttccttacaattgg	5014	8033	1	5
SEQ ID NO: 2939	ttaccaccagtttgaagtt	13097	13116	SEQ ID NO: 4277	tgggctaaacgtttaattg	10479	10498	1	5
SEQ ID NO: 2940	ttgcagtgtatcggacaga	13218	13237	SEQ ID NO: 4278	tattgaaatatgatttttt	13163	13202	1	5
SEQ ID NO: 2941	cattcagaaccagggaaga	13437	13450	SEQ ID NO: 4279	agcttgtttgccagtgggc	12932	12047	1	5
SEQ ID NO: 2942	acaaactgtttatagtccc	13712	13731	SEQ ID NO: 4280	tgaaatcattgaaaattta	7465	7484	1	5
SEQ ID NO: 2943	ggatttcaatacatttcac	13719	13738	SEQ ID NO: 4281	caggaggctttaagttgtc	7230	7258	1	5
SEQ ID NO: 2944	ttgtagaaatgaaagtaaa	13882	13901	SEQ ID NO: 4282	agcttgtttgccagtgggc	8380	8399	1	5
SEQ ID NO: 2945	ccaaaatttctctcggctt	14011	14030	SEQ ID NO: 4283	taaagccattcaggtctct	3013	3025	1	5
SEQ ID NO: 2946	caaattttctctgctaaaa	14020	14039	SEQ ID NO: 4284	ggacaaggcccagaatctg	9566	9585	1	5
SEQ ID NO: 2947	tctgctggaaacaacaacg	1627	1646	SEQ ID NO: 4285	attcttaacgggattcctt	8641	8660	3	4
SEQ ID NO: 2948	cagaagctaagcaatgtcc	1866	1975	SEQ ID NO: 4286	aattgttgaaagaaaaact	12564	12583	3	4
SEQ ID NO: 2949	taagattaaatatttactt	6435	6454	SEQ ID NO: 4287	agcttgtttgccagtgggc	11409	11426	3	4
SEQ ID NO: 2950	aaagattactttgagaaat	6488	6507	SEQ ID NO: 4288	ttaatctttcataagtcaa	7429	7448	3	4
SEQ ID NO: 2951	aggaaatagttggattacc	26	45	SEQ ID NO: 4289	agcttgtttgccagtgggc	8039	8058	2	4
SEQ ID NO: 2952	attttgatgatgctggccc	253	272	SEQ ID NO: 4290	agcttgtttgccagtgggc	13184	13203	2	4
SEQ ID NO: 2953	gaattatcttttaaacatt	316	335	SEQ ID NO: 4291	attccttccttacaattgg	13415	13434	2	4
SEQ ID NO: 2954	ttaccaccagtttgaagtt	483	502	SEQ ID NO: 4292	agcttgtttgccagtgggc	5889	5908	2	4
SEQ ID NO: 2955	taagattaaatatttactt	555	574	SEQ ID NO: 4293	agcttgtttgccagtgggc	10498	15174	2	4
SEQ ID NO: 2956	aaagattactttgagaaat	1095	1114	SEQ ID NO: 4294	attccttccttacaattgg	13191	13210	2	4
SEQ ID NO: 2957	aggaaatagttggattacc	1210	1220	SEQ ID NO: 4295	tgggctaaacgtttaattg	9237	9256	2	4
SEQ ID NO: 2958	attttgatgatgctggccc	1211	1230	SEQ ID NO: 4296	tattgaaatatgatttttt	9236	9255	2	4
SEQ ID NO: 2959	gaattatcttttaaacatt	1231	1250	SEQ ID NO: 4297	agcttgtttgccagtgggc	5216	5235	2	4
SEQ ID NO: 2960	ttaccaccagtttgaagtt	1628	1647	SEQ ID NO: 4298	tgaaatcattgaaaattta	5918	5994	2	4
SEQ ID NO: 2961	ctgaaaaagttagtgaaag	1948	1988	SEQ ID NO: 4299	caggaggctttaagttgtc	10631	10660	2	4
SEQ ID NO: 2962	tttttcccagacagtgtca	2246	2205	SEQ ID NO: 4300	agcttgtttgccagtgggc	9730	9749	2	4
SEQ ID NO: 2963	ttttcccagacagtgtcca	2247	2268	SEQ ID NO: 4301	taaagccattcaggtctct	9729	9746	2	4
SEQ ID NO: 2964	cattcagaaccagggaaga	3403	3422	SEQ ID NO: 4302	cttcatcgttcatttgagt	10414	10433	2	4
SEQ ID NO: 2965	acaaactgtttatagtccc	3628	3647	SEQ ID NO: 4303	cttatgggatttcctaagg	10902	10921	2	4
SEQ ID NO: 2966	ggatttcaatacatttcac	3844	3663	SEQ ID NO: 4304	cttcatcgttcatttgagt	11815	11834	2	4
SEQ ID NO: 2967	ttgtagaaatgaaagtaaa	4407	4426	SEQ ID NO: 4305	ttccatcacaaatcctttg	7377	7395	2	4
SEQ ID NO: 2968	ccaaaatttctctcggctt	4412	4431	SEQ ID NO: 4306	tctcaaagagttacaacag	7162	7181	2	4
SEQ ID NO: 2969	caaattttctctgctaaaa	5083	5102	SEQ ID NO: 4307	tattgaaatatgatttttt	11384	11403	2	4
SEQ ID NO: 2970	tctgctggaaacaacaacg	5325	5344	SEQ ID NO: 4308	aattgttgaaagaaaaact	7382	7401	2	4
SEQ ID NO: 2971	cagaagctaagcaatgtcc	6074	6093	SEQ ID NO: 4309	ggacaaggcccagaatctg	13876	13896	2	4
SEQ ID NO: 2972	taagattaaatatttactt	6088	6107	SEQ ID NO: 4310	aaagaaacctatggcctta	10087	10704	2	4
SEQ ID NO: 2973	aaagattactttgagaaat	6421	6440	SEQ ID NO: 4311	attcttaacgggattcctt	11456	11505	2	4
SEQ ID NO: 2974	aggaaatagttggattacc	7059	7878	SEQ ID NO: 4312	taaagccattcaggtctct	9381	9400	2	4
SEQ ID NO: 2975	attttgatgatgctggccc	7227	7246	SEQ ID NO: 4313	gacatgtttgaagggtgga	13446	13465	2	4
SEQ ID NO: 2976	gaattatcttttaaacatt	7929	7948	SEQ ID NO: 4314	tgggctaaacgtttaattg	12312	1331	2	4
SEQ ID NO: 2977	ttaccaccagtttgaagtt	8787	8805	SEQ ID NO: 4315	tattgaaatatgatttttt	12533	12552	2	4
SEQ ID NO: 2978	taagattaaatatttactt	10167	10186	SEQ ID NO: 4316	agcttgtttgccagtgggc	13191	13210	2	4
SEQ ID NO: 2979	aaagattactttgagaaat	12898	12917	SEQ ID NO: 4317	tgaaatcattgaaaattta	13640	136659	2	4
SEQ ID NO: 2980	aggaaatagttggattacc	13680	12699	SEQ ID NO: 4318	caggaggctttaagttgtc	13613	13832	2	4
SEQ ID NO: 2981	attttgatgatgctggccc	19	38	SEQ ID NO: 4319	agcttgtttgccagtgggc	84	103	1	4
SEQ ID NO: 2982	aggtttaaatggataattt	20	39	SEQ ID NO: 4320	taaagccattcaggtctct	83	102	1	4
SEQ ID NO: 2983	cagaagctaagcaatgtcc	22	41	SEQ ID NO: 4321	aattgttgaaagaaaaact	11667	11576	1	4
SEQ ID NO: 2984	taagattaaatatttactt	33	52	SEQ ID NO: 4322	taaagccattcaggtctct	2188	2167	1	4
SEQ ID NO: 2985	aaagattactttgagaaat	90	109	SEQ ID NO: 4323	ttaatctttcataagtcaa	376	395	1	4
SEQ ID NO: 2986	aggaaatagttggattacc	151	170	SEQ ID NO: 4324	ggacaaggcccagaatctg	4252	4271	1	4
SEQ ID NO: 2987	attttgatgatgctggccc	177	196	SEQ ID NO: 4325	attcttaacgggattcctt	11186	11202	1	4
SEQ ID NO: 2988	gaattatcttttaaacatt	227	246	SEQ ID NO: 4326	aattgttgaaagaaaaact	388	407	1	4
SEQ ID NO: 2989	ttaccaccagtttgaagtt	291	310	SEQ ID NO: 4327	agcttgtttgccagtgggc	3391	3410	1	4
SEQ ID NO: 2990	ttgcagtgtatcggacaga	269	316	SEQ ID NO: 4328	ttaatctttcataagtcaa	3863	2702	1	4
SEQ ID NO: 2991	cattcagaaccagggaaga	354	373	SEQ ID NO: 4329	agcttgtttgccagtgggc	5736	4755	1	4
SEQ ID NO: 2992	acaaactgtttatagtccc	358	377	SEQ ID NO: 4330	tgaaatcattgaaaattta	9171	9190	1	4
SEQ ID NO: 2993	ggatttcaatacatttcac	375	397	SEQ ID NO: 4331	caggaggctttaagttgtc	3269	3288	1	4
SEQ ID NO: 2994	ttgtagaaatgaaagtaaa	402	421	SEQ ID NO: 4332	agcttgtttgccagtgggc	5802	5821	1	4
SEQ ID NO: 2995	ctgaacagtgagctgcagt	472	491	SEQ ID NO: 4333	taaagccattcaggtctct	6348	6367	1	4
SEQ ID NO: 2996	aatccaatctcctctttgc	600	519	SEQ ID NO: 4334	cttcatcgttcatttgagt	12724	12743	1	4
SEQ ID NO: 2997	atttgattttcaagcaaag	543	582	SEQ ID NO: 4335	cttatgggatttcctaagg	2227	2246	1	4
SEQ ID NO: 2998	ttttgattttcaagcaaat	583	602	SEQ ID NO: 4336	cttcatcgttcatttgagt	2266	2304	1	4
SEQ ID NO: 2999	tgatttcaagcaaatgcag	509	628	SEQ ID NO: 4337	ttccatcacaaatcctttg	13996	14015	1	4
SEQ ID NO: 3000	atgctgtttgggaaattgg	630	649	SEQ ID NO: 4338	tctcaaagagttacaacag	14050	14099	1	4
SEQ ID NO: 3001	tgctgttttttggaaatgc	638	857	SEQ ID NO: 4339	tattgaaatatgatttttt	11050	11049	1	4
SEQ ID NO: 3002	aaaaaaatacactggacgt	652	671	SEQ ID NO: 4340	aattgttgaaagaaaaact	11364	11383	1	4
SEQ ID NO: 3003	actggagcttagtaagggc	678	697	SEQ ID NO: 4341	ggacaaggcccagaatctg	6825	6844	1	4
SEQ ID NO: 3004	cttctggaaagagggtcat	701	720	SEQ ID NO: 4342	aaagaaacctatggcctta	3013	3032	1	4
SEQ ID NO: 3005	ggaaaagggtcatggaaat	708	727	SEQ ID NO: 4343	attcttaacgggattcctt	3466	3507	1	4
SEQ ID NO: 3006	gggcctgccccagatttct	709	728	SEQ ID NO: 4344	taaagccattcaggtctct	13834	13853	1	4
SEQ ID NO: 3007	ttctcagataggggaacac	714	733	SEQ ID NO: 4345	gacatgtttgaagggtgga	1895	1914	1	4
SEQ ID NO: 3008	gatgaaagaattaagggga	737	758	SEQ ID NO: 4346	tgggctaaacgtttaattg	2935	2987	1	4
SEQ ID NO: 3009	cttggactgtcaaataagt	752	771	SEQ ID NO: 4349	tattgaaatatgatttttt	5218	5327	1	4
SEQ ID NO: 3010	ggaaaagggtcatggaaat	783	772	SEQ ID NO: 4350	agcttgtttgccagtgggc	5217	5226	1	4
SEQ ID NO: 3011	gggcctgccccagatttct	784	803	SEQ ID NO: 4351	tgaaatcattgaaaattta	8152	8171	1	4
SEQ ID NO: 3012	cttggactgtcaaataagt	794	813	SEQ ID NO: 4352	tgaaatcattgaaaattta	10345	10364	1	4
SEQ ID NO: 3013	ggatttcaatacatttcac	819	838	SEQ ID NO: 4353	aaagaaacctatggcctta	12453	12472	1	4
SEQ ID NO: 3014	ttgtagaaatgaaagtaaa	820	839	SEQ ID NO: 4354	attcttaacgggattcctt	12452	12471	1	4
SEQ ID NO: 3015	ccaaaatttctctcggctt	892	911	SEQ ID NO: 4355	taaagccattcaggtctct	3813	5832	1	4
SEQ ID NO: 3016	caaattttctctgctaaaa	893	912	SEQ ID NO: 4356	gacatgtttgaagggtgga	3812	3831	1	4
SEQ ID NO: 3017	tctgctggaaacaacaacg	916	935	SEQ ID NO: 4357	tgggctaaacgtttaattg	9481	9480	1	4
SEQ ID NO: 3018	cagaagctaagcaatgtcc	924	943	SEQ ID NO: 4358	tattgaaatatgatttttt	13655	13675	1	4
SEQ ID NO: 3019	taagattaaatatttactt	997	1016	SEQ ID NO: 4359	agcttgtttgccagtgggc	1669	1688	1	4
SEQ ID NO: 3020	aaagattactttgagaaat	998	1017	SEQ ID NO: 4360	tgaaatcattgaaaattta	1668	1687	1	4
SEQ ID NO: 3021	aggaaatagttggattacc	1002	1021	SEQ ID NO: 4361	caggaggctttaagttgtc	9675	9694	1	4
SEQ ID NO: 3022	attttgatgatgctggccc	1031	1050	SEQ ID NO: 4362	agcttgtttgccagtgggc	2077	2096	1	4
SEQ ID NO: 3023	gaattatcttttaaacatt	1090	1109	SEQ ID NO: 4363	taaagccattcaggtctct	5495	5514	1	4
SEQ ID NO: 3024	ttaccaccagtttgaagtt	1094	1113	SEQ ID NO: 4364	aattgttgaaagaaaaact	13192	13211	1	4
SEQ ID NO: 3025	taagattaaatatttactt	1110	1129	SEQ ID NO: 4365	taaagccattcaggtctct	14014	14033	1	4
SEQ ID NO: 3026	aaagattactttgagaaat	1112	1131	SEQ ID NO: 4366	ttaatctttcataagtcaa	4572	4591	1	4
SEQ ID NO: 3027	aggaaatagttggattacc	1117	1136	SEQ ID NO: 4367	ggacaaggcccagaatctg	2578	2597	1	4
SEQ ID NO: 3028	attttgatgatgctggccc	1132	1151	SEQ ID NO: 4368	attcttaacgggattcctt	12208	12228	1	4
SEQ ID NO: 3029	gaattatcttttaaacatt	1162	1181	SEQ ID NO: 4369	aattgttgaaagaaaaact	12273	12292	1	4
SEQ ID NO: 3030	ttaccaccagtttgaagtt	1174	1193	SEQ ID NO: 4370	agcttgtttgccagtgggc	10337	10356	1	4
SEQ ID NO: 3031	ctgaaaaagttagtgaaag	1188	1207	SEQ ID NO: 4371	ttaatctttcataagtcaa	12580	12599	1	4
SEQ ID NO: 3032	tttttcccagacagtgtca	1204	1223	SEQ ID NO: 4372	agcttgtttgccagtgggc	8866	8885	1	4
SEQ ID NO: 3033	ttttcccagacagtgtcca	1210	1229	SEQ ID NO: 4373	tgaaatcattgaaaattta	2169	2166	1	4
SEQ ID NO: 3034	cattcagaaccagggaaga	1211	1230	SEQ ID NO: 4374	caggaggctttaagttgtc	6168	2167	1	4
SEQ ID NO: 3035	acaaactgtttatagtccc	1218	1237	SEQ ID NO: 4375	agcttgtttgccagtgggc	13963	13982	1	4
SEQ ID NO: 3036	ggatttcaatacatttcac	1219	1236	SEQ ID NO: 4376	taaagccattcaggtctct	11248	11267	1	4
SEQ ID NO: 3037	ttgtagaaatgaaagtaaa	1248	1267	SEQ ID NO: 4377	cttcatcgttcatttgagt	8148	8167	1	4
SEQ ID NO: 3038	ccaaaatttctctcggctt	1332	1351	SEQ ID NO: 4378	cttatgggatttcctaagg	10824	10643	1	4
SEQ ID NO: 3039	caaattttctctgctaaaa	1349	1368	SEQ ID NO: 4379	cttcatcgttcatttgagt	3439	3458	1	4
SEQ ID NO: 3040	tctgctggaaacaacaacg	1440	1459	SEQ ID NO: 4380	ttccatcacaaatcctttg	5596	5505	1	4
SEQ ID NO: 3041	cagaagctaagcaatgtcc	1480	1499	SEQ ID NO: 4381	tctcaaagagttacaacag	12355	12374	1	4
SEQ ID NO: 3042	taagattaaatatttactt	1516	1535	SEQ ID NO: 4382	tattgaaatatgatttttt	9938	9957	1	4
SEQ ID NO: 3043	aaagattactttgagaaat	1546	1565	SEQ ID NO: 4383	aattgttgaaagaaaaact	10917	10936	1	4
SEQ ID NO: 3044	aggaaatagttggattacc	1548	1567	SEQ ID NO: 4384	ggacaaggcccagaatctg	8007	6026	1	4
SEQ ID NO: 3045	attttgatgatgctggccc	1600	1579	SEQ ID NO: 4385	aaagaaacctatggcctta	8118	8137	1	4
SEQ ID NO: 3046	gaattatcttttaaacatt	1610	1028	SEQ ID NO: 4386	attcttaacgggattcctt	9024	9043	1	4
SEQ ID NO: 3047	ttaccaccagtttgaagtt	1610	1637	SEQ ID NO: 4387	taaagccattcaggtctct	13727	13748	1	4
SEQ ID NO: 3048	taagattaaatatttactt	1629	1648	SEQ ID NO: 4388	gacatgtttgaagggtgga	1933	1952	1	4
SEQ ID NO: 3049	aaagattactttgagaaat	1703	1722	SEQ ID NO: 4389	tgggctaaacgtttaattg	2485	2504	1	4
SEQ ID NO: 3050	aggaaatagttggattacc	1738	1757	SEQ ID NO: 4390	tattgaaatatgatttttt	5519	5538	1	4
SEQ ID NO: 3051	attttgatgatgctggccc	1744	1783	SEQ ID NO: 4391	agcttgtttgccagtgggc	4439	4458	1	4
SEQ ID NO: 3052	aggtttaaatggataattt	1759	1778	SEQ ID NO: 4392	aaagaaacctatggcctta	8893	6912	1	4
SEQ ID NO: 3053	cagaagctaagcaatgtcc	1781	1800	SEQ ID NO: 4393	attcttaacgggattcctt	9952	9971	1	4
SEQ ID NO: 3054	taagattaaatatttactt	1798	7815	SEQ ID NO: 4394	taaagccattcaggtctct	12045	12084	1	4
SEQ ID NO: 3055	aaagattactttgagaaat	1890	1909	SEQ ID NO: 4395	gacatgtttgaagggtgga	10737	10758	1	4
SEQ ID NO: 3056	aggaaatagttggattacc	1910	1929	SEQ ID NO: 4396	tgggctaaacgtttaattg	14000	14019	1	4
SEQ ID NO: 3057	attttgatgatgctggccc	1913	1932	SEQ ID NO: 4397	tattgaaatatgatttttt	13378	13397	1	4
SEQ ID NO: 3058	gaattatcttttaaacatt	1924	1943	SEQ ID NO: 4398	agcttgtttgccagtgggc	7273	7292	1	4
SEQ ID NO: 3059	ttaccaccagtttgaagtt	1928	1948	SEQ ID NO: 4399	ggacaaggcccagaatctg	13825	13844	1	4
SEQ ID NO: 3060	ttgcagtgtatcggacaga	1930	1949	SEQ ID NO: 4400	aaagaaacctatggcctta	13824	13843	1	4
SEQ ID NO: 3061	cattcagaaccagggaaga	1935	1954	SEQ ID NO: 4401	attcttaacgggattcctt	8325	8344	1	4
SEQ ID NO: 3062	acaaactgtttatagtccc	1938	1957	SEQ ID NO: 4402	taaagccattcaggtctct	10185	10204	1	4
SEQ ID NO: 3063	ttaccaccagtttgaagtt	1939	1956	SEQ ID NO: 4403	aattgttgaaagaaaaact	10184	10203	1	4
SEQ ID NO: 3064	taagattaaatatttactt	1943	1982	SEQ ID NO: 4404	ggacaaggcccagaatctg	4973	4982	1	4
SEQ ID NO: 3065	aaagattactttgagaaat	1944	1953	SEQ ID NO: 4405	aaagaaacctatggcctta	4972	4991	1	4
SEQ ID NO: 3066	aggaaatagttggattacc	1950	1969	SEQ ID NO: 4406	attcttaacgggattcctt	10630	10649	1	4
SEQ ID NO: 3067	attttgatgatgctggccc	1990	2009	SEQ ID NO: 4407	taaagccattcaggtctct	13945	13964	1	4
SEQ ID NO: 3068	aggtttaaatggataattt	2007	2026	SEQ ID NO: 4408	gacatgtttgaagggtgga	9501	9520	1	4
SEQ ID NO: 3069	cagaagctaagcaatgtcc	2052	2071	SEQ ID NO: 4409	tgggctaaacgtttaattg	8441	9460	1	4
SEQ ID NO: 3070	taagattaaatatttactt	2065	2084	SEQ ID NO: 4410	tattgaaatatgatttttt	7820	7839	1	4
SEQ ID NO: 3071	aaagattactttgagaaat	2096	2087	SEQ ID NO: 4411	agcttgtttgccagtgggc	7822	7841	1	4
SEQ ID NO: 3072	aggaaatagttggattacc	2091	2110	SEQ ID NO: 4412	tgaaatcattgaaaattta	11621	11840	1	4
SEQ ID NO: 3073	attttgatgatgctggccc	2092	2111	SEQ ID NO: 4413	caggaggctttaagttgtc	14019	14038	1	4
SEQ ID NO: 3074	gaattatcttttaaacatt	117	2136	SEQ ID NO: 4414	agcttgtttgccagtgggc	3614	3633	1	4
SEQ ID NO: 3075	ttaccaccagtttgaagtt	2121	2140	SEQ ID NO: 4415	taaagccattcaggtctct	6694	6713	1	4
SEQ ID NO: 3076	ttgcagtgtatcggacaga	2122	2141	SEQ ID NO: 4416	aattgttgaaagaaaaact	9490	9509	1	4
SEQ ID NO: 3077	cattcagaaccagggaaga	2183	2202	SEQ ID NO: 4417	taaagccattcaggtctct	11709	11728	1	4
SEQ ID NO: 3078	acaaactgtttatagtccc	2206	2225	SEQ ID NO: 4418	ttaatctttcataagtcaa	11066	11087	1	4
SEQ ID NO: 3079	ggatttcaatacatttcac	2253	2272	SEQ ID NO: 4419	ggacaaggcccagaatctg	6142	6161	1	4
SEQ ID NO: 3080	ttgtagaaatgaaagtaaa	2257	2276	SEQ ID NO: 4420	attcttaacgggattcctt	9857	9675	1	4
SEQ ID NO: 3081	ctgaacagtgagctgcagt	2258	2277	SEQ ID NO: 4421	aattgttgaaagaaaaact	4359	4378	1	4
SEQ ID NO: 3082	aatccaatctcctctttgc	2298	2317	SEQ ID NO: 4422	agcttgtttgccagtgggc	3333	3352	1	4
SEQ ID NO: 3083	atttgattttcaagcaaag	2299	2318	SEQ ID NO: 4423	ttaatctttcataagtcaa	8831	8850	1	4
SEQ ID NO: 3084	ttttgattttcaagcaaat	2351	2370	SEQ ID NO: 4424	agcttgtttgccagtgggc	6796	6815	1	4
SEQ ID NO: 3085	tgatttcaagcaaatgcag	2395	2414	SEQ ID NO: 4425	tgaaatcattgaaaattta	6287	5306	1	4
SEQ ID NO: 3086	atgctgtttgggaaattgg	2405	2424	SEQ ID NO: 4426	caggaggctttaagttgtc	7614	7633	1	4
SEQ ID NO: 3087	tgctgttttttggaaatgc	2518	2537	SEQ ID NO: 4427	agcttgtttgccagtgggc	7983	8002	1	4
SEQ ID NO: 3088	aaaaaaatacactggacgt	2540	2559	SEQ ID NO: 4428	taaagccattcaggtctct	9083	9102	1	4
SEQ ID NO: 3089	actggagcttagtaagggc	2593	2612	SEQ ID NO: 4429	cttcatcgttcatttgagt	10382	10401	1	4
SEQ ID NO: 3090	cttctggaaagagggtcat	2596	2615	SEQ ID NO: 4430	cttatgggatttcctaagg	3615	3634	1	4
SEQ ID NO: 3091	ggaaaagggtcatggaaat	2603	2622	SEQ ID NO: 4431	cttcatcgttcatttgagt	9445	9464	1	4
SEQ ID NO: 3092	gggcctgccccagatttct	2609	2828	SEQ ID NO: 4432	ttccatcacaaatcctttg	8607	6628	1	4
SEQ ID NO: 3093	ttctcagataggggaacac	2610	2629	SEQ ID NO: 4433	tctcaaagagttacaacag	13116	13135	1	4
SEQ ID NO: 3094	gatgaaagaattaagggga	2624	2643	SEQ ID NO: 4434	tattgaaatatgatttttt	9842	9661	1	4
SEQ ID NO: 3095	cttggactgtcaaataagt	2625	2644	SEQ ID NO: 4435	aattgttgaaagaaaaact	9641	9660	1	4
SEQ ID NO: 3096	ggaaaagggtcatggaaat	2526	2645	SEQ ID NO: 4436	ggacaaggcccagaatctg	9640	9859	1	4
SEQ ID NO: 3097	gggcctgccccagatttct	2635	2054	SEQ ID NO: 4437	aaagaaacctatggcctta	9344	9363	1	4
SEQ ID NO: 3098	cttggactgtcaaataagt	2638	2655	SEQ ID NO: 4438	attcttaacgggattcctt	9343	9362	1	4
SEQ ID NO: 3099	ggatttcaatacatttcac	2652	2671	SEQ ID NO: 4439	taaagccattcaggtctct	5699	6618	1	4
SEQ ID NO: 3100	ttgtagaaatgaaagtaaa	2653	2872	SEQ ID NO: 4440	gacatgtttgaagggtgga	6598	6617	1	4
SEQ ID NO: 3101	ccaaaatttctctcggctt	2658	2877	SEQ ID NO: 4441	tgggctaaacgtttaattg	14004	14023	1	4
SEQ ID NO: 3102	caaattttctctgctaaaa	2705	2722	SEQ ID NO: 4442	tattgaaatatgatttttt	7600	7619	1	4
SEQ ID NO: 3103	tctgctggaaacaacaacg	2728	2747	SEQ ID NO: 4443	agcttgtttgccagtgggc	9228	9247	1	4
SEQ ID NO: 3104	cagaagctaagcaatgtcc	2758	2777	SEQ ID NO: 4444	tgaaatcattgaaaattta	8538	8557	1	4
SEQ ID NO: 3105	taagattaaatatttactt	2786	2785	SEQ ID NO: 4445	tgaaatcattgaaaattta	9521	9540	1	4
SEQ ID NO: 3106	aaagattactttgagaaat	2819	2838	SEQ ID NO: 4446	aaagaaacctatggcctta	2877	2896	1	4
SEQ ID NO: 3107	aggaaatagttggattacc	2833	2852	SEQ ID NO: 4447	attcttaacgggattcctt	5235	5254	1	4
SEQ ID NO: 3108	attttgatgatgctggccc	2840	2859	SEQ ID NO: 4448	taaagccattcaggtctct	6140	6169	1	4
SEQ ID NO: 3109	gaattatcttttaaacatt	2866	2985	SEQ ID NO: 4449	gacatgtttgaagggtgga	12120	13139	1	4
SEQ ID NO: 3110	ttaccaccagtttgaagtt	2872	2981	SEQ ID NO: 4450	tgggctaaacgtttaattg	5463	5482	1	4
SEQ ID NO: 3111	taagattaaatatttactt	3114	3133	SEQ ID NO: 4451	tattgaaatatgatttttt	12726	12746	1	4
SEQ ID NO: 3112	aaagattactttgagaaat	3206	3227	SEQ ID NO: 4452	agcttgtttgccagtgggc	8393	88412	1	4
SEQ ID NO: 3113	aggaaatagttggattacc	3252	3271	SEQ ID NO: 4453	tgaaatcattgaaaattta	3817	3836	1	4
SEQ ID NO: 3114	attttgatgatgctggccc	3297	3316	SEQ ID NO: 4454	caggaggctttaagttgtc	8357	6376	1	4
SEQ ID NO: 3115	gaattatcttttaaacatt	3313	3332	SEQ ID NO: 4455	agcttgtttgccagtgggc	8938	8957	1	4
SEQ ID NO: 3116	ttaccaccagtttgaagtt	3315	3334	SEQ ID NO: 4456	taaagccattcaggtctct	13207	13226	1	4
SEQ ID NO: 3117	ctgaaaaagttagtgaaag	3337	3356	SEQ ID NO: 4457	aattgttgaaagaaaaact	4211	4230	1	4
SEQ ID NO: 3118	tttttcccagacagtgtca	3345	3364	SEQ ID NO: 4458	taaagccattcaggtctct	7593	7612	1	4
SEQ ID NO: 3119	ttttcccagacagtgtcca	3392	3411	SEQ ID NO: 4459	ttaatctttcataagtcaa	12439	12458	1	4
SEQ ID NO: 3120	cattcagaaccagggaaga	3403	3422	SEQ ID NO: 4460	ggacaaggcccagaatctg	8104	8123	1	4
SEQ ID NO: 3121	acaaactgtttatagtccc	3422	3441	SEQ ID NO: 4461	attcttaacgggattcctt	10924	10943	1	4
SEQ ID NO: 3122	ggatttcaatacatttcac	3486	3505	SEQ ID NO: 4462	aattgttgaaagaaaaact	13800	13899	1	4
SEQ ID NO: 3123	ttgtagaaatgaaagtaaa	3501	3520	SEQ ID NO: 4463	agcttgtttgccagtgggc	5408	5427	1	4
SEQ ID NO: 3124	ccaaaatttctctcggctt	3502	3521	SEQ ID NO: 4464	ggacaaggcccagaatctg	5272	5291	1	4
SEQ ID NO: 3125	caaattttctctgctaaaa	3503	3522	SEQ ID NO: 4465	aaagaaacctatggcctta	5271	5290	1	4
SEQ ID NO: 3126	tctgctggaaacaacaacg	3554	3573	SEQ ID NO: 4466	attcttaacgggattcctt	4567	4536	1	4
SEQ ID NO: 3127	cagaagctaagcaatgtcc	3577	3526	SEQ ID NO: 4467	taaagccattcaggtctct	7809	7626	1	4
SEQ ID NO: 3128	taagattaaatatttactt	3581	3600	SEQ ID NO: 4468	gacatgtttgaagggtgga	5897	6916	1	4
SEQ ID NO: 3129	aaagattactttgagaaat	3600	3619	SEQ ID NO: 4469	tgggctaaacgtttaattg	8877	8895	1	4
SEQ ID NO: 3130	aggaaatagttggattacc	3603	3622	SEQ ID NO: 4470	tattgaaatatgatttttt	9361	8380	1	4
SEQ ID NO: 3131	attttgatgatgctggccc	3811	3630	SEQ ID NO: 4471	agcttgtttgccagtgggc	8071	8090	1	4
SEQ ID NO: 3132	gaattatcttttaaacatt	3625	3644	SEQ ID NO: 4472	tgaaatcattgaaaattta	7871	7880	1	4
SEQ ID NO: 3133	ttaccaccagtttgaagtt	6289	3646	SEQ ID NO: 4473	tgaaatcattgaaaattta	5287	5308	1	4
SEQ ID NO: 3134	taagattaaatatttactt	3632	3651	SEQ ID NO: 4474	aaagaaacctatggcctta	8278	8297	1	4
SEQ ID NO: 3135	aaagattactttgagaaat	3844	3003	SEQ ID NO: 4475	attcttaacgggattcctt	11236	11255	1	4
SEQ ID NO: 3136	aggaaatagttggattacc	3658	3677	SEQ ID NO: 4476	taaagccattcaggtctct	8591	5610	1	4
SEQ ID NO: 3137	attttgatgatgctggccc	3776	3695	SEQ ID NO: 4477	gacatgtttgaagggtgga	8366	8385	1	4
SEQ ID NO: 3138	aggtttaaatggataattt	3878	3696	SEQ ID NO: 4478	tgggctaaacgtttaattg	8355	8384	1	4
SEQ ID NO: 3139	ggatttcaatacatttcac	3678	3697	SEQ ID NO: 4479	tattgaaatatgatttttt	8406	8425	1	4
SEQ ID NO: 3140	ttgtagaaatgaaagtaaa	3760	3779	SEQ ID NO: 4480	agcttgtttgccagtgggc	4090	4109	1	4
SEQ ID NO: 3141	ctgaacagtgagctgcagt	3803	3822	SEQ ID NO: 4481	tgaaatcattgaaaattta	13186	13205	1	4
SEQ ID NO: 3142	aatccaatctcctctttgc	3899	3918	SEQ ID NO: 4482	caggaggctttaagttgtc	8142	8161	1	4
SEQ ID NO: 3143	atttgattttcaagcaaag	3915	3934	SEQ ID NO: 4483	agcttgtttgccagtgggc	8003	8922	1	4
SEQ ID NO: 3144	ttttgattttcaagcaaat	3994	4013	SEQ ID NO: 4484	taaagccattcaggtctct	9541	9550	1	4
SEQ ID NO: 3145	acaaactgtttatagtccc	4000	4019	SEQ ID NO: 4485	aattgttgaaagaaaaact	13604	13713	1	4
SEQ ID NO: 3146	ggatttcaatacatttcac	4015	4034	SEQ ID NO: 4486	taaagccattcaggtctct	6359	6378	1	4
SEQ ID NO: 3147	ttgtagaaatgaaagtaaa	4036	4055	SEQ ID NO: 4487	ttaatctttcataagtcaa	7504	7613	1	4
SEQ ID NO: 3148	ttaccaccagtttgaagtt	4045	4064	SEQ ID NO: 4488	ggacaaggcccagaatctg	10033	10052	1	4
SEQ ID NO: 3149	ttgcagtgtatcggacaga	4062	4111	SEQ ID NO: 4489	attcttaacgggattcctt	9735	9754	1	4
SEQ ID NO: 3150	cattcagaaccagggaaga	4104	4123	SEQ ID NO: 4490	aattgttgaaagaaaaact	8558	8576	1	4
SEQ ID NO: 3151	acaaactgtttatagtccc	4112	4131	SEQ ID NO: 4491	agcttgtttgccagtgggc	8215	8234	1	4
SEQ ID NO: 3152	ggatttcaatacatttcac	4126	4145	SEQ ID NO: 4492	ttaatctttcataagtcaa	6088	6105	1	4
SEQ ID NO: 3153	ttgtagaaatgaaagtaaa	4133	4152	SEQ ID NO: 4493	agcttgtttgccagtgggc	8109	8128	1	4
SEQ ID NO: 3154	ccaaaatttctctcggctt	4141	4160	SEQ ID NO: 4494	tgaaatcattgaaaattta	12009	12028	1	4
SEQ ID NO: 3155	caaattttctctgctaaaa	4284	4303	SEQ ID NO: 4495	caggaggctttaagttgtc	9024	9043	1	4
SEQ ID NO: 3156	tctgctggaaacaacaacg	4317	4336	SEQ ID NO: 4496	agcttgtttgccagtgggc	9503	9822	1	4
SEQ ID NO: 3157	cagaagctaagcaatgtcc	4338	4357	SEQ ID NO: 4497	taaagccattcaggtctct	11035	11054	1	4
SEQ ID NO: 3158	taagattaaatatttactt	4376	4397	SEQ ID NO: 4498	cttcatcgttcatttgagt	8585	6584	1	4
SEQ ID NO: 3159	aaagattactttgagaaat	4380	4399	SEQ ID NO: 4499	cttatgggatttcctaagg	12133	12152	1	4
SEQ ID NO: 3160	aggaaatagttggattacc	4407	4426	SEQ ID NO: 4500	cttcatcgttcatttgagt	7300	7327	1	4
SEQ ID NO: 3161	attttgatgatgctggccc	4499	4518	SEQ ID NO: 4501	ttccatcacaaatcctttg	8334	8353	1	4
SEQ ID NO: 3162	gaattatcttttaaacatt	4644	4663	SEQ ID NO: 4502	tctcaaagagttacaacag	8954	7009	1	4
SEQ ID NO: 3163	ttaccaccagtttgaagtt	4645	4664	SEQ ID NO: 4503	tattgaaatatgatttttt	6963	7002	1	4
SEQ ID NO: 3164	taagattaaatatttactt	4646	4665	SEQ ID NO: 4504	aattgttgaaagaaaaact	6982	7001	1	4
SEQ ID NO: 3165	aaagattactttgagaaat	4650	4889	SEQ ID NO: 4505	ggacaaggcccagaatctg	12073	12092	1	4
SEQ ID NO: 3166	aggaaatagttggattacc	4713	4732	SEQ ID NO: 4506	aaagaaacctatggcctta	10210	10229	1	4
SEQ ID NO: 3167	attttgatgatgctggccc	4745	4764	SEQ ID NO: 4507	attcttaacgggattcctt	8184	8203	1	4
SEQ ID NO: 3168	gaattatcttttaaacatt	4818	4837	SEQ ID NO: 4508	taaagccattcaggtctct	7930	7949	1	4
SEQ ID NO: 3169	ttaccaccagtttgaagtt	4820	4839	SEQ ID NO: 4509	gacatgtttgaagggtgga	8740	8759	1	4
SEQ ID NO: 3170	ctgaaaaagttagtgaaag	4873	4892	SEQ ID NO: 4510	tgggctaaacgtttaattg	6843	6862	1	4
SEQ ID NO: 3171	tttttcccagacagtgtca	4909	4926	SEQ ID NO: 4511	tattgaaatatgatttttt	9350	9369	1	4
SEQ ID NO: 3172	ttttcccagacagtgtcca	4913	4932	SEQ ID NO: 4512	agcttgtttgccagtgggc	8511	6630	1	4
SEQ ID NO: 3173	cattcagaaccagggaaga	4976	4995	SEQ ID NO: 4513	aaagaaacctatggcctta	12272	12291	1	4
SEQ ID NO: 3174	acaaactgtttatagtccc	4984	5003	SEQ ID NO: 4514	attcttaacgggattcctt	10809	10828	1	4
SEQ ID NO: 3175	ggatttcaatacatttcac	4988	5007	SEQ ID NO: 4515	taaagccattcaggtctct	10042	10061	1	4
SEQ ID NO: 3176	ttgtagaaatgaaagtaaa	4989	5008	SEQ ID NO: 4516	gacatgtttgaagggtgga	10041	10080	1	4
SEQ ID NO: 3177	ccaaaatttctctcggctt	4990	5009	SEQ ID NO: 4517	tgggctaaacgtttaattg	4733	4752	1	4
SEQ ID NO: 3178	caaattttctctgctaaaa	8091	5113	SEQ ID NO: 4518	tattgaaatatgatttttt	12267	12266	1	4
SEQ ID NO: 3179	tctgctggaaacaacaacg	5100	5119	SEQ ID NO: 4519	agcttgtttgccagtgggc	12514	12533	1	4
SEQ ID NO: 3180	cagaagctaagcaatgtcc	5114	5133	SEQ ID NO: 4520	ggacaaggcccagaatctg	7734	7753	1	4
SEQ ID NO: 3181	taagattaaatatttactt	5151	5170	SEQ ID NO: 4521	aaagaaacctatggcctta	13579	13598	1	4
SEQ ID NO: 3182	aaagattactttgagaaat	5178	5107	SEQ ID NO: 4522	attcttaacgggattcctt	8649	8668	1	4
SEQ ID NO: 3183	aggaaatagttggattacc	5207	5220	SEQ ID NO: 4523	taaagccattcaggtctct	9727	9746	1	4
SEQ ID NO: 3184	attttgatgatgctggccc	5265	5284	SEQ ID NO: 4524	aattgttgaaagaaaaact	5838	5857	1	4
SEQ ID NO: 3185	gaattatcttttaaacatt	5289	5308	SEQ ID NO: 4525	ggacaaggcccagaatctg	6993	7012	1	4
SEQ ID NO: 3186	ttaccaccagtttgaagtt	5324	5434	SEQ ID NO: 4526	aaagaaacctatggcctta	7255	7274	1	4
SEQ ID NO: 3187	taagattaaatatttactt	5325	5344	SEQ ID NO: 4527	attcttaacgggattcctt	7254	7273	1	4
SEQ ID NO: 3188	aaagattactttgagaaat	5341	6360	SEQ ID NO: 4528	taaagccattcaggtctct	7624	7643	1	4
SEQ ID NO: 3189	aggaaatagttggattacc	5346	6365	SEQ ID NO: 4529	gacatgtttgaagggtgga	12942	12961	1	4
SEQ ID NO: 3190	attttgatgatgctggccc	5407	6428	SEQ ID NO: 4530	tgggctaaacgtttaattg	8380	3399	1	4
SEQ ID NO: 3191	aggtttaaatggataattt	5412	6431	SEQ ID NO: 4531	tattgaaatatgatttttt	8504	5523	1	4
SEQ ID NO: 3192	cagaagctaagcaatgtcc	5445	5464	SEQ ID NO: 4532	agcttgtttgccagtgggc	9593	9712	1	4
SEQ ID NO: 3193	taagattaaatatttactt	5450	5469	SEQ ID NO: 4533	tgaaatcattgaaaattta	4429	4448	1	4
SEQ ID NO: 3194	aaagattactttgagaaat	5465	5485	SEQ ID NO: 4534	caggaggctttaagttgtc	6388	6407	1	4
SEQ ID NO: 3195	aggaaatagttggattacc	5474	5493	SEQ ID NO: 4535	agcttgtttgccagtgggc	6425	6444	1	4
SEQ ID NO: 3196	attttgatgatgctggccc	5494	8513	SEQ ID NO: 4536	taaagccattcaggtctct	11820	11839	1	4
SEQ ID NO: 3197	gaattatcttttaaacatt	5506	8825	SEQ ID NO: 4537	aattgttgaaagaaaaact	7032	7051	1	4
SEQ ID NO: 3198	ttaccaccagtttgaagtt	5507	8826	SEQ ID NO: 4538	taaagccattcaggtctct	10434	10453	1	4
SEQ ID NO: 3199	ttgcagtgtatcggacaga	5512	8831	SEQ ID NO: 4539	ttaatctttcataagtcaa	6131	6150	1	4
SEQ ID NO: 3200	cattcagaaccagggaaga	5554	5803	SEQ ID NO: 4540	ggacaaggcccagaatctg	10765	10784	1	4
SEQ ID NO: 3201	acaaactgtttatagtccc	5857	5676	SEQ ID NO: 4541	attcttaacgggattcctt	12764	12783	1	4
SEQ ID NO: 3202	ggatttcaatacatttcac	5692	5711	SEQ ID NO: 4542	aattgttgaaagaaaaact	9014	9033	1	4
SEQ ID NO: 3203	ttgtagaaatgaaagtaaa	5775	5794	SEQ ID NO: 4543	agcttgtttgccagtgggc	8437	8456	1	4
SEQ ID NO: 3204	ctgaacagtgagctgcagt	5776	5795	SEQ ID NO: 4544	ttaatctttcataagtcaa	13312	13331	1	4
SEQ ID NO: 3205	aatccaatctcctctttgc	5848	5867	SEQ ID NO: 4545	taaagccattcaggtctct	11176	11195	1	4
SEQ ID NO: 3206	atttgattttcaagcaaag	5896	5915	SEQ ID NO: 4546	aattgttgaaagaaaaact	10916	10938	1	4
SEQ ID NO: 3207	ttttgattttcaagcaaat	66043	6062	SEQ ID NO: 4547	taaagccattcaggtctct	8341	8360	1	4
SEQ ID NO: 3208	tgatttcaagcaaatgcag	6053	6072	SEQ ID NO: 4548	ttaatctttcataagtcaa	11221	11240	1	4
SEQ ID NO: 3209	atgctgtttgggaaattgg	8088	6107	SEQ ID NO: 4549	ggacaaggcccagaatctg	9330	8398	1	4
SEQ ID NO: 3210	tgctgttttttggaaatgc	6089	6108	SEQ ID NO: 4550	attcttaacgggattcctt	10656	10687	1	4
SEQ ID NO: 3211	aaaaaaatacactggacgt	8147	6165	SEQ ID NO: 4551	aattgttgaaagaaaaact	7930	7958	1	4
SEQ ID NO: 3212	actggagcttagtaagggc	8201	6220	SEQ ID NO: 4552	agcttgtttgccagtgggc	8876	6897	1	4
SEQ ID NO: 3213	cttctggaaagagggtcat	6241	6260	SEQ ID NO: 4553	ttaatctttcataagtcaa	10209	10228	1	4
SEQ ID NO: 3214	ggaaaagggtcatggaaat	6277	6296	SEQ ID NO: 4554	agcttgtttgccagtgggc	9064	9083	1	4
SEQ ID NO: 3215	gggcctgccccagatttct	6285	6304	SEQ ID NO: 4555	tgaaatcattgaaaattta	13318	13337	1	4
SEQ ID NO: 3216	ttctcagataggggaacac	287	6306	SEQ ID NO: 4556	caggaggctttaagttgtc	8421	8440	1	4
SEQ ID NO: 3217	gatgaaagaattaagggga	6320	6339	SEQ ID NO: 4557	agcttgtttgccagtgggc	10727	10746	1	4
SEQ ID NO: 3218	cttggactgtcaaataagt	6321	6340	SEQ ID NO: 4558	taaagccattcaggtctct	8893	8912	1	4
SEQ ID NO: 3219	ggaaaagggtcatggaaat	6346	6365	SEQ ID NO: 4559	cttcatcgttcatttgagt	8528	8547	1	4
SEQ ID NO: 3220	aggaaatagttggattacc	6352	6371	SEQ ID NO: 4560	cttatgggatttcctaagg	11708	11727	1	4
SEQ ID NO: 3221	attttgatgatgctggccc	6380	6399	SEQ ID NO: 4561	cttcatcgttcatttgagt	10435	10454	1	4
SEQ ID NO: 3222	gaattatcttttaaacatt	6415	6434	SEQ ID NO: 4562	ttccatcacaaatcctttg	7999	8018	1	4
SEQ ID NO: 3223	ttaccaccagtttgaagtt	3420	6439	SEQ ID NO: 4563	tctcaaagagttacaacag	11467	11508	1	4
SEQ ID NO: 3224	ctgaaaaagttagtgaaag	6455	6484	SEQ ID NO: 4564	tattgaaatatgatttttt	10000	10079	1	4
SEQ ID NO: 3225	tttttcccagacagtgtca	6494	6613	SEQ ID NO: 4565	aattgttgaaagaaaaact	11371	11390	1	4
SEQ ID NO: 3226	ttttcccagacagtgtcca	6405	6514	SEQ ID NO: 4566	ggacaaggcccagaatctg	11370	11359	1	4
SEQ ID NO: 3227	cattcagaaccagggaaga	6540	6559	SEQ ID NO: 4567	aaagaaacctatggcctta	7967	8008	1	4
SEQ ID NO: 3228	acaaactgtttatagtccc	6558	6577	SEQ ID NO: 4568	attcttaacgggattcctt	9050	9069	1	4
SEQ ID NO: 3229	ggatttcaatacatttcac	6611	6630	SEQ ID NO: 4569	taaagccattcaggtctct	6811	6830	1	4
SEQ ID NO: 3230	ttgtagaaatgaaagtaaa	6694	5713	SEQ ID NO: 4570	gacatgtttgaagggtgga	6710	8735	1	4
SEQ ID NO: 3231	ccaaaatttctctcggctt	8898	5715	SEQ ID NO: 4571	tgggctaaacgtttaattg	10441	10460	1	4
SEQ ID NO: 3232	caaattttctctgctaaaa	8710	5729	SEQ ID NO: 4572	tattgaaatatgatttttt	10159	10178	1	4
SEQ ID NO: 3233	tctgctggaaacaacaacg	8737	5766	SEQ ID NO: 4573	agcttgtttgccagtgggc	11161	11180	1	4
SEQ ID NO: 3234	cagaagctaagcaatgtcc	8762	6781	SEQ ID NO: 4574	aaagaaacctatggcctta	7281	7300	1	4
SEQ ID NO: 3235	taagattaaatatttactt	8823	6842	SEQ ID NO: 4575	attcttaacgggattcctt	11404	11423	1	4
SEQ ID NO: 3236	aaagattactttgagaaat	8914	6933	SEQ ID NO: 4576	taaagccattcaggtctct	10699	10716	1	4
SEQ ID NO: 3237	aggaaatagttggattacc	8973	6992	SEQ ID NO: 4577	gacatgtttgaagggtgga	10571	10690	1	4
SEQ ID NO: 3238	attttgatgatgctggccc	7059	7078	SEQ ID NO: 4578	tgggctaaacgtttaattg	7104	7123	1	4
SEQ ID NO: 3239	gaattatcttttaaacatt	7100	7119	SEQ ID NO: 4579	tattgaaatatgatttttt	8421	8440	1	4
SEQ ID NO: 3240	ttaccaccagtttgaagtt	7199	7218	SEQ ID NO: 4580	agcttgtttgccagtgggc	7776	7796	1	4
SEQ ID NO: 3241	taagattaaatatttactt	7227	7245	SEQ ID NO: 4581	ggacaaggcccagaatctg	8526	8545	1	4
SEQ ID NO: 3242	aaagattactttgagaaat	7232	7251	SEQ ID NO: 4582	aaagaaacctatggcctta	10301	10320	1	4
SEQ ID NO: 3243	aggaaatagttggattacc	7293	7312	SEQ ID NO: 4583	attcttaacgggattcctt	7320	7339	1	4
SEQ ID NO: 3244	attttgatgatgctggccc	7327	7346	SEQ ID NO: 4584	taaagccattcaggtctct	13209	13228	1	4
SEQ ID NO: 3245	aggtttaaatggataattt	7365	7334	SEQ ID NO: 4585	taaagccattcaggtctct	7254	7273	1	4
SEQ ID NO: 3246	cagaagctaagcaatgtcc	7394	7413	SEQ ID NO: 4586	gacatgtttgaagggtgga	8723	9742	1	4
SEQ ID NO: 3247	taagattaaatatttactt	7484	7433	SEQ ID NO: 4587	tgggctaaacgtttaattg	8076	9095	1	4
SEQ ID NO: 3248	aaagattactttgagaaat	7475	7494	SEQ ID NO: 4588	tattgaaatatgatttttt	8526	8544	1	4
SEQ ID NO: 3249	aggaaatagttggattacc	7492	7511	SEQ ID NO: 4589	agcttgtttgccagtgggc	10645	10064	1	4
SEQ ID NO: 3250	attttgatgatgctggccc	7576	7597	SEQ ID NO: 4590	ggacaaggcccagaatctg	10950	10096	1	4
SEQ ID NO: 3251	gaattatcttttaaacatt	7551	7600	SEQ ID NO: 4591	aaagaaacctatggcctta	10749	10768	1	4
SEQ ID NO: 3252	ttaccaccagtttgaagtt	7815	7634	SEQ ID NO: 4592	attcttaacgggattcctt	9480	9499	1	4
SEQ ID NO: 3253	ttgcagtgtatcggacaga	7739	7758	SEQ ID NO: 4593	taaagccattcaggtctct	7805	7884	1	4
SEQ ID NO: 3254	cattcagaaccagggaaga	7870	7889	SEQ ID NO: 4594	tattgaaatatgatttttt	8922	8941	1	4
SEQ ID NO: 3255	acaaactgtttatagtccc	7873	7892	SEQ ID NO: 4595	agcttgtttgccagtgggc	8006	9027	1	4
SEQ ID NO: 3256	ggatttcaatacatttcac	7874	7993	SEQ ID NO: 4596	ggacaaggcccagaatctg	6007	9026	1	4
SEQ ID NO: 3257	ttgtagaaatgaaagtaaa	7909	7928	SEQ ID NO: 4597	aaagaaacctatggcctta	12580	12599	1	4
SEQ ID NO: 3258	ctgaacagtgagctgcagt	7980	7999	SEQ ID NO: 4598	taaagccattcaggtctct	13222	13241	1	4
SEQ ID NO: 3259	aatccaatctcctctttgc	8080	8069	SEQ ID NO: 4599	taaagccattcaggtctct	8418	9437	1	4
SEQ ID NO: 3260	atttgattttcaagcaaag	8136	8155	SEQ ID NO: 4600	gacatgtttgaagggtgga	12438	12457	1	4
SEQ ID NO: 3261	ttttgattttcaagcaaat	8226	8245	SEQ ID NO: 4601	tgggctaaacgtttaattg	12301	12320	1	4
SEQ ID NO: 3262	tgatttcaagcaaatgcag	8299	6318	SEQ ID NO: 4602	tgggctaaacgtttaattg	12579	12508	1	4
SEQ ID NO: 3263	atgctgtttgggaaattgg	8328	8347	SEQ ID NO: 4603	attcttaacgggattcctt	10026	10045	1	4
SEQ ID NO: 3264	tgctgttttttggaaatgc	8436	8457	SEQ ID NO: 4604	aattgttgaaagaaaaact	10005	10024	1	4
SEQ ID NO: 3265	aaaaaaatacactggacgt	8439	8458	SEQ ID NO: 4605	agcttgtttgccagtgggc	9245	9254	1	4
SEQ ID NO: 3266	ctgaaaaagttagtgaaag	8458	8477	SEQ ID NO: 4606	ttaatctttcataagtcaa	9291	9310	1	4
SEQ ID NO: 3267	tttttcccagacagtgtca	8476	8495	SEQ ID NO: 4607	agcttgtttgccagtgggc	10972	10991	1	4
SEQ ID NO: 3268	ttttcccagacagtgtcca	8551	8570	SEQ ID NO: 4608	tgaaatcattgaaaattta	13501	13520	1	4
SEQ ID NO: 3269	cattcagaaccagggaaga	8592	8611	SEQ ID NO: 4609	caggaggctttaagttgtc	13788	13807	1	4
SEQ ID NO: 3270	acaaactgtttatagtccc	8652	8671	SEQ ID NO: 4610	agcttgtttgccagtgggc	13194	13213	1	4
SEQ ID NO: 3271	ggatttcaatacatttcac	8729	8748	SEQ ID NO: 4611	taaagccattcaggtctct	9231	9250	1	4
SEQ ID NO: 3272	ttgtagaaatgaaagtaaa	8741	8760	SEQ ID NO: 4612	cttcatcgttcatttgagt	4819	4838	1	4
SEQ ID NO: 3273	ccaaaatttctctcggctt	8787	8808	SEQ ID NO: 4613	cttatgggatttcctaagg	11172	11191	1	4
SEQ ID NO: 3274	caaattttctctgctaaaa	8795	8814	SEQ ID NO: 4614	cttcatcgttcatttgagt	12603	12622	1	4
SEQ ID NO: 3275	tctgctggaaacaacaacg	8722	8818	SEQ ID NO: 4615	ttccatcacaaatcctttg	9090	9109	1	4
SEQ ID NO: 3276	cagaagctaagcaatgtcc	8814	8833	SEQ ID NO: 4616	tctcaaagagttacaacag	11010	11009	1	4
SEQ ID NO: 3277	taagattaaatatttactt	8944	8983	SEQ ID NO: 4617	tattgaaatatgatttttt	12817	12836	1	4
SEQ ID NO: 3278	aaagattactttgagaaat	8988	8957	SEQ ID NO: 4618	aattgttgaaagaaaaact	12292	12311	1	4
SEQ ID NO: 3279	aggaaatagttggattacc	8988	9007	SEQ ID NO: 4619	ggacaaggcccagaatctg	11634	11653	1	4
SEQ ID NO: 3280	attttgatgatgctggccc	8992	9011	SEQ ID NO: 4620	aaagaaacctatggcctta	13891	13910	1	4
SEQ ID NO: 3281	gaattatcttttaaacatt	8933	9012	SEQ ID NO: 4621	attcttaacgggattcctt	13890	13909	1	4
SEQ ID NO: 3282	ttaccaccagtttgaagtt	9041	9060	SEQ ID NO: 4622	taaagccattcaggtctct	12255	12274	1	4
SEQ ID NO: 3283	taagattaaatatttactt	9045	9064	SEQ ID NO: 4623	gacatgtttgaagggtgga	11667	11686	1	4
SEQ ID NO: 3284	aaagattactttgagaaat	9059	9078	SEQ ID NO: 4624	tgggctaaacgtttaattg	11653	11672	1	4
SEQ ID NO: 3285	aggaaatagttggattacc	9129	9148	SEQ ID NO: 4625	tattgaaatatgatttttt	9749	9768	1	4
SEQ ID NO: 3286	attttgatgatgctggccc	9132	9151	SEQ ID NO: 4626	agcttgtttgccagtgggc	11077	11096	1	4
SEQ ID NO: 3287	aggtttaaatggataattt	9262	9281	SEQ ID NO: 4627	aaagaaacctatggcctta	7977	7993	1	4
SEQ ID NO: 3288	cagaagctaagcaatgtcc	9279	9298	SEQ ID NO: 4628	attcttaacgggattcctt	9555	9574	1	4
SEQ ID NO: 3289	taagattaaatatttactt	9332	9351	SEQ ID NO: 4629	taaagccattcaggtctct	12838	12855	1	4
SEQ ID NO: 3290	aaagattactttgagaaat	9432	9451	SEQ ID NO: 4630	gacatgtttgaagggtgga	13608	13627	1	4
SEQ ID NO: 3291	aggaaatagttggattacc	9599	9818	SEQ ID NO: 4631	tgggctaaacgtttaattg	11085	11084	1	4
SEQ ID NO: 3292	attttgatgatgctggccc	9548	9557	SEQ ID NO: 4632	tattgaaatatgatttttt	9890	9709	1	4
SEQ ID NO: 3293	gaattatcttttaaacatt	9554	9673	SEQ ID NO: 4633	agcttgtttgccagtgggc	9280	9299	1	4
SEQ ID NO: 3294	ttaccaccagtttgaagtt	9887	9696	SEQ ID NO: 4634	ggacaaggcccagaatctg	10971	10990	1	4
SEQ ID NO: 3295	ttgcagtgtatcggacaga	9740	9759	SEQ ID NO: 4635	aaagaaacctatggcctta	12933	12952	1	4
SEQ ID NO: 3296	cattcagaaccagggaaga	9757	9776	SEQ ID NO: 4636	attcttaacgggattcctt	10279	10298	1	4
SEQ ID NO: 3297	acaaactgtttatagtccc	9817	9326	SEQ ID NO: 4637	taaagccattcaggtctct	11438	11457	1	4
SEQ ID NO: 3298	ggatttcaatacatttcac	9863	9832	SEQ ID NO: 4638	aattgttgaaagaaaaact	8810	8829	1	4
SEQ ID NO: 3299	ttgtagaaatgaaagtaaa	9890	9909	SEQ ID NO: 4639	ggacaaggcccagaatctg	9046	9065	1	4
SEQ ID NO: 3300	ctgaacagtgagctgcagt	9964	9933	SEQ ID NO: 4640	aaagaaacctatggcctta	6893	6912	1	4
SEQ ID NO: 3301	aatccaatctcctctttgc	9905	9984	SEQ ID NO: 4641	attcttaacgggattcctt	6892	6911	1	4
SEQ ID NO: 3302	atttgattttcaagcaaag	10019	10036	SEQ ID NO: 4642	taaagccattcaggtctct	13305	13324	1	4
SEQ ID NO: 3303	ttttgattttcaagcaaat	10177	10196	SEQ ID NO: 4643	gacatgtttgaagggtgga	11156	11205	1	4
SEQ ID NO: 3304	tgatttcaagcaaatgcag	10214	10233	SEQ ID NO: 4644	tgggctaaacgtttaattg	13772	13791	1	4
SEQ ID NO: 3305	atgctgtttgggaaattgg	10234	10253	SEQ ID NO: 4645	tattgaaatatgatttttt	1060	12099	1	4
SEQ ID NO: 3306	ttgtagaaatgaaagtaaa	10240	10259	SEQ ID NO: 4646	agcttgtttgccagtgggc	11668	11635	1	4
SEQ ID NO: 3307	ctgaacagtgagctgcagt	10309	10328	SEQ ID NO: 4647	tgaaatcattgaaaattta	10547	10566	1	4
SEQ ID NO: 3308	aatccaatctcctctttgc	10408	10427	SEQ ID NO: 4648	caggaggctttaagttgtc	13971	13990	1	4
SEQ ID NO: 3309	atttgattttcaagcaaag	10424	10443	SEQ ID NO: 4649	agcttgtttgccagtgggc	13216	13235	1	4
SEQ ID NO: 3310	ttttgattttcaagcaaat	10469	10488	SEQ ID NO: 4650	taaagccattcaggtctct	12591	12610	1	4
SEQ ID NO: 3311	tgatttcaagcaaatgcag	10583	10602	SEQ ID NO: 4651	aattgttgaaagaaaaact	12363	12382	1	4
SEQ ID NO: 3312	atgctgtttgggaaattgg	10918	10937	SEQ ID NO: 4652	taaagccattcaggtctct	5001	5020	1	4
SEQ ID NO: 3313	cagaagctaagcaatgtcc	10926	10945	SEQ ID NO: 4653	ttaatctttcataagtcaa	14002	14021	1	4
SEQ ID NO: 3314	taagattaaatatttactt	10983	11002	SEQ ID NO: 4654	ggacaaggcccagaatctg	8526	8545	1	4
SEQ ID NO: 3315	aaagattactttgagaaat	11004	11023	SEQ ID NO: 4655	attcttaacgggattcctt	12173	12192	1	4
SEQ ID NO: 3316	aggaaatagttggattacc	11264	11283	SEQ ID NO: 4656	aattgttgaaagaaaaact	11632	11651	1	4
SEQ ID NO: 3317	attttgatgatgctggccc	11268	11307	SEQ ID NO: 4657	agcttgtttgccagtgggc	9489	9508	1	4
SEQ ID NO: 3318	gaattatcttttaaacatt	11200	11309	SEQ ID NO: 4658	ttaatctttcataagtcaa	9047	9066	1	4
SEQ ID NO: 3319	ttaccaccagtttgaagtt	11315	11335	SEQ ID NO: 4659	taaagccattcaggtctct	11035	11054	1	4
SEQ ID NO: 3320	taagattaaatatttactt	11413	11432	SEQ ID NO: 4660	aattgttgaaagaaaaact	8873	8892	1	4
SEQ ID NO: 3321	aaagattactttgagaaat	11480	11499	SEQ ID NO: 4662	taaagccattcaggtctct	1206	12325	1	4
SEQ ID NO: 3322	aggaaatagttggattacc	11545	11584	SEQ ID NO: 4663	ttaatctttcataagtcaa	13723	13742	1	4
SEQ ID NO: 3323	attttgatgatgctggccc	11735	11754	SEQ ID NO: 4664	ggacaaggcccagaatctg	12102	12121	1	4
SEQ ID NO: 3324	gaattatcttttaaacatt	11796	11814	SEQ ID NO: 4665	attcttaacgggattcctt	13104	13123	1	4
SEQ ID NO: 3325	ttaccaccagtttgaagtt	11859	11876	SEQ ID NO: 4666	aattgttgaaagaaaaact	6254	6273	1	4
SEQ ID NO: 3326	ctgaaaaagttagtgaaag	11992	12011	SEQ ID NO: 4667	agcttgtttgccagtgggc	9220	9230	1	4
SEQ ID NO: 3327	tttttcccagacagtgtca	12050	12069	SEQ ID NO: 4668	ttaatctttcataagtcaa	12669	12666	1	4
SEQ ID NO: 3328	ttttcccagacagtgtcca	12143	12162	SEQ ID NO: 4669	agcttgtttgccagtgggc	9071	8000	1	4
SEQ ID NO: 3329	cattcagaaccagggaaga	12179	12198	SEQ ID NO: 4670	tgaaatcattgaaaattta	10088	10107	1	4
SEQ ID NO: 3330	acaaactgtttatagtccc	12219	12238	SEQ ID NO: 4671	caggaggctttaagttgtc	6993	7012	1	4
SEQ ID NO: 3331	ggatttcaatacatttcac	12428	13447	SEQ ID NO: 4672	agcttgtttgccagtgggc	13447	13466	1	4
SEQ ID NO: 3332	ttgtagaaatgaaagtaaa	12447	12466	SEQ ID NO: 4673	taaagccattcaggtctct	14056	14105	1	4
SEQ ID NO: 3333	ccaaaatttctctcggctt	12448	12487	SEQ ID NO: 4674	cttcatcgttcatttgagt	14085	14104	1	4
SEQ ID NO: 3334	caaattttctctgctaaaa	12477	12498	SEQ ID NO: 4675	cttatgggatttcctaagg	13392	13411	1	4
SEQ ID NO: 3335	tctgctggaaacaacaacg	12897	12916	SEQ ID NO: 4676	cttcatcgttcatttgagt	13641	13680	1	4
SEQ ID NO: 3336	cagaagctaagcaatgtcc	13039	13058	SEQ ID NO: 4677	ttccatcacaaatcctttg	14052	14071	1	4
SEQ ID NO: 3337	taagattaaatatttactt	13094	13114	SEQ ID NO: 4678	tctcaaagagttacaacag	15800	13819	1	4
SEQ ID NO: 3338	aaagattactttgagaaat	13151	13170	SEQ ID NO: 4679	tattgaaatatgatttttt	7032	7051	1	4
SEQ ID NO: 3339	aggaaatagttggattacc	13190	13209	SEQ ID NO: 4680	aattgttgaaagaaaaact	10168	10187	1	4
SEQ ID NO: 3340	attttgatgatgctggccc	13326	13346	SEQ ID NO: 4681	ggacaaggcccagaatctg	12407	12428	1	4
SEQ ID NO: 3341	gaattatcttttaaacatt	13377	13398	SEQ ID NO: 4682	aaagaaacctatggcctta	10737	10756	1	4
SEQ ID NO: 3342	ttaccaccagtttgaagtt	13451	13470	SEQ ID NO: 4683	attcttaacgggattcctt	11488	11307	1	4
SEQ ID NO: 3343	taagattaaatatttactt	13580	13579	SEQ ID NO: 4684	taaagccattcaggtctct	10103	10122	1	4
SEQ ID NO: 3344	aaagattactttgagaaat	13607	13626	SEQ ID NO: 4685	gacatgtttgaagggtgga	13873	13892	1	4
SEQ ID NO: 3345	aggaaatagttggattacc	13637	13858	SEQ ID NO: 4686	tgggctaaacgtttaattg	6436	6455	1	4
SEQ ID NO: 3346	attttgatgatgctggccc	13726	13745	SEQ ID NO: 4687	tattgaaatatgatttttt	9952	9971	1	4
SEQ ID NO: 3347	aggtttaaatggataattt	13773	13792	SEQ ID NO: 4688	agcttgtttgccagtgggc	10213	10232	1	4
SEQ ID NO: 3348	cagaagctaagcaatgtcc	13055	13974	SEQ ID NO: 4689	aaagaaacctatggcctta	7856	7875	1	4
SEQ ID NO: 3349	taagattaaatatttactt	14058	14077	SEQ ID NO: 4690	attcttaacgggattcctt	7230	4249	1	4
SEQ ID NO: 3350	aaagattactttgagaaat	4412	4431	SEQ ID NO: 4691	taaagccattcaggtctct	9603	9622	3	3
SEQ ID NO: 3351	aggaaatagttggattacc	4551	4570	SEQ ID NO: 4692	gacatgtttgaagggtgga	9063	9082	3	3
SEQ ID NO: 3352	attttgatgatgctggccc	33	52	SEQ ID NO: 4693	tgggctaaacgtttaattg	9236	9256	2	3
SEQ ID NO: 3353	gaattatcttttaaacatt	47	66	SEQ ID NO: 4694	tattgaaatatgatttttt	11185	11204	2	3
SEQ ID NO: 3354	ttaccaccagtttgaagtt	227	246	SEQ ID NO: 4695	agcttgtttgccagtgggc	8134	8153	2	3
SEQ ID NO: 3355	ttgcagtgtatcggacaga	291	310	SEQ ID NO: 4696	ggacaaggcccagaatctg	7465	7484	2	3
SEQ ID NO: 3356	cattcagaaccagggaaga	404	423	SEQ ID NO: 4697	aaagaaacctatggcctta	8977	8996	2	3
SEQ ID NO: 3357	acaaactgtttatagtccc	472	491	SEQ ID NO: 4698	attcttaacgggattcctt	11399	11418	2	3
SEQ ID NO: 3358	ggatttcaatacatttcac	582	601	SEQ ID NO: 4699	taaagccattcaggtctct	7050	7099	2	3
SEQ ID NO: 3359	ttgtagaaatgaaagtaaa	830	849	SEQ ID NO: 4700	taaagccattcaggtctct	8809	8825	2	3
SEQ ID NO: 3360	ctgaacagtgagctgcagt	864	884	SEQ ID NO: 4701	gacatgtttgaagggtgga	11354	11373	2	3
SEQ ID NO: 3361	aatccaatctcctctttgc	1087	1106	SEQ ID NO: 4702	tgggctaaacgtttaattg	12411	12430	2	3
SEQ ID NO: 3362	atttgattttcaagcaaag	1113	1132	SEQ ID NO: 4703	tattgaaatatgatttttt	13112	13131	2	3
SEQ ID NO: 3363	ttttgattttcaagcaaat	1176	1195	SEQ ID NO: 4704	agcttgtttgccagtgggc	3035	3054	2	3
SEQ ID NO: 3364	tgatttcaagcaaatgcag	1311	1330	SEQ ID NO: 4705	ggacaaggcccagaatctg	7856	7876	2	3
SEQ ID NO: 3365	atgctgtttgggaaattgg	1440	1459	SEQ ID NO: 4706	aaagaaacctatggcctta	9138	9157	2	3
SEQ ID NO: 3366	tgctgttttttggaaatgc	1500	1519	SEQ ID NO: 4707	attcttaacgggattcctt	10926	10947	2	3
SEQ ID NO: 3367	aaaaaaatacactggacgt	1575	1594	SEQ ID NO: 4708	aattgttgaaagaaaaact	7428	7447	2	3
SEQ ID NO: 3368	actggagcttagtaagggc	1627	1646	SEQ ID NO: 4709	ggacaaggcccagaatctg	5520	5539	2	3
SEQ ID NO: 3369	cttctggaaagagggtcat	1744	1783	SEQ ID NO: 4710	aaagaaacctatggcctta	10455	10474	2	3
SEQ ID NO: 3370	ggaaaagggtcatggaaat	1810	1829	SEQ ID NO: 4711	attcttaacgggattcctt	13734	13753	2	3
SEQ ID NO: 3371	gggcctgccccagatttct	1941	1960	SEQ ID NO: 4712	taaagccattcaggtctct	3552	3571	2	3
SEQ ID NO: 3372	ttctcagataggggaacac	1956	1976	SEQ ID NO: 4713	gacatgtttgaagggtgga	8275	8294	2	3
SEQ ID NO: 3373	gatgaaagaattaagggga	2084	2103	SEQ ID NO: 4714	tgggctaaacgtttaattg	10467	10486	2	3
SEQ ID NO: 3374	cttggactgtcaaataagt	2221	2240	SEQ ID NO: 4715	tattgaaatatgatttttt	8324	8343	2	3
SEQ ID NO: 3375	ggaaaagggtcatggaaat	2374	2393	SEQ ID NO: 4716	agcttgtttgccagtgggc	9668	9575	2	3
SEQ ID NO: 3376	aggaaatagttggattacc	2408	2427	SEQ ID NO: 4717	tgaaatcattgaaaattta	10037	10095	2	3
SEQ ID NO: 3377	attttgatgatgctggccc	2417	2436	SEQ ID NO: 4718	caggaggctttaagttgtc	4951	4970	2	3
SEQ ID NO: 3378	gaattatcttttaaacatt	2570	2569	SEQ ID NO: 4719	agcttgtttgccagtgggc	10020	10039	2	3
SEQ ID NO: 3379	ttaccaccagtttgaagtt	2583	2602	SEQ ID NO: 4720	taaagccattcaggtctct	12160	12179	2	3
SEQ ID NO: 3380	ctgaaaaagttagtgaaag	2765	2784	SEQ ID NO: 4721	aattgttgaaagaaaaact	11815	11834	2	3
SEQ ID NO: 3381	tttttcccagacagtgtca	3252	3271	SEQ ID NO: 4722	taaagccattcaggtctct	10903	10922	2	3
SEQ ID NO: 3382	ttttcccagacagtgtcca	3000	3667	SEQ ID NO: 4723	ttaatctttcataagtcaa	11450	11469	2	3
SEQ ID NO: 3383	cattcagaaccagggaaga	3681	3700	SEQ ID NO: 4724	ggacaaggcccagaatctg	12927	12946	2	3
SEQ ID NO: 3384	ttgcagtgtatcggacaga	3683	3702	SEQ ID NO: 4725	attcttaacgggattcctt	13785	13804	2	3
SEQ ID NO: 3385	cattcagaaccagggaaga	4104	4123	SEQ ID NO: 4726	aattgttgaaagaaaaact	9547	9566	2	3
SEQ ID NO: 3386	acaaactgtttatagtccc	4551	4570	SEQ ID NO: 4727	agcttgtttgccagtgggc	8247	8266	2	3
SEQ ID NO: 3387	ggatttcaatacatttcac	5135	5154	SEQ ID NO: 4728	ttaatctttcataagtcaa	13518	13537	2	3
SEQ ID NO: 3388	ttgtagaaatgaaagtaaa	5353	5372	SEQ ID NO: 4729	taaagccattcaggtctct	13874	13893	2	3
SEQ ID NO: 3389	ctgaacagtgagctgcagt	5420	5439	SEQ ID NO: 4730	aattgttgaaagaaaaact	8780	8799	2	3
SEQ ID NO: 3390	aatccaatctcctctttgc	5512	5331	SEQ ID NO: 4731	taaagccattcaggtctct	8794	8813	2	3
SEQ ID NO: 3391	atttgattttcaagcaaag	5848	5367	SEQ ID NO: 4732	ttaatctttcataagtcaa	11229	11248	2	3
SEQ ID NO: 3392	ttttgattttcaagcaaat	6285	5304	SEQ ID NO: 4733	ggacaaggcccagaatctg	13656	13676	2	3
SEQ ID NO: 3393	tgatttcaagcaaatgcag	6288	6307	SEQ ID NO: 4734	attcttaacgggattcctt	8538	8558	2	3
SEQ ID NO: 3394	atgctgtttgggaaattgg	6320	6339	SEQ ID NO: 4735	aattgttgaaagaaaaact	10756	10775	2	3
SEQ ID NO: 3395	tgctgttttttggaaatgc	6488	6507	SEQ ID NO: 4736	agcttgtttgccagtgggc	7162	7181	2	3
SEQ ID NO: 3396	aaaaaaatacactggacgt	6506	6525	SEQ ID NO: 4737	ttaatctttcataagtcaa	11324	11343	2	3
SEQ ID NO: 3397	ctgaaaaagttagtgaaag	6696	6715	SEQ ID NO: 4738	agcttgtttgccagtgggc	13441	13460	2	3
SEQ ID NO: 3398	tttttcccagacagtgtca	7060	7079	SEQ ID NO: 4739	tgaaatcattgaaaattta	12971	12990	2	3
SEQ ID NO: 3399	ttttcccagacagtgtcca	7356	7375	SEQ ID NO: 4740	caggaggctttaagttgtc	12660	12679	2	3
SEQ ID NO: 3400	cattcagaaccagggaaga	7753	7772	SEQ ID NO: 4741	agcttgtttgccagtgggc	13276	13295	2	3
SEQ ID NO: 3401	acaaactgtttatagtccc	8080	8069	SEQ ID NO: 4742	taaagccattcaggtctct	9481	9500	2	3
SEQ ID NO: 3402	ggatttcaatacatttcac	9408	9424	SEQ ID NO: 4743	cttcatcgttcatttgagt	14083	14102	2	3
SEQ ID NO: 3403	ttgtagaaatgaaagtaaa	8	27	SEQ ID NO: 4744	cttatgggatttcctaagg	11571	11590	1	3
SEQ ID NO: 3404	ccaaaatttctctcggctt	25	44	SEQ ID NO: 4745	cttcatcgttcatttgagt	12078	12697	1	3
SEQ ID NO: 3405	caaattttctctgctaaaa	47	86	SEQ ID NO: 4746	ttccatcacaaatcctttg	1225	1244	1	3
SEQ ID NO: 3406	tctgctggaaacaacaacg	50	69	SEQ ID NO: 4747	tctcaaagagttacaacag	3752	3771	1	3
SEQ ID NO: 3407	cagaagctaagcaatgtcc	72	91	SEQ ID NO: 4748	tattgaaatatgatttttt	1383	1382	1	3
SEQ ID NO: 3408	taagattaaatatttactt	89	108	SEQ ID NO: 4749	aattgttgaaagaaaaact	2692	2701	1	3
SEQ ID NO: 3409	aaagattactttgagaaat	104	123	SEQ ID NO: 4750	ggacaaggcccagaatctg	1086	1107	1	3
SEQ ID NO: 3410	aggaaatagttggattacc	200	210	SEQ ID NO: 4751	aaagaaacctatggcctta	8309	8328	1	3
SEQ ID NO: 3411	attttgatgatgctggccc	237	256	SEQ ID NO: 4752	attcttaacgggattcctt	7085	7104	1	3
SEQ ID NO: 3412	gaattatcttttaaacatt	253	272	SEQ ID NO: 4753	taaagccattcaggtctct	10067	10086	1	3
SEQ ID NO: 3413	ttaccaccagtttgaagtt	297	316	SEQ ID NO: 4754	gacatgtttgaagggtgga	8610	8629	1	3
SEQ ID NO: 3414	taagattaaatatttactt	316	335	SEQ ID NO: 4755	tgggctaaacgtttaattg	13324	13345	1	3
SEQ ID NO: 3415	aaagattactttgagaaat	333	352	SEQ ID NO: 4756	tattgaaatatgatttttt	10704	10723	1	3
SEQ ID NO: 3416	aggaaatagttggattacc	343	362	SEQ ID NO: 4757	agcttgtttgccagtgggc	4748	4767	1	3
SEQ ID NO: 3417	attttgatgatgctggccc	348	367	SEQ ID NO: 4758	aaagaaacctatggcctta	1289	1305	1	3
SEQ ID NO: 3418	aggtttaaatggataattt	373	392	SEQ ID NO: 4759	attcttaacgggattcctt	1343	1382	1	3
SEQ ID NO: 3419	cagaagctaagcaatgtcc	383	402	SEQ ID NO: 4760	taaagccattcaggtctct	5112	5131	1	3
SEQ ID NO: 3420	taagattaaatatttactt	385	404	SEQ ID NO: 4761	gacatgtttgaagggtgga	2698	2715	1	3
SEQ ID NO: 3421	aaagattactttgagaaat	399	418	SEQ ID NO: 4762	tgggctaaacgtttaattg	1539	1588	1	3
SEQ ID NO: 3422	aggaaatagttggattacc	404	423	SEQ ID NO: 4763	tattgaaatatgatttttt	5230	5249	1	3
SEQ ID NO: 3423	attttgatgatgctggccc	427	446	SEQ ID NO: 4764	agcttgtttgccagtgggc	3533	3652	1	3
SEQ ID NO: 3424	gaattatcttttaaacatt	430	449	SEQ ID NO: 4765	ggacaaggcccagaatctg	2207	2226	1	3
SEQ ID NO: 3425	ttaccaccagtttgaagtt	431	450	SEQ ID NO: 4766	aaagaaacctatggcctta	9727	9746	1	3
SEQ ID NO: 3426	ttgcagtgtatcggacaga	451	470	SEQ ID NO: 4767	attcttaacgggattcctt	9074	9093	1	3
SEQ ID NO: 3427	cattcagaaccagggaaga	453	472	SEQ ID NO: 4768	taaagccattcaggtctct	5647	5666	1	3
SEQ ID NO: 3428	acaaactgtttatagtccc	483	503	SEQ ID NO: 4769	taaagccattcaggtctct	3004	3023	1	3
SEQ ID NO: 3429	ggatttcaatacatttcac	484	503	SEQ ID NO: 4770	gacatgtttgaagggtgga	3003	3022	1	3
SEQ ID NO: 3430	ttgtagaaatgaaagtaaa	490	509	SEQ ID NO: 4771	tgggctaaacgtttaattg	1293	1312	1	3
SEQ ID NO: 3431	ctgaacagtgagctgcagt	507	526	SEQ ID NO: 4772	tattgaaatatgatttttt	7489	7508	1	3
SEQ ID NO: 3432	aatccaatctcctctttgc	526	545	SEQ ID NO: 4773	agcttgtttgccagtgggc	13821	13840	1	3
SEQ ID NO: 3433	atttgattttcaagcaaag	628	5547	SEQ ID NO: 4774	ggacaaggcccagaatctg	7572	7591	1	3
SEQ ID NO: 3434	ttttgattttcaagcaaat	555	574	SEQ ID NO: 4775	aaagaaacctatggcctta	4852	4871	1	3
SEQ ID NO: 3435	tgatttcaagcaaatgcag	575	594	SEQ ID NO: 4776	attcttaacgggattcctt	7977	7995	1	3
SEQ ID NO: 3436	atgctgtttgggaaattgg	576	595	SEQ ID NO: 4777	taaagccattcaggtctct	7976	7995	1	3
SEQ ID NO: 3437	ttgtagaaatgaaagtaaa	576	597	SEQ ID NO: 4778	tattgaaatatgatttttt	7908	7927	1	3
SEQ ID NO: 3438	ctgaacagtgagctgcagt	581	600	SEQ ID NO: 4779	agcttgtttgccagtgggc	5298	6317	1	3
SEQ ID NO: 3439	aatccaatctcctctttgc	582	601	SEQ ID NO: 4780	ggacaaggcccagaatctg	6297	8316	1	3
SEQ ID NO: 3440	atttgattttcaagcaaag	597	616	SEQ ID NO: 4781	aaagaaacctatggcctta	7413	7432	1	3
SEQ ID NO: 3441	ttttgattttcaagcaaat	615	634	SEQ ID NO: 4782	taaagccattcaggtctct	10066	10085	1	3
SEQ ID NO: 3442	tgatttcaagcaaatgcag	629	648	SEQ ID NO: 4783	taaagccattcaggtctct	8079	8098	1	3
SEQ ID NO: 3443	atgctgtttgggaaattgg	647	666	SEQ ID NO: 4784	gacatgtttgaagggtgga	8775	8794	1	3
SEQ ID NO: 3444	cagaagctaagcaatgtcc	663	682	SEQ ID NO: 4785	tgggctaaacgtttaattg	2928	2947	1	3
SEQ ID NO: 3445	taagattaaatatttactt	670	689	SEQ ID NO: 4786	tgggctaaacgtttaattg	4540	4559	1	3
SEQ ID NO: 3446	aaagattactttgagaaat	660	699	SEQ ID NO: 4787	attcttaacgggattcctt	10580	10599	1	3
SEQ ID NO: 3447	aggaaatagttggattacc	665	704	SEQ ID NO: 4788	aattgttgaaagaaaaact	2491	2510	1	3
SEQ ID NO: 3448	attttgatgatgctggccc	687	706	SEQ ID NO: 4789	agcttgtttgccagtgggc	11177	11198	1	3
SEQ ID NO: 3449	gaattatcttttaaacatt	698	717	SEQ ID NO: 4790	ggacaaggcccagaatctg	5964	5983	1	3
SEQ ID NO: 3450	ttaccaccagtttgaagtt	699	718	SEQ ID NO: 4791	aaagaaacctatggcctta	3515	3534	1	3
SEQ ID NO: 3451	taagattaaatatttactt	700	719	SEQ ID NO: 4792	attcttaacgggattcctt	3476	2495	1	3
SEQ ID NO: 3452	aaagattactttgagaaat	702	721	SEQ ID NO: 4793	taaagccattcaggtctct	13364	13383	1	3
SEQ ID NO: 3453	aggaaatagttggattacc	703	722	SEQ ID NO: 4794	taaagccattcaggtctct	1809	1828	1	3
SEQ ID NO: 3454	attttgatgatgctggccc	777	798	SEQ ID NO: 4795	gacatgtttgaagggtgga	3019	3038	1	3
SEQ ID NO: 3455	gaattatcttttaaacatt	776	797	SEQ ID NO: 4796	tgggctaaacgtttaattg	3018	3037	1	3
SEQ ID NO: 3456	ttaccaccagtttgaagtt	783	802	SEQ ID NO: 4797	tattgaaatatgatttttt	12668	12687	1	3
SEQ ID NO: 3457	ctgaaaaagttagtgaaag	823	842	SEQ ID NO: 4798	agcttgtttgccagtgggc	4819	4838	1	3
SEQ ID NO: 3458	tttttcccagacagtgtca	885	884	SEQ ID NO: 4799	ggacaaggcccagaatctg	8339	9358	1	3
SEQ ID NO: 3459	ttttcccagacagtgtcca	902	921	SEQ ID NO: 4800	aaagaaacctatggcctta	13465	13484	1	3
SEQ ID NO: 3460	cattcagaaccagggaaga	903	922	SEQ ID NO: 4801	attcttaacgggattcctt	13464	13483	1	3
SEQ ID NO: 3461	acaaactgtttatagtccc	908	927	SEQ ID NO: 4802	taaagccattcaggtctct	4292	4311	1	3
SEQ ID NO: 3462	ggatttcaatacatttcac	909	928	SEQ ID NO: 4803	tattgaaatatgatttttt	1075	1094	1	3
SEQ ID NO: 3463	ttgtagaaatgaaagtaaa	933	952	SEQ ID NO: 4804	agcttgtttgccagtgggc	5794	6813	1	3
SEQ ID NO: 3464	ccaaaatttctctcggctt	934	953	SEQ ID NO: 4805	ggacaaggcccagaatctg	1246	1265	1	3
SEQ ID NO: 3465	caaattttctctgctaaaa	954	973	SEQ ID NO: 4806	ggacaaggcccagaatctg	4592	4611	1	3
SEQ ID NO: 3466	tctgctggaaacaacaacg	955	974	SEQ ID NO: 4807	attcttaacgggattcctt	4591	4610	1	3
SEQ ID NO: 3467	ttgtagaaatgaaagtaaa	956	975	SEQ ID NO: 4808	aattgttgaaagaaaaact	5099	5116	1	3
SEQ ID NO: 3468	ccaaaatttctctcggctt	978	997	SEQ ID NO: 4809	agcttgtttgccagtgggc	7987	8006	1	3
SEQ ID NO: 3469	caaattttctctgctaaaa	1008	1027	SEQ ID NO: 4810	ttaatctttcataagtcaa	8372	8391	1	3
SEQ ID NO: 3470	tctgctggaaacaacaacg	1012	1031	SEQ ID NO: 4811	taaagccattcaggtctct	11167	11186	1	3
SEQ ID NO: 3471	cagaagctaagcaatgtcc	1024	1043	SEQ ID NO: 4812	aattgttgaaagaaaaact	10337	10366	1	3
SEQ ID NO: 3472	taagattaaatatttactt	1045	1065	SEQ ID NO: 4813	taaagccattcaggtctct	10380	10399	1	3
SEQ ID NO: 3473	aaagattactttgagaaat	1050	1069	SEQ ID NO: 4814	ttaatctttcataagtcaa	4876	4695	1	3
SEQ ID NO: 3474	aggaaatagttggattacc	1087	1106	SEQ ID NO: 4815	ggacaaggcccagaatctg	5880	5899	1	3
SEQ ID NO: 3475	attttgatgatgctggccc	1113	1132	SEQ ID NO: 4816	attcttaacgggattcctt	7112	7131	1	3
SEQ ID NO: 3476	gaattatcttttaaacatt	1114	1133	SEQ ID NO: 4817	aattgttgaaagaaaaact	11052	11071	1	3
SEQ ID NO: 3477	ttaccaccagtttgaagtt	1130	1149	SEQ ID NO: 4818	agcttgtttgccagtgggc	12260	12279	1	3
SEQ ID NO: 3478	taagattaaatatttactt	1156	1175	SEQ ID NO: 4819	ttaatctttcataagtcaa	6326	6345	1	3
SEQ ID NO: 3479	aaagattactttgagaaat	1176	1196	SEQ ID NO: 4820	agcttgtttgccagtgggc	1387	1386	1	3
SEQ ID NO: 3480	aggaaatagttggattacc	1198	1217	SEQ ID NO: 4821	tgaaatcattgaaaattta	7112	7131	1	3
SEQ ID NO: 3481	attttgatgatgctggccc	1200	1219	SEQ ID NO: 4822	caggaggctttaagttgtc	12297	12316	1	3
SEQ ID NO: 3482	aggtttaaatggataattt	1212	1231	SEQ ID NO: 4823	agcttgtttgccagtgggc	12948	12965	1	3
SEQ ID NO: 3483	cagaagctaagcaatgtcc	1215	1234	SEQ ID NO: 4824	taaagccattcaggtctct	12279	12298	1	3
SEQ ID NO: 3484	taagattaaatatttactt	1216	1235	SEQ ID NO: 4825	cttcatcgttcatttgagt	8528	8547	1	3
SEQ ID NO: 3485	aaagattactttgagaaat	1231	1250	SEQ ID NO: 4826	cttatgggatttcctaagg	4733	4752	1	3
SEQ ID NO: 3486	aggaaatagttggattacc	1267	1286	SEQ ID NO: 4827	cttcatcgttcatttgagt	5001	5020	1	3
SEQ ID NO: 3487	attttgatgatgctggccc	1296	1315	SEQ ID NO: 4828	ttccatcacaaatcctttg	7239	7258	1	3
SEQ ID NO: 3488	gaattatcttttaaacatt	1385	1404	SEQ ID NO: 4829	tctcaaagagttacaacag	12323	12342	1	3
SEQ ID NO: 3489	ttaccaccagtttgaagtt	1388	1407	SEQ ID NO: 4830	tattgaaatatgatttttt	13051	13070	1	3
SEQ ID NO: 3490	ttgcagtgtatcggacaga	1415	1434	SEQ ID NO: 4831	aattgttgaaagaaaaact	5718	5737	1	3
SEQ ID NO: 3491	cattcagaaccagggaaga	1478	1497	SEQ ID NO: 4832	ggacaaggcccagaatctg	3936	3955	1	3
SEQ ID NO: 3492	acaaactgtttatagtccc	1499	1518	SEQ ID NO: 4833	aaagaaacctatggcctta	4660	4679	1	3
SEQ ID NO: 3493	ggatttcaatacatttcac	1500	1519	SEQ ID NO: 4834	attcttaacgggattcctt	7664	7883	1	3
SEQ ID NO: 3494	ttgtagaaatgaaagtaaa	1505	1524	SEQ ID NO: 4835	taaagccattcaggtctct	12287	12306	1	3
SEQ ID NO: 3495	ctgaacagtgagctgcagt	1565	1584	SEQ ID NO: 4836	gacatgtttgaagggtgga	6353	6372	1	3
SEQ ID NO: 3496	aatccaatctcctctttgc	1575	1594	SEQ ID NO: 4837	tgggctaaacgtttaattg	7161	7180	1	3
SEQ ID NO: 3497	atttgattttcaagcaaag	1582	1801	SEQ ID NO: 4838	tattgaaatatgatttttt	6436	8455	1	3
SEQ ID NO: 3498	ttttgattttcaagcaaat	1609	1826	SEQ ID NO: 4839	agcttgtttgccagtgggc	9962	9981	1	3
SEQ ID NO: 3499	tgatttcaagcaaatgcag	1615	1834	SEQ ID NO: 4840	aaagaaacctatggcctta	8400	8419	1	3
SEQ ID NO: 3500	atgctgtttgggaaattgg	1625	1844	SEQ ID NO: 4841	attcttaacgggattcctt	11654	11673	1	3
SEQ ID NO: 3501	tgctgttttttggaaatgc	1627	1845	SEQ ID NO: 4842	taaagccattcaggtctct	1920	1939	1	3
SEQ ID NO: 3502	aaaaaaatacactggacgt	1663	1682	SEQ ID NO: 4843	gacatgtttgaagggtgga	4429	4448	1	3
SEQ ID NO: 3503	actggagcttagtaagggc	1672	1691	SEQ ID NO: 4844	tgggctaaacgtttaattg	10883	10902	1	3
SEQ ID NO: 3504	cttctggaaagagggtcat	1673	1692	SEQ ID NO: 4845	tattgaaatatgatttttt	13999	14016	1	3
SEQ ID NO: 3505	ggaaaagggtcatggaaat	1685	1704	SEQ ID NO: 4846	agcttgtttgccagtgggc	11173	11192	1	3
SEQ ID NO: 3506	gggcctgccccagatttct	1688	1707	SEQ ID NO: 4847	ggacaaggcccagaatctg	5518	5537	1	3
SEQ ID NO: 3507	ttctcagataggggaacac	1731	1750	SEQ ID NO: 4848	aaagaaacctatggcctta	4542	4561	1	3
SEQ ID NO: 3508	gatgaaagaattaagggga	1734	1753	SEQ ID NO: 4849	attcttaacgggattcctt	7242	7261	1	3
SEQ ID NO: 3509	cttggactgtcaaataagt	1737	1756	SEQ ID NO: 4850	taaagccattcaggtctct	4290	4309	1	3
SEQ ID NO: 3510	ggaaaagggtcatggaaat	1750	1789	SEQ ID NO: 4851	taaagccattcaggtctct	2567	2566	1	3
SEQ ID NO: 3511	aggaaatagttggattacc	1752	1771	SEQ ID NO: 4852	gacatgtttgaagggtgga	11307	11326	1	3
SEQ ID NO: 3512	attttgatgatgctggccc	1810	1829	SEQ ID NO: 4853	tgggctaaacgtttaattg	9046	9065	1	3
SEQ ID NO: 3513	gaattatcttttaaacatt	1824	1843	SEQ ID NO: 4854	tattgaaatatgatttttt	7148	7167	1	3
SEQ ID NO: 3514	ttaccaccagtttgaagtt	1825	1844	SEQ ID NO: 4855	agcttgtttgccagtgggc	7147	7155	1	3
SEQ ID NO: 3515	ctgaaaaagttagtgaaag	1831	1850	SEQ ID NO: 4856	ggacaaggcccagaatctg	6589	6608	1	3
SEQ ID NO: 3516	tttttcccagacagtgtca	1836	1855	SEQ ID NO: 4857	aaagaaacctatggcctta	2760	2779	1	3
SEQ ID NO: 3517	ttttcccagacagtgtcca	1842	1861	SEQ ID NO: 4858	attcttaacgggattcctt	4542	4561	1	3
SEQ ID NO: 3518	cattcagaaccagggaaga	1877	1896	SEQ ID NO: 4859	aattgttgaaagaaaaact	5334	5353	1	3
SEQ ID NO: 3519	ttgcagtgtatcggacaga	1883	1902	SEQ ID NO: 4860	ggacaaggcccagaatctg	12677	12698	1	3
SEQ ID NO: 3520	cattcagaaccagggaaga	1887	1900	SEQ ID NO: 4861	aaagaaacctatggcctta	8909	5928	1	3
SEQ ID NO: 3521	acaaactgtttatagtccc	1892	1911	SEQ ID NO: 4862	attcttaacgggattcctt	8998	9017	1	3
SEQ ID NO: 3522	ggatttcaatacatttcac	1896	1915	SEQ ID NO: 4863	taaagccattcaggtctct	7631	7850	1	3
SEQ ID NO: 3523	ttgtagaaatgaaagtaaa	1900	1919	SEQ ID NO: 4864	gacatgtttgaagggtgga	12745	12764	1	3
SEQ ID NO: 3524	ctgaacagtgagctgcagt	1901	1920	SEQ ID NO: 4865	tgggctaaacgtttaattg	12276	12296	1	3
SEQ ID NO: 3525	aatccaatctcctctttgc	1908	1927	SEQ ID NO: 4866	tattgaaatatgatttttt	9352	9371	1	3
SEQ ID NO: 3526	atttgattttcaagcaaag	1934	1953	SEQ ID NO: 4867	agcttgtttgccagtgggc	9400	9419	1	3
SEQ ID NO: 3527	ttttgattttcaagcaaat	1941	1960	SEQ ID NO: 4868	tgaaatcattgaaaattta	1986	2005	1	3
SEQ ID NO: 3528	tgatttcaagcaaatgcag	1949	1968	SEQ ID NO: 4869	caggaggctttaagttgtc	7915	7934	1	3
SEQ ID NO: 3529	atgctgtttgggaaattgg	1955	1975	SEQ ID NO: 4870	agcttgtttgccagtgggc	1978	1997	1	3
SEQ ID NO: 3530	tgctgttttttggaaatgc	1980	1999	SEQ ID NO: 4871	taaagccattcaggtctct	12350	12369	1	3
SEQ ID NO: 3531	aaaaaaatacactggacgt	1997	2016	SEQ ID NO: 4872	aattgttgaaagaaaaact	7036	7055	1	3
SEQ ID NO: 3532	ctgaaaaagttagtgaaag	2029	2048	SEQ ID NO: 4873	taaagccattcaggtctct	6976	6997	1	3
SEQ ID NO: 3533	tttttcccagacagtgtca	2031	2050	SEQ ID NO: 4874	ttaatctttcataagtcaa	10284	10303	1	3
SEQ ID NO: 3534	ttttcccagacagtgtcca	2079	2098	SEQ ID NO: 4875	ggacaaggcccagaatctg	5457	5476	1	3
SEQ ID NO: 3535	cattcagaaccagggaaga	2086	2102	SEQ ID NO: 4876	attcttaacgggattcctt	4900	4919	1	3
SEQ ID NO: 3536	acaaactgtttatagtccc	2084	2103	SEQ ID NO: 4877	aattgttgaaagaaaaact	5287	5305	1	3
SEQ ID NO: 3537	ggatttcaatacatttcac	2101	2120	SEQ ID NO: 4878	agcttgtttgccagtgggc	3461	3480	1	3
SEQ ID NO: 3538	ttgtagaaatgaaagtaaa	2158	2177	SEQ ID NO: 4879	ttaatctttcataagtcaa	10932	10951	1	3
SEQ ID NO: 3539	ccaaaatttctctcggctt	2162	2181	SEQ ID NO: 4880	taaagccattcaggtctct	12265	12284	1	3
SEQ ID NO: 3540	caaattttctctgctaaaa	2191	2210	SEQ ID NO: 4881	aattgttgaaagaaaaact	7850	7869	1	3
SEQ ID NO: 3541	tctgctggaaacaacaacg	2193	2212	SEQ ID NO: 4882	taaagccattcaggtctct	4086	4105	1	3
SEQ ID NO: 3542	cagaagctaagcaatgtcc	2204	2223	SEQ ID NO: 4883	attcttaacgggattcctt	12956	12977	1	3
SEQ ID NO: 3543	taagattaaatatttactt	2209	2228	SEQ ID NO: 4884	taaagccattcaggtctct	13521	13540	1	3
SEQ ID NO: 3544	aaagattactttgagaaat	2210	2229	SEQ ID NO: 4885	gacatgtttgaagggtgga	13520	13530	1	3
SEQ ID NO: 3545	aggaaatagttggattacc	2215	2234	SEQ ID NO: 4886	tgggctaaacgtttaattg	4539	4558	1	3
SEQ ID NO: 3546	attttgatgatgctggccc	2220	2239	SEQ ID NO: 4887	tattgaaatatgatttttt	3785	3804	1	3
SEQ ID NO: 3547	gaattatcttttaaacatt	2226	2245	SEQ ID NO: 4888	agcttgtttgccagtgggc	7796	7817	1	3
SEQ ID NO: 3548	ttaccaccagtttgaagtt	2229	2248	SEQ ID NO: 4889	tgaaatcattgaaaattta	6730	5749	1	3
SEQ ID NO: 3549	taagattaaatatttactt	2247	2266	SEQ ID NO: 4890	caggaggctttaagttgtc	6723	6742	1	3
SEQ ID NO: 3550	aaagattactttgagaaat	2331	2350	SEQ ID NO: 4891	agcttgtttgccagtgggc	2576	2696	1	3
SEQ ID NO: 3551	aggaaatagttggattacc	2337	2366	SEQ ID NO: 4892	taaagccattcaggtctct	6280	6299	1	3
SEQ ID NO: 3552	attttgatgatgctggccc	2357	2376	SEQ ID NO: 4893	aattgttgaaagaaaaact	4262	4281	1	3
SEQ ID NO: 3553	aggtttaaatggataattt	2365	2385	SEQ ID NO: 4894	taaagccattcaggtctct	9954	9973	1	3
SEQ ID NO: 3554	cagaagctaagcaatgtcc	2367	2398	SEQ ID NO: 4895	ttaatctttcataagtcaa	10809	10525	1	3
SEQ ID NO: 3555	taagattaaatatttactt	2370	2389	SEQ ID NO: 4896	ggacaaggcccagaatctg	13067	13086	1	3
SEQ ID NO: 3556	aaagattactttgagaaat	2374	2393	SEQ ID NO: 4897	attcttaacgggattcctt	7981	8000	1	3
SEQ ID NO: 3557	aggaaatagttggattacc	2385	2404	SEQ ID NO: 4898	aattgttgaaagaaaaact	9542	9501	1	3
SEQ ID NO: 3558	attttgatgatgctggccc	2386	2405	SEQ ID NO: 4899	agcttgtttgccagtgggc	9541	9550	1	3
SEQ ID NO: 3559	gaattatcttttaaacatt	2387	2406	SEQ ID NO: 4900	ttaatctttcataagtcaa	9540	9560	1	3
SEQ ID NO: 3560	ttaccaccagtttgaagtt	2401	2420	SEQ ID NO: 4901	taaagccattcaggtctct	6874	6893	1	3
SEQ ID NO: 3561	ttgcagtgtatcggacaga	1408	2427	SEQ ID NO: 4902	aattgttgaaagaaaaact	8248	8267	1	3
SEQ ID NO: 3562	cattcagaaccagggaaga	1409	2428	SEQ ID NO: 4903	taaagccattcaggtctct	8247	8266	1	3
SEQ ID NO: 3563	acaaactgtttatagtccc	2416	2435	SEQ ID NO: 4904	agcttgtttgccagtgggc	3598	3707	1	3
SEQ ID NO: 3564	ggatttcaatacatttcac	2417	2436	SEQ ID NO: 4905	ggacaaggcccagaatctg	3887	3706	1	3
SEQ ID NO: 3565	ttgtagaaatgaaagtaaa	2438	2457	SEQ ID NO: 4906	aaagaaacctatggcctta	8571	8590	1	3
SEQ ID NO: 3566	ctgaacagtgagctgcagt	2439	2458	SEQ ID NO: 4907	attcttaacgggattcctt	8570	8559	1	3
SEQ ID NO: 3567	aatccaatctcctctttgc	2465	2474	SEQ ID NO: 4908	taaagccattcaggtctct	12527	12545	1	3
SEQ ID NO: 3568	atttgattttcaagcaaag	2464	2483	SEQ ID NO: 4909	tattgaaatatgatttttt	2734	2763	1	3
SEQ ID NO: 3569	ttttgattttcaagcaaat	2469	2488	SEQ ID NO: 4910	agcttgtttgccagtgggc	10771	10790	1	3
SEQ ID NO: 3570	tgatttcaagcaaatgcag	2479	2468	SEQ ID NO: 4911	ggacaaggcccagaatctg	3538	3887	1	3
SEQ ID NO: 3571	atgctgtttgggaaattgg	3483	2502	SEQ ID NO: 4912	ggacaaggcccagaatctg	2672	2691	1	3
SEQ ID NO: 3572	ttgtagaaatgaaagtaaa	2601	2520	SEQ ID NO: 4913	attcttaacgggattcctt	7939	7958	1	3
SEQ ID NO: 3573	ctgaacagtgagctgcagt	2551	2580	SEQ ID NO: 4914	aattgttgaaagaaaaact	3514	3533	1	3
SEQ ID NO: 3574	aatccaatctcctctttgc	2582	2601	SEQ ID NO: 4915	agcttgtttgccagtgggc	3836	3854	1	3
SEQ ID NO: 3575	atttgattttcaagcaaag	2590	2509	SEQ ID NO: 4916	ttaatctttcataagtcaa	6107	5128	1	3
SEQ ID NO: 3576	ttttgattttcaagcaaat	2605	2624	SEQ ID NO: 4917	taaagccattcaggtctct	10718	10737	1	3
SEQ ID NO: 3577	tgatttcaagcaaatgcag	2606	2627	SEQ ID NO: 4918	aattgttgaaagaaaaact	12281	12300	1	3
SEQ ID NO: 3578	atgctgtttgggaaattgg	2618	2637	SEQ ID NO: 4919	taaagccattcaggtctct	5737	5759	1	3
SEQ ID NO: 3579	cagaagctaagcaatgtcc	2621	2640	SEQ ID NO: 4920	ttaatctttcataagtcaa	5383	5402	1	3
SEQ ID NO: 3580	taagattaaatatttactt	2692	2711	SEQ ID NO: 4921	ggacaaggcccagaatctg	5080	6099	1	3
SEQ ID NO: 3581	cttggactgtcaaataagt	2698	2717	SEQ ID NO: 4922	attcttaacgggattcctt	3059	076	1	3
SEQ ID NO: 3582	ggaaaagggtcatggaaat	2710	2729	SEQ ID NO: 4923	aattgttgaaagaaaaact	4106	4125	1	3
SEQ ID NO: 3583	aggaaatagttggattacc	2765	2734	SEQ ID NO: 4924	agcttgtttgccagtgggc	11236	11255	1	3
SEQ ID NO: 3584	attttgatgatgctggccc	2767	2785	SEQ ID NO: 4925	ttaatctttcataagtcaa	4385	4404	1	3
SEQ ID NO: 3585	gaattatcttttaaacatt	2794	2813	SEQ ID NO: 4926	agcttgtttgccagtgggc	12341	12380	1	3
SEQ ID NO: 3586	ttaccaccagtttgaagtt	2808	2827	SEQ ID NO: 4927	tgaaatcattgaaaattta	4179	4198	1	3
SEQ ID NO: 3587	ctgaaaaagttagtgaaag	2834	2853	SEQ ID NO: 4928	caggaggctttaagttgtc	12562	12581	1	3
SEQ ID NO: 3588	tttttcccagacagtgtca	2841	2850	SEQ ID NO: 4929	agcttgtttgccagtgggc	4807	4828	1	3
SEQ ID NO: 3589	ttttcccagacagtgtcca	2843	2882	SEQ ID NO: 4930	taaagccattcaggtctct	7639	7665	1	3
SEQ ID NO: 3590	cattcagaaccagggaaga	2869	2888	SEQ ID NO: 4931	cttcatcgttcatttgagt	7951	7970	1	3
SEQ ID NO: 3591	ttgcagtgtatcggacaga	2879	2898	SEQ ID NO: 4932	cttatgggatttcctaagg	6269	6288	1	3
SEQ ID NO: 3592	cattcagaaccagggaaga	2908	2927	SEQ ID NO: 4933	cttcatcgttcatttgagt	13214	13233	1	3
SEQ ID NO: 3593	acaaactgtttatagtccc	2934	2953	SEQ ID NO: 4934	ttccatcacaaatcctttg	10191	10210	1	3
SEQ ID NO: 3594	ggatttcaatacatttcac	2939	2958	SEQ ID NO: 4935	tctcaaagagttacaacag	11889	11908	1	3
SEQ ID NO: 3595	ttgtagaaatgaaagtaaa	2940	2959	SEQ ID NO: 4936	tattgaaatatgatttttt	7106	7125	1	3
SEQ ID NO: 3596	ctgaacagtgagctgcagt	2954	2983	SEQ ID NO: 4937	aattgttgaaagaaaaact	3205	3224	1	3
SEQ ID NO: 3597	aatccaatctcctctttgc	2987	3006	SEQ ID NO: 4938	ggacaaggcccagaatctg	12031	12050	1	3
SEQ ID NO: 3598	atttgattttcaagcaaag	2989	3006	SEQ ID NO: 4939	aaagaaacctatggcctta	8341	8360	1	3
SEQ ID NO: 3599	ttttgattttcaagcaaat	3043	3062	SEQ ID NO: 4940	attcttaacgggattcctt	8440	6459	1	3
SEQ ID NO: 3600	tgatttcaagcaaatgcag	3074	3093	SEQ ID NO: 4941	taaagccattcaggtctct	3192	3211	1	3
SEQ ID NO: 3601	atgctgtttgggaaattgg	3101	3120	SEQ ID NO: 4942	gacatgtttgaagggtgga	12259	12278	1	3
SEQ ID NO: 3602	tgctgttttttggaaatgc	3120	3139	SEQ ID NO: 4943	tgggctaaacgtttaattg	5144	5163	1	3
SEQ ID NO: 3603	aaaaaaatacactggacgt	3175	3194	SEQ ID NO: 4944	tattgaaatatgatttttt	6549	6668	1	3
SEQ ID NO: 3604	ctgaaaaagttagtgaaag	3202	3221	SEQ ID NO: 4945	agcttgtttgccagtgggc	13217	13236	1	3
SEQ ID NO: 3605	tttttcccagacagtgtca	3222	3241	SEQ ID NO: 4946	aaagaaacctatggcctta	9630	9549	1	3
SEQ ID NO: 3606	ttttcccagacagtgtcca	3225	3244	SEQ ID NO: 4947	attcttaacgggattcctt	5955	6974	1	3
SEQ ID NO: 3607	cattcagaaccagggaaga	3233	3252	SEQ ID NO: 4948	taaagccattcaggtctct	4091	4110	1	3
SEQ ID NO: 3608	acaaactgtttatagtccc	3234	3253	SEQ ID NO: 4949	gacatgtttgaagggtgga	4090	4109	1	3
SEQ ID NO: 3609	ggatttcaatacatttcac	3288	3307	SEQ ID NO: 4950	tgggctaaacgtttaattg	10851	10670	1	3
SEQ ID NO: 3610	ttgtagaaatgaaagtaaa	3289	3308	SEQ ID NO: 4951	tattgaaatatgatttttt	10850	10869	1	3
SEQ ID NO: 3611	ccaaaatttctctcggctt	3305	3324	SEQ ID NO: 4952	agcttgtttgccagtgggc	10052	10071	1	3
SEQ ID NO: 3612	caaattttctctgctaaaa	3371	3390	SEQ ID NO: 4953	ggacaaggcccagaatctg	4215	4234	1	3
SEQ ID NO: 3613	tctgctggaaacaacaacg	3372	3391	SEQ ID NO: 4954	aaagaaacctatggcctta	4062	4081	1	3
SEQ ID NO: 3614	cagaagctaagcaatgtcc	3390	3409	SEQ ID NO: 4955	attcttaacgggattcctt	9076	9095	1	3
SEQ ID NO: 3615	taagattaaatatttactt	3396	3417	SEQ ID NO: 4956	taaagccattcaggtctct	9226	9245	1	3
SEQ ID NO: 3616	aaagattactttgagaaat	3435	3454	SEQ ID NO: 4957	taaagccattcaggtctct	5835	9654	1	3
SEQ ID NO: 3617	aggaaatagttggattacc	3438	3457	SEQ ID NO: 4958	gacatgtttgaagggtgga	6316	6335	1	3
SEQ ID NO: 3618	attttgatgatgctggccc	3449	3468	SEQ ID NO: 4959	tgggctaaacgtttaattg	13838	13857	1	3
SEQ ID NO: 3619	gaattatcttttaaacatt	3454	3473	SEQ ID NO: 4960	tattgaaatatgatttttt	10220	10239	1	3
SEQ ID NO: 3620	ttaccaccagtttgaagtt	3455	3474	SEQ ID NO: 4961	agcttgtttgccagtgggc	10219	10238	1	3
SEQ ID NO: 3621	taagattaaatatttactt	3463	3482	SEQ ID NO: 4962	ggacaaggcccagaatctg	11348	11967	1	3
SEQ ID NO: 3622	aaagattactttgagaaat	3473	3492	SEQ ID NO: 4963	aaagaaacctatggcctta	7084	7103	1	3
SEQ ID NO: 3623	aggaaatagttggattacc	3491	3510	SEQ ID NO: 4964	attcttaacgggattcctt	8858	5877	1	3
SEQ ID NO: 3624	attttgatgatgctggccc	3504	3523	SEQ ID NO: 4965	aattgttgaaagaaaaact	5185	5204	1	3
SEQ ID NO: 3625	aggtttaaatggataattt	3510	3529	SEQ ID NO: 4966	ggacaaggcccagaatctg	5633	5652	1	3
SEQ ID NO: 3626	cagaagctaagcaatgtcc	3523	3542	SEQ ID NO: 4967	aaagaaacctatggcctta	8341	8360	1	3
SEQ ID NO: 3627	taagattaaatatttactt	3540	3559	SEQ ID NO: 4968	attcttaacgggattcctt	13947	13968	1	3
SEQ ID NO: 3628	aaagattactttgagaaat	3555	3574	SEQ ID NO: 4969	taaagccattcaggtctct	9701	9720	1	3
SEQ ID NO: 3629	aggaaatagttggattacc	3507	3588	SEQ ID NO: 4970	gacatgtttgaagggtgga	8167	8188	1	3
SEQ ID NO: 3630	attttgatgatgctggccc	3578	3597	SEQ ID NO: 4971	tgggctaaacgtttaattg	8211	8230	1	3
SEQ ID NO: 3631	gaattatcttttaaacatt	3610	3629	SEQ ID NO: 4972	tattgaaatatgatttttt	8947	8999	1	3
SEQ ID NO: 3632	ttaccaccagtttgaagtt	3631	3650	SEQ ID NO: 4973	agcttgtttgccagtgggc	7713	7732	1	3
SEQ ID NO: 3633	ttgcagtgtatcggacaga	3657	3676	SEQ ID NO: 4974	tgaaatcattgaaaattta	3736	3755	1	3
SEQ ID NO: 3634	cattcagaaccagggaaga	3685	3704	SEQ ID NO: 4975	caggaggctttaagttgtc	5195	5214	1	3
SEQ ID NO: 3635	acaaactgtttatagtccc	3764	3783	SEQ ID NO: 4976	agcttgtttgccagtgggc	12777	12796	1	3
SEQ ID NO: 3636	ggatttcaatacatttcac	3770	3789	SEQ ID NO: 4977	taaagccattcaggtctct	7022	7041	1	3
SEQ ID NO: 3637	ttgtagaaatgaaagtaaa	3809	3828	SEQ ID NO: 4978	aattgttgaaagaaaaact	6693	6712	1	3
SEQ ID NO: 3638	ctgaacagtgagctgcagt	3829	3848	SEQ ID NO: 4979	taaagccattcaggtctct	12957	12976	1	3
SEQ ID NO: 3639	aatccaatctcctctttgc	3895	3914	SEQ ID NO: 4980	ttaatctttcataagtcaa	6314	6333	1	3
SEQ ID NO: 3640	atttgattttcaagcaaag	3940	3959	SEQ ID NO: 4981	ggacaaggcccagaatctg	12179	12192	1	3
SEQ ID NO: 3641	taagattaaatatttactt	3942	3961	SEQ ID NO: 4982	attcttaacgggattcctt	9213	6232	1	3
SEQ ID NO: 3642	aaagattactttgagaaat	3555	3974	SEQ ID NO: 4983	aattgttgaaagaaaaact	6343	6263	1	3
SEQ ID NO: 3643	aggaaatagttggattacc	3971	3990	SEQ ID NO: 4984	agcttgtttgccagtgggc	7363	7382	1	3
SEQ ID NO: 3644	attttgatgatgctggccc	3984	4003	SEQ ID NO: 4985	ttaatctttcataagtcaa	8310	8329	1	3
SEQ ID NO: 3645	gaattatcttttaaacatt	3995	4014	SEQ ID NO: 4986	taaagccattcaggtctct	6047	9066	1	3
SEQ ID NO: 3646	ttaccaccagtttgaagtt	3996	4015	SEQ ID NO: 4987	aattgttgaaagaaaaact	10454	10473	1	3
SEQ ID NO: 3647	ttgcagtgtatcggacaga	3958	4017	SEQ ID NO: 4988	taaagccattcaggtctct	12030	12049	1	3
SEQ ID NO: 3648	cattcagaaccagggaaga	4046	4065	SEQ ID NO: 4989	attcttaacgggattcctt	10545	10564	1	3
SEQ ID NO: 3649	acaaactgtttatagtccc	4052	4071	SEQ ID NO: 4990	taaagccattcaggtctct	8073	8092	1	3
SEQ ID NO: 3650	ggatttcaatacatttcac	4074	4093	SEQ ID NO: 4991	gacatgtttgaagggtgga	8813	8832	1	3
SEQ ID NO: 3651	ttgtagaaatgaaagtaaa	4075	4094	SEQ ID NO: 4992	tgggctaaacgtttaattg	5728	6747	1	3
SEQ ID NO: 3652	ctgaacagtgagctgcagt	4102	4121	SEQ ID NO: 4993	tattgaaatatgatttttt	5330	5349	1	3
SEQ ID NO: 3653	aatccaatctcctctttgc	4142	4161	SEQ ID NO: 4994	agcttgtttgccagtgggc	5254	6273	1	3
SEQ ID NO: 3654	atttgattttcaagcaaag	4156	4175	SEQ ID NO: 4995	tgaaatcattgaaaattta	8843	8862	1	3
SEQ ID NO: 3655	ttttgattttcaagcaaat	4176	1497	SEQ ID NO: 4996	caggaggctttaagttgtc	4735	4754	1	3
SEQ ID NO: 3656	tgatttcaagcaaatgcag	4192	4211	SEQ ID NO: 4997	agcttgtttgccagtgggc	5588	8807	1	3
SEQ ID NO: 3657	atgctgtttgggaaattgg	4195	4214	SEQ ID NO: 4998	taaagccattcaggtctct	11671	11690	1	3
SEQ ID NO: 3658	ttgtagaaatgaaagtaaa	4196	4215	SEQ ID NO: 4999	aattgttgaaagaaaaact	11670	11880	1	3
SEQ ID NO: 3659	ctgaacagtgagctgcagt	4232	4251	SEQ ID NO: 5000	ttaatctttcataagtcaa	13359	13378	1	3
SEQ ID NO: 3660	ggatttcaatacatttcac	4280	4299	SEQ ID NO: 5001	taaagccattcaggtctct	8611	8630	1	3
SEQ ID NO: 3661	ttgtagaaatgaaagtaaa	4291	4310	SEQ ID NO: 5002	aattgttgaaagaaaaact	13189	13188	1	3
SEQ ID NO: 3662	ctgaacagtgagctgcagt	4303	4322	SEQ ID NO: 5003	taaagccattcaggtctct	6966	6984	1	3
SEQ ID NO: 3663	ctgaaaaagttagtgaaag	4312	4331	SEQ ID NO: 5004	agcttgtttgccagtgggc	11066	11107	1	3
SEQ ID NO: 3664	tttttcccagacagtgtca	4313	4332	SEQ ID NO: 5005	taaagccattcaggtctct	11087	11106	1	3
SEQ ID NO: 3665	ttttcccagacagtgtcca	4319	4338	SEQ ID NO: 5006	aattgttgaaagaaaaact	8577	8596	1	3
SEQ ID NO: 3666	cattcagaaccagggaaga	4352	4371	SEQ ID NO: 5007	taaagccattcaggtctct	9984	10003	1	3
SEQ ID NO: 3667	acaaactgtttatagtccc	4353	4372	SEQ ID NO: 5008	ttaatctttcataagtcaa	9933	10002	1	3
SEQ ID NO: 3668	ggatttcaatacatttcac	4363	4382	SEQ ID NO: 5009	ggacaaggcccagaatctg	6487	8506	1	3
SEQ ID NO: 3669	ttgtagaaatgaaagtaaa	4406	4425	SEQ ID NO: 5010	attcttaacgggattcctt	6685	6704	1	3
SEQ ID NO: 3670	ccaaaatttctctcggctt	4412	4431	SEQ ID NO: 5011	aattgttgaaagaaaaact	7102	7121	1	3
SEQ ID NO: 3671	caaattttctctgctaaaa	4449	4468	SEQ ID NO: 5012	agcttgtttgccagtgggc	9214	9233	1	3
SEQ ID NO: 3672	tctgctggaaacaacaacg	4456	4475	SEQ ID NO: 5013	ttaatctttcataagtcaa	11302	11321	1	3
SEQ ID NO: 3673	ttgtagaaatgaaagtaaa	4462	4481	SEQ ID NO: 5014	taaagccattcaggtctct	12231	12250	1	3
SEQ ID NO: 3674	ccaaaatttctctcggctt	4463	4482	SEQ ID NO: 5015	aattgttgaaagaaaaact	12230	12249	1	3
SEQ ID NO: 3675	caaattttctctgctaaaa	4465	4484	SEQ ID NO: 5016	taaagccattcaggtctct	9280	9299	1	3
SEQ ID NO: 3676	tctgctggaaacaacaacg	4543	4562	SEQ ID NO: 5017	attcttaacgggattcctt	11214	11233	1	3
SEQ ID NO: 3677	cagaagctaagcaatgtcc	4551	4570	SEQ ID NO: 5018	taaagccattcaggtctct	8022	8041	1	3
SEQ ID NO: 3678	taagattaaatatttactt	4559	4588	SEQ ID NO: 5019	gacatgtttgaagggtgga	13422	13441	1	3
SEQ ID NO: 3679	aaagattactttgagaaat	4588	4605	SEQ ID NO: 5020	tgggctaaacgtttaattg	11995	12014	1	3
SEQ ID NO: 3680	aggaaatagttggattacc	4588	4607	SEQ ID NO: 5021	tattgaaatatgatttttt	8873	8882	1	3
SEQ ID NO: 3681	attttgatgatgctggccc	4505	4624	SEQ ID NO: 5022	agcttgtttgccagtgggc	9245	9264	1	3
SEQ ID NO: 3682	gaattatcttttaaacatt	4614	4633	SEQ ID NO: 5023	tgaaatcattgaaaattta	9372	9391	1	3
SEQ ID NO: 3683	ttaccaccagtttgaagtt	4667	4686	SEQ ID NO: 5024	caggaggctttaagttgtc	6190	6209	1	3
SEQ ID NO: 3684	taagattaaatatttactt	4668	4587	SEQ ID NO: 5025	agcttgtttgccagtgggc	9308	9327	1	3
SEQ ID NO: 3685	aaagattactttgagaaat	4892	4711	SEQ ID NO: 5026	taaagccattcaggtctct	9376	9385	1	3
SEQ ID NO: 3686	aggaaatagttggattacc	4730	4749	SEQ ID NO: 5027	aattgttgaaagaaaaact	12291	12310	1	3
SEQ ID NO: 3687	attttgatgatgctggccc	4782	4781	SEQ ID NO: 5028	taaagccattcaggtctct	12263	12282	1	3
SEQ ID NO: 3688	aggtttaaatggataattt	4783	4782	SEQ ID NO: 5029	ttaatctttcataagtcaa	12262	12281	1	3
SEQ ID NO: 3689	cagaagctaagcaatgtcc	4793	4812	SEQ ID NO: 5030	ggacaaggcccagaatctg	5977	5996	1	3
SEQ ID NO: 3690	taagattaaatatttactt	4804	4823	SEQ ID NO: 5031	attcttaacgggattcctt	8620	6939	1	3
SEQ ID NO: 3691	aaagattactttgagaaat	4858	4887	SEQ ID NO: 5032	aattgttgaaagaaaaact	13032	13051	1	3
SEQ ID NO: 3692	aggaaatagttggattacc	4907	4926	SEQ ID NO: 5033	agcttgtttgccagtgggc	6901	6920	1	3
SEQ ID NO: 3693	attttgatgatgctggccc	4940	4959	SEQ ID NO: 5034	ttaatctttcataagtcaa	5348	5367	1	3
SEQ ID NO: 3694	gaattatcttttaaacatt	4963	4982	SEQ ID NO: 5035	taaagccattcaggtctct	12549	12568	1	3
SEQ ID NO: 3695	ttaccaccagtttgaagtt	4981	5000	SEQ ID NO: 5036	aattgttgaaagaaaaact	10103	10122	1	3
SEQ ID NO: 3696	ttgcagtgtatcggacaga	5007	5026	SEQ ID NO: 5037	taaagccattcaggtctct	14019	14038	1	3
SEQ ID NO: 3697	cattcagaaccagggaaga	5040	5059	SEQ ID NO: 5038	agcttgtttgccagtgggc	6794	6813	1	3
SEQ ID NO: 3698	acaaactgtttatagtccc	5083	5102	SEQ ID NO: 5039	ggacaaggcccagaatctg	5185	5204	1	3
SEQ ID NO: 3699	ggatttcaatacatttcac	5092	5111	SEQ ID NO: 5040	aaagaaacctatggcctta	2239	6258	1	3
SEQ ID NO: 3700	ttgtagaaatgaaagtaaa	5115	5135	SEQ ID NO: 5041	attcttaacgggattcctt	9222	9241	1	3
SEQ ID NO: 3701	ctgaacagtgagctgcagt	5135	5154	SEQ ID NO: 5042	taaagccattcaggtctct	6899	6918	1	3
SEQ ID NO: 3702	aatccaatctcctctttgc	5149	5167	SEQ ID NO: 5043	tattgaaatatgatttttt	6499	6518	1	3
SEQ ID NO: 3703	atttgattttcaagcaaag	5150	5189	SEQ ID NO: 5044	agcttgtttgccagtgggc	6436	6455	1	3
SEQ ID NO: 3704	ttttgattttcaagcaaat	5193	5212	SEQ ID NO: 5045	ggacaaggcccagaatctg	7506	7525	1	3
SEQ ID NO: 3705	tgatttcaagcaaatgcag	5231	5250	SEQ ID NO: 5046	ggacaaggcccagaatctg	8149	8168	1	3
SEQ ID NO: 3706	atgctgtttgggaaattgg	5247	5266	SEQ ID NO: 5047	attcttaacgggattcctt	11358	11375	1	3
SEQ ID NO: 3707	tgctgttttttggaaatgc	5303	5322	SEQ ID NO: 5048	aattgttgaaagaaaaact	11977	11966	1	3
SEQ ID NO: 3708	aaaaaaatacactggacgt	5336	5355	SEQ ID NO: 5049	agcttgtttgccagtgggc	6486	6605	1	3
SEQ ID NO: 3709	actggagcttagtaagggc	5349	5366	SEQ ID NO: 5050	ttaatctttcataagtcaa	5746	5765	1	3
SEQ ID NO: 3710	cttctggaaagagggtcat	5353	5372	SEQ ID NO: 5051	taaagccattcaggtctct	6702	6721	1	3
SEQ ID NO: 3711	ggaaaagggtcatggaaat	5386	5405	SEQ ID NO: 5052	aattgttgaaagaaaaact	10996	11015	1	3
SEQ ID NO: 3712	gggcctgccccagatttct	5389	5408	SEQ ID NO: 5053	taaagccattcaggtctct	12490	12509	1	3
SEQ ID NO: 3713	ttctcagataggggaacac	5395	5414	SEQ ID NO: 5054	ttaatctttcataagtcaa	11852	11871	1	3
SEQ ID NO: 3714	gatgaaagaattaagggga	5420	5439	SEQ ID NO: 5055	ggacaaggcccagaatctg	7370	7389	1	3
SEQ ID NO: 3715	cttggactgtcaaataagt	5435	5484	SEQ ID NO: 5056	attcttaacgggattcctt	8022	8041	1	3
SEQ ID NO: 3716	ggaaaagggtcatggaaat	5443	5482	SEQ ID NO: 5057	aattgttgaaagaaaaact	10674	10693	1	3
SEQ ID NO: 3717	aggaaatagttggattacc	5468	5487	SEQ ID NO: 5058	agcttgtttgccagtgggc	5578	5597	1	3
SEQ ID NO: 3718	attttgatgatgctggccc	5491	5510	SEQ ID NO: 5059	ttaatctttcataagtcaa	7275	7294	1	3
SEQ ID NO: 3719	gaattatcttttaaacatt	5596	5615	SEQ ID NO: 5060	agcttgtttgccagtgggc	9375	9395	1	3
SEQ ID NO: 3720	ttaccaccagtttgaagtt	5599	5618	SEQ ID NO: 5061	tgaaatcattgaaaattta	6271	6290	1	3
SEQ ID NO: 3721	ctgaaaaagttagtgaaag	5602	5621	SEQ ID NO: 5062	caggaggctttaagttgtc	10127	10148	1	3
SEQ ID NO: 3722	tttttcccagacagtgtca	6816	5635	SEQ ID NO: 5063	agcttgtttgccagtgggc	7065	7084	1	3
SEQ ID NO: 3723	ttttcccagacagtgtcca	5620	5845	SEQ ID NO: 5064	taaagccattcaggtctct	12158	12177	1	3
SEQ ID NO: 3724	cattcagaaccagggaaga	6586	5705	SEQ ID NO: 5065	cttcatcgttcatttgagt	9087	9106	1	3
SEQ ID NO: 3725	ttgcagtgtatcggacaga	5705	5724	SEQ ID NO: 5066	cttatgggatttcctaagg	7450	7489	1	3
SEQ ID NO: 3726	cattcagaaccagggaaga	5740	5759	SEQ ID NO: 5067	cttcatcgttcatttgagt	8598	9816	1	3
SEQ ID NO: 3727	acaaactgtttatagtccc	5790	5809	SEQ ID NO: 5068	ttccatcacaaatcctttg	8828	8647	1	3
SEQ ID NO: 3728	ggatttcaatacatttcac	5791	5810	SEQ ID NO: 5069	tctcaaagagttacaacag	8627	8646	1	3
SEQ ID NO: 3729	ttgtagaaatgaaagtaaa	5792	5811	SEQ ID NO: 5070	tattgaaatatgatttttt	8626	8645	1	3
SEQ ID NO: 3730	ctgaacagtgagctgcagt	5794	5813	SEQ ID NO: 5071	aattgttgaaagaaaaact	12412	12431	1	3
SEQ ID NO: 3731	aatccaatctcctctttgc	5800	5819	SEQ ID NO: 5072	ggacaaggcccagaatctg	6501	8520	1	3
SEQ ID NO: 3732	atttgattttcaagcaaag	5801	5820	SEQ ID NO: 5073	aaagaaacctatggcctta	12975	12994	1	3
SEQ ID NO: 3733	ttttgattttcaagcaaat	5859	5878	SEQ ID NO: 5074	attcttaacgggattcctt	12213	12232	1	3
SEQ ID NO: 3734	tgatttcaagcaaatgcag	5879	5898	SEQ ID NO: 5075	taaagccattcaggtctct	1088	1107	1	3
SEQ ID NO: 3735	atgctgtttgggaaattgg	5914	5933	SEQ ID NO: 5076	gacatgtttgaagggtgga	8274	8293	1	3
SEQ ID NO: 3736	tgctgttttttggaaatgc	5983	6002	SEQ ID NO: 5077	tgggctaaacgtttaattg	13612	13631	1	3
SEQ ID NO: 3737	aaaaaaatacactggacgt	5993	6012	SEQ ID NO: 5078	tattgaaatatgatttttt	13351	13370	1	3
SEQ ID NO: 3738	ctgaaaaagttagtgaaag	6009	6028	SEQ ID NO: 5079	agcttgtttgccagtgggc	6418	6437	1	3
SEQ ID NO: 3739	tttttcccagacagtgtca	6046	6085	SEQ ID NO: 5080	aaagaaacctatggcctta	11610	11629	1	3
SEQ ID NO: 3740	ttttcccagacagtgtcca	6061	6080	SEQ ID NO: 5081	attcttaacgggattcctt	8037	8058	1	3
SEQ ID NO: 3741	cattcagaaccagggaaga	6084	6103	SEQ ID NO: 5082	taaagccattcaggtctct	9882	9701	1	3
SEQ ID NO: 3742	acaaactgtttatagtccc	6103	6122	SEQ ID NO: 5083	gacatgtttgaagggtgga	8599	8618	1	3
SEQ ID NO: 3743	ggatttcaatacatttcac	6132	6151	SEQ ID NO: 5084	tgggctaaacgtttaattg	8273	8292	1	3
SEQ ID NO: 3744	ttgtagaaatgaaagtaaa	6198	6217	SEQ ID NO: 5085	tattgaaatatgatttttt	8988	7007	1	3
SEQ ID NO: 3745	ccaaaatttctctcggctt	6235	6254	SEQ ID NO: 5086	agcttgtttgccagtgggc	9177	9196	1	3
SEQ ID NO: 3746	caaattttctctgctaaaa	6257	6276	SEQ ID NO: 5087	ggacaaggcccagaatctg	12970	12989	1	3
SEQ ID NO: 3747	tctgctggaaacaacaacg	6276	6295	SEQ ID NO: 5088	aaagaaacctatggcctta	10187	10208	1	3
SEQ ID NO: 3748	cagaagctaagcaatgtcc	6266	6305	SEQ ID NO: 5089	attcttaacgggattcctt	13655	13374	1	3
SEQ ID NO: 3749	taagattaaatatttactt	6288	6307	SEQ ID NO: 5090	taaagccattcaggtctct	6358	6377	1	3
SEQ ID NO: 3750	aaagattactttgagaaat	6315	6334	SEQ ID NO: 5091	taaagccattcaggtctct	9576	9595	1	3
SEQ ID NO: 3751	aggaaatagttggattacc	6337	6358	SEQ ID NO: 5092	gacatgtttgaagggtgga	9556	9586	1	3
SEQ ID NO: 3752	attttgatgatgctggccc	6344	6353	SEQ ID NO: 5093	tgggctaaacgtttaattg	12948	12967	1	3
SEQ ID NO: 3753	gaattatcttttaaacatt	6423	6442	SEQ ID NO: 5094	tattgaaatatgatttttt	11829	11848	1	3
SEQ ID NO: 3754	ttaccaccagtttgaagtt	6451	6470	SEQ ID NO: 5095	agcttgtttgccagtgggc	13622	13641	1	3
SEQ ID NO: 3755	taagattaaatatttactt	3460	6479	SEQ ID NO: 5096	ggacaaggcccagaatctg	7724	7743	1	3
SEQ ID NO: 3756	aaagattactttgagaaat	6489	6480	SEQ ID NO: 5097	aaagaaacctatggcctta	12869	12888	1	3
SEQ ID NO: 3757	aggaaatagttggattacc	8497	6516	SEQ ID NO: 5098	attcttaacgggattcctt	10479	10498	1	3
SEQ ID NO: 3758	attttgatgatgctggccc	6503	6522	SEQ ID NO: 5099	aattgttgaaagaaaaact	11809	11827	1	3
SEQ ID NO: 3759	aggtttaaatggataattt	6534	6553	SEQ ID NO: 5100	ggacaaggcccagaatctg	11163	11182	1	3
SEQ ID NO: 3760	cagaagctaagcaatgtcc	6541	6680	SEQ ID NO: 5101	aaagaaacctatggcctta	8380	8399	1	3
SEQ ID NO: 3761	taagattaaatatttactt	6544	6563	SEQ ID NO: 5102	attcttaacgggattcctt	9695	9714	1	3
SEQ ID NO: 3762	aaagattactttgagaaat	6641	6650	SEQ ID NO: 5103	taaagccattcaggtctct	1933	1962	1	3
SEQ ID NO: 3763	aggaaatagttggattacc	6857	6678	SEQ ID NO: 5104	gacatgtttgaagggtgga	13815	13834	1	3
SEQ ID NO: 3764	attttgatgatgctggccc	6883	6702	SEQ ID NO: 5105	tgggctaaacgtttaattg	13128	13147	1	3
SEQ ID NO: 3765	gaattatcttttaaacatt	6697	6716	SEQ ID NO: 5106	tattgaaatatgatttttt	13854	13683	1	3
SEQ ID NO: 3766	ttaccaccagtttgaagtt	6899	6717	SEQ ID NO: 5107	agcttgtttgccagtgggc	13883	13882	1	3
SEQ ID NO: 3767	ttgcagtgtatcggacaga	6703	6722	SEQ ID NO: 5108	tgaaatcattgaaaattta	14084	14103	1	3
SEQ ID NO: 3768	cattcagaaccagggaaga	6719	6738	SEQ ID NO: 5109	caggaggctttaagttgtc	8268	8287	1	3
SEQ ID NO: 3769	acaaactgtttatagtccc	6747	6766	SEQ ID NO: 5110	agcttgtttgccagtgggc	12864	12883	1	3
SEQ ID NO: 3770	ggatttcaatacatttcac	6748	6767	SEQ ID NO: 5111	taaagccattcaggtctct	8789	8788	1	3
SEQ ID NO: 3771	ttgtagaaatgaaagtaaa	6753	6772	SEQ ID NO: 5112	aattgttgaaagaaaaact	13101	13120	1	3
SEQ ID NO: 3772	ctgaacagtgagctgcagt	6777	6796	SEQ ID NO: 5113	taaagccattcaggtctct	11702	11721	1	3
SEQ ID NO: 3773	aatccaatctcctctttgc	6760	6799	SEQ ID NO: 5114	ttaatctttcataagtcaa	11810	11828	1	3
SEQ ID NO: 3774	atttgattttcaagcaaag	6805	6824	SEQ ID NO: 5115	ggacaaggcccagaatctg	11175	11194	1	3
SEQ ID NO: 3775	ttttgattttcaagcaaat	6524	6843	SEQ ID NO: 5116	attcttaacgggattcctt	9671	9890	1	3
SEQ ID NO: 3776	tgatttcaagcaaatgcag	6828	6847	SEQ ID NO: 5117	aattgttgaaagaaaaact	8014	8033	1	3
SEQ ID NO: 3777	atgctgtttgggaaattgg	6888	6907	SEQ ID NO: 5118	agcttgtttgccagtgggc	10088	10107	1	3
SEQ ID NO: 3778	ttgtagaaatgaaagtaaa	6894	6913	SEQ ID NO: 5119	ttaatctttcataagtcaa	9065	9084	1	3
SEQ ID NO: 3779	attttgatgatgctggccc	6924	6943	SEQ ID NO: 5120	taaagccattcaggtctct	9491	9510	1	3
SEQ ID NO: 3780	gaattatcttttaaacatt	6950	6969	SEQ ID NO: 5121	aattgttgaaagaaaaact	3688	3767	1	3
SEQ ID NO: 3781	ttaccaccagtttgaagtt	7080	7079	SEQ ID NO: 5122	taaagccattcaggtctct	6191	6210	1	3
SEQ ID NO: 3782	ttgcagtgtatcggacaga	7081	7080	SEQ ID NO: 5123	attcttaacgggattcctt	5334	5353	1	3
SEQ ID NO: 3783	cattcagaaccagggaaga	7070	7089	SEQ ID NO: 5124	taaagccattcaggtctct	13133	13152	1	3
SEQ ID NO: 3784	acaaactgtttatagtccc	7111	7130	SEQ ID NO: 5125	gacatgtttgaagggtgga	12029	12048	1	3
SEQ ID NO: 3785	ggatttcaatacatttcac	7160	7179	SEQ ID NO: 5126	tgggctaaacgtttaattg	10331	10350	1	3
SEQ ID NO: 3786	ttgtagaaatgaaagtaaa	7164	7183	SEQ ID NO: 5127	tattgaaatatgatttttt	12158	12175	1	3
SEQ ID NO: 3787	ctgaacagtgagctgcagt	7223	7242	SEQ ID NO: 5128	agcttgtttgccagtgggc	10168	10187	1	3
SEQ ID NO: 3788	aatccaatctcctctttgc	7264	7283	SEQ ID NO: 5129	tgaaatcattgaaaattta	11334	11353	1	3
SEQ ID NO: 3789	atttgattttcaagcaaag	7271	7290	SEQ ID NO: 5130	caggaggctttaagttgtc	2077	2096	1	3
SEQ ID NO: 3790	ttttgattttcaagcaaat	7280	7299	SEQ ID NO: 5131	agcttgtttgccagtgggc	9069	9088	1	3
SEQ ID NO: 3791	tgatttcaagcaaatgcag	7288	7307	SEQ ID NO: 5132	taaagccattcaggtctct	7443	7462	1	3
SEQ ID NO: 3792	atgctgtttgggaaattgg	7292	7311	SEQ ID NO: 5133	aattgttgaaagaaaaact	10419	10438	1	3
SEQ ID NO: 3793	tgctgttttttggaaatgc	7300	7319	SEQ ID NO: 5134	ttaatctttcataagtcaa	9953	9972	1	3
SEQ ID NO: 3794	aaaaaaatacactggacgt	7353	7372	SEQ ID NO: 5135	taaagccattcaggtctct	7422	7441	1	3
SEQ ID NO: 3795	actggagcttagtaagggc	7354	7373	SEQ ID NO: 5136	aattgttgaaagaaaaact	7421	7440	1	3
SEQ ID NO: 3796	cttctggaaagagggtcat	7355	7374	SEQ ID NO: 5137	ttaatctttcataagtcaa	10495	10514	1	3
SEQ ID NO: 3797	ggaaaagggtcatggaaat	7356	7375	SEQ ID NO: 5138	ggacaaggcccagaatctg	10494	10513	1	3
SEQ ID NO: 3798	gggcctgccccagatttct	7357	7376	SEQ ID NO: 5139	attcttaacgggattcctt	10493	10512	1	3
SEQ ID NO: 3799	ttctcagataggggaacac	7380	7399	SEQ ID NO: 5140	aattgttgaaagaaaaact	11487	11606	1	3
SEQ ID NO: 3800	gatgaaagaattaagggga	7406	7425	SEQ ID NO: 5141	agcttgtttgccagtgggc	8791	8810	1	3
SEQ ID NO: 3801	cttggactgtcaaataagt	7441	7460	SEQ ID NO: 5142	ttaatctttcataagtcaa	7972	7991	1	3
SEQ ID NO: 3802	ggaaaagggtcatggaaat	7442	7461	SEQ ID NO: 5143	agcttgtttgccagtgggc	8536	8687	1	3
SEQ ID NO: 3803	aggaaatagttggattacc	7452	7471	SEQ ID NO: 5144	tgaaatcattgaaaattta	10731	10760	1	3
SEQ ID NO: 3804	attttgatgatgctggccc	7473	7492	SEQ ID NO: 5145	caggaggctttaagttgtc	8409	8428	1	3
SEQ ID NO: 3805	gaattatcttttaaacatt	7547	7566	SEQ ID NO: 5146	agcttgtttgccagtgggc	8540	8559	1	3
SEQ ID NO: 3806	aatccaatctcctctttgc	7616	7635	SEQ ID NO: 5147	taaagccattcaggtctct	10173	10192	1	3
SEQ ID NO: 3807	atttgattttcaagcaaag	7641	7660	SEQ ID NO: 5148	cttcatcgttcatttgagt	10213	10232	1	3
SEQ ID NO: 3808	ttttgattttcaagcaaat	7684	7703	SEQ ID NO: 5149	cttatgggatttcctaagg	11021	11040	1	3
SEQ ID NO: 3809	tgatttcaagcaaatgcag	7700	7719	SEQ ID NO: 5150	cttcatcgttcatttgagt	6254	8273	1	3
SEQ ID NO: 3810	atgctgtttgggaaattgg	7722	7741	SEQ ID NO: 5151	ttccatcacaaatcctttg	9377	9396	1	3
SEQ ID NO: 3811	tgctgttttttggaaatgc	7723	7742	SEQ ID NO: 5152	tctcaaagagttacaacag	8810	8829	1	3
SEQ ID NO: 3812	aaaaaaatacactggacgt	7732	7761	SEQ ID NO: 5153	tattgaaatatgatttttt	10535	10554	1	3
SEQ ID NO: 3813	actggagcttagtaagggc	7738	7757	SEQ ID NO: 5154	aattgttgaaagaaaaact	11025	11044	1	3
SEQ ID NO: 3814	cttctggaaagagggtcat	7742	7781	SEQ ID NO: 5155	ggacaaggcccagaatctg	13681	13700	1	3
SEQ ID NO: 3815	ggaaaagggtcatggaaat	7755	7772	SEQ ID NO: 5156	aaagaaacctatggcctta	10283	10302	1	3
SEQ ID NO: 3816	gggcctgccccagatttct	7770	7789	SEQ ID NO: 5157	attcttaacgggattcctt	14026	14045	1	3
SEQ ID NO: 3817	ttctcagataggggaacac	7847	7866	SEQ ID NO: 5158	taaagccattcaggtctct	10208	10227	1	3
SEQ ID NO: 3818	gatgaaagaattaagggga	7846	7857	SEQ ID NO: 5159	gacatgtttgaagggtgga	10207	10226	1	3
SEQ ID NO: 3819	cttggactgtcaaataagt	7858	7867	SEQ ID NO: 5160	tgggctaaacgtttaattg	8959	8978	1	3
SEQ ID NO: 3820	ggaaaagggtcatggaaat	7887	7906	SEQ ID NO: 5161	tattgaaatatgatttttt	7973	7992	1	3
SEQ ID NO: 3821	ttttgattttcaagcaaat	7897	7916	SEQ ID NO: 5162	agcttgtttgccagtgggc	13978	13997	1	3
SEQ ID NO: 3822	tgatttcaagcaaatgcag	7929	7948	SEQ ID NO: 5163	aaagaaacctatggcctta	9588	9507	1	3
SEQ ID NO: 3823	atgctgtttgggaaattgg	8004	8023	SEQ ID NO: 5164	attcttaacgggattcctt	13183	13202	1	3
SEQ ID NO: 3824	tgctgttttttggaaatgc	8913	8032	SEQ ID NO: 5165	taaagccattcaggtctct	6829	6848	1	3
SEQ ID NO: 3825	aaaaaaatacactggacgt	8039	8058	SEQ ID NO: 5166	gacatgtttgaagggtgga	9472	9491	1	3
SEQ ID NO: 3826	actggagcttagtaagggc	8053	8082	SEQ ID NO: 5167	tgggctaaacgtttaattg	9887	9908	1	3
SEQ ID NO: 3827	cttctggaaagagggtcat	8089	8108	SEQ ID NO: 5168	tattgaaatatgatttttt	10414	10433	1	3
SEQ ID NO: 3828	ggaaaagggtcatggaaat	8145	8164	SEQ ID NO: 5169	agcttgtttgccagtgggc	12932	12861	1	3
SEQ ID NO: 3829	gggcctgccccagatttct	8233	8232	SEQ ID NO: 5170	ggacaaggcccagaatctg	8522	8541	1	3
SEQ ID NO: 3830	ttctcagataggggaacac	8320	8339	SEQ ID NO: 5171	aaagaaacctatggcctta	8884	8903	1	3
SEQ ID NO: 3831	gatgaaagaattaagggga	8339	8358	SEQ ID NO: 5172	attcttaacgggattcctt	8921	8940	1	3
SEQ ID NO: 3832	cttggactgtcaaataagt	8387	8408	SEQ ID NO: 5173	taaagccattcaggtctct	11612	11631	1	3
SEQ ID NO: 3833	ggaaaagggtcatggaaat	8300	8418	SEQ ID NO: 5174	taaagccattcaggtctct	11552	11571	1	3
SEQ ID NO: 3834	aggaaatagttggattacc	8422	8441	SEQ ID NO: 5175	gacatgtttgaagggtgga	9738	9757	1	3
SEQ ID NO: 3835	attttgatgatgctggccc	8436	8459	SEQ ID NO: 5176	tgggctaaacgtttaattg	9670	9889	1	3
SEQ ID NO: 3836	gaattatcttttaaacatt	8508	8527	SEQ ID NO: 5177	tattgaaatatgatttttt	13410	13429	1	3
SEQ ID NO: 3837	ttaccaccagtttgaagtt	8509	8528	SEQ ID NO: 5178	agcttgtttgccagtgggc	13409	13428	1	3
SEQ ID NO: 3838	ctgaaaaagttagtgaaag	8527	8546	SEQ ID NO: 5179	ggacaaggcccagaatctg	11537	11556	1	3
SEQ ID NO: 3839	tttttcccagacagtgtca	8530	8549	SEQ ID NO: 5180	aaagaaacctatggcctta	13650	13679	1	3
SEQ ID NO: 3840	ttttcccagacagtgtcca	8549	8655	SEQ ID NO: 5181	attcttaacgggattcctt	12460	12479	1	3
SEQ ID NO: 3841	cattcagaaccagggaaga	8604	8623	SEQ ID NO: 5182	aattgttgaaagaaaaact	113688	11407	1	3
SEQ ID NO: 3842	ttgcagtgtatcggacaga	8615	8635	SEQ ID NO: 5183	ggacaaggcccagaatctg	13780	13799	1	3
SEQ ID NO: 3843	cattcagaaccagggaaga	8678	8697	SEQ ID NO: 5184	aaagaaacctatggcctta	13422	13441	1	3
SEQ ID NO: 3844	acaaactgtttatagtccc	8751	8770	SEQ ID NO: 5185	attcttaacgggattcctt	13380	13399	1	3
SEQ ID NO: 3845	ggatttcaatacatttcac	8765	8784	SEQ ID NO: 5186	taaagccattcaggtctct	12702	12721	1	3
SEQ ID NO: 3846	ttgtagaaatgaaagtaaa	8829	8848	SEQ ID NO: 5187	gacatgtttgaagggtgga	12259	12278	1	3
SEQ ID NO: 3847	ctgaacagtgagctgcagt	8929	8948	SEQ ID NO: 5188	tgggctaaacgtttaattg	10036	10057	1	3
SEQ ID NO: 3848	aatccaatctcctctttgc	9060	9079	SEQ ID NO: 5189	tattgaaatatgatttttt	12618	12837	1	3
SEQ ID NO: 3849	atttgattttcaagcaaag	9067	9066	SEQ ID NO: 5190	agcttgtttgccagtgggc	7308	7327	1	3
SEQ ID NO: 3850	ttttgattttcaagcaaat	9077	9998	SEQ ID NO: 5191	tgaaatcattgaaaattta	13585	13585	1	3
SEQ ID NO: 3851	tgatttcaagcaaatgcag	9141	9160	SEQ ID NO: 5192	caggaggctttaagttgtc	12506	12525	1	3
SEQ ID NO: 3852	atgctgtttgggaaattgg	9142	9161	SEQ ID NO: 5193	agcttgtttgccagtgggc	2098	2917	1	3
SEQ ID NO: 3853	tgctgttttttggaaatgc	9221	9240	SEQ ID NO: 5194	taaagccattcaggtctct	13638	13557	1	3
SEQ ID NO: 3854	aaaaaaatacactggacgt	9230	6249	SEQ ID NO: 5195	aattgttgaaagaaaaact	13404	13513	1	3
SEQ ID NO: 3855	ctgaaaaagttagtgaaag	9283	9602	SEQ ID NO: 5196	taaagccattcaggtctct	10380	10399	1	3
SEQ ID NO: 3856	tttttcccagacagtgtca	9312	9331	SEQ ID NO: 5197	ttaatctttcataagtcaa	9848	9667	1	3
SEQ ID NO: 3857	ttttcccagacagtgtcca	9405	9424	SEQ ID NO: 5198	ggacaaggcccagaatctg	14038	14057	1	3
SEQ ID NO: 3858	cattcagaaccagggaaga	9435	9454	SEQ ID NO: 5199	attcttaacgggattcctt	13380	13399	1	3
SEQ ID NO: 3859	acaaactgtttatagtccc	9463	9482	SEQ ID NO: 5200	aattgttgaaagaaaaact	11734	11753	1	3
SEQ ID NO: 3860	ggatttcaatacatttcac	9476	9497	SEQ ID NO: 5201	agcttgtttgccagtgggc	11719	11738	1	3
SEQ ID NO: 3861	ttgtagaaatgaaagtaaa	9502	9521	SEQ ID NO: 5202	ttaatctttcataagtcaa	13206	13225	1	3
SEQ ID NO: 3862	ccaaaatttctctcggctt	9505	9524	SEQ ID NO: 5203	taaagccattcaggtctct	7621	9540	1	3
SEQ ID NO: 3863	caaattttctctgctaaaa	9534	9553	SEQ ID NO: 5204	aattgttgaaagaaaaact	11211	11230	1	3
SEQ ID NO: 3864	tctgctggaaacaacaacg	9561	9580	SEQ ID NO: 5205	taaagccattcaggtctct	3936	3955	1	3
SEQ ID NO: 3865	cagaagctaagcaatgtcc	9578	9597	SEQ ID NO: 5206	attcttaacgggattcctt	13021	13040	1	3
SEQ ID NO: 3866	taagattaaatatttactt	9590	9809	SEQ ID NO: 5207	taaagccattcaggtctct	10422	10441	1	3
SEQ ID NO: 3867	aaagattactttgagaaat	9591	9610	SEQ ID NO: 5208	gacatgtttgaagggtgga	11291	11310	1	3
SEQ ID NO: 3868	aggaaatagttggattacc	9640	9659	SEQ ID NO: 5209	tgggctaaacgtttaattg	9805	9824	1	3
SEQ ID NO: 3869	attttgatgatgctggccc	9720	9739	SEQ ID NO: 5210	tattgaaatatgatttttt	11688	11707	1	3
SEQ ID NO: 3870	gaattatcttttaaacatt	9769	9608	SEQ ID NO: 5211	agcttgtttgccagtgggc	12762	12781	1	3
SEQ ID NO: 3871	ttaccaccagtttgaagtt	9859	9878	SEQ ID NO: 5212	tgaaatcattgaaaattta	8938	8957	1	3
SEQ ID NO: 3872	taagattaaatatttactt	9876	9595	SEQ ID NO: 5213	caggaggctttaagttgtc	13207	13226	1	3
SEQ ID NO: 3873	aaagattactttgagaaat	9594	9913	SEQ ID NO: 5214	agcttgtttgccagtgggc	4842	4861	1	3
SEQ ID NO: 3874	aggaaatagttggattacc	9950	9969	SEQ ID NO: 5215	taaagccattcaggtctct	11080	11099	1	3
SEQ ID NO: 3875	attttgatgatgctggccc	10113	10132	SEQ ID NO: 5216	aattgttgaaagaaaaact	5440	8469	1	3
SEQ ID NO: 3876	aggtttaaatggataattt	10167	10186	SEQ ID NO: 5217	ttaatctttcataagtcaa	7224	7243	1	3
SEQ ID NO: 3877	cagaagctaagcaatgtcc	10201	10220	SEQ ID NO: 5218	tattgaaatatgatttttt	12889	12906	1	3
SEQ ID NO: 3878	taagattaaatatttactt	10204	10223	SEQ ID NO: 5219	agcttgtttgccagtgggc	12431	12450	1	3
SEQ ID NO: 3879	aaagattactttgagaaat	10232	10251	SEQ ID NO: 5220	tgaaatcattgaaaattta	13928	13947	1	3
SEQ ID NO: 3880	aggaaatagttggattacc	10305	10324	SEQ ID NO: 5221	caggaggctttaagttgtc	8195	8214	1	3
SEQ ID NO: 3881	attttgatgatgctggccc	10330	10349	SEQ ID NO: 5222	agcttgtttgccagtgggc	7161	7180	1	3
SEQ ID NO: 3882	gaattatcttttaaacatt	10377	10306	SEQ ID NO: 5223	taaagccattcaggtctct	10778	10797	1	3
SEQ ID NO: 3883	ttaccaccagtttgaagtt	10453	10472	SEQ ID NO: 5224	aattgttgaaagaaaaact	13023	13042	1	3
SEQ ID NO: 3884	ttgcagtgtatcggacaga	10463	10452	SEQ ID NO: 5225	taaagccattcaggtctct	12859	12878	1	3
SEQ ID NO: 3885	cattcagaaccagggaaga	10476	10494	SEQ ID NO: 5226	ttaatctttcataagtcaa	10824	10943	1	3
SEQ ID NO: 3886	acaaactgtttatagtccc	10482	10501	SEQ ID NO: 5227	ggacaaggcccagaatctg	7148	7167	1	3
SEQ ID NO: 3887	ggatttcaatacatttcac	10512	10531	SEQ ID NO: 5228	attcttaacgggattcctt	12242	1226+1	1	3
SEQ ID NO: 3888	ttgtagaaatgaaagtaaa	10648	10567	SEQ ID NO: 5231	aattgttgaaagaaaaact	12036	12055	1	3
SEQ ID NO: 3889	ctgaacagtgagctgcagt	10573	10592	SEQ ID NO: 5232	agcttgtttgccagtgggc	10649	10668	1	3
SEQ ID NO: 3890	aatccaatctcctctttgc	10710	10729	SEQ ID NO: 5233	ttaatctttcataagtcaa	12289	12308	1	3
SEQ ID NO: 3891	atttgattttcaagcaaag	10723	10742	SEQ ID NO: 5234	taaagccattcaggtctct	12745	12764	1	3
SEQ ID NO: 3892	ttttgattttcaagcaaat	10880	10879	SEQ ID NO: 5235	aattgttgaaagaaaaact	2467	2486	1	3
SEQ ID NO: 3893	tgatttcaagcaaatgcag	10897	10916	SEQ ID NO: 5236	taaagccattcaggtctct	12950	12979	1	3
SEQ ID NO: 3894	atgctgtttgggaaattgg	10898	10917	SEQ ID NO: 5237	attcttaacgggattcctt	12959	12978	1	3
SEQ ID NO: 3895	ttgtagaaatgaaagtaaa	10914	10933	SEQ ID NO: 5238	taaagccattcaggtctct	14066	14085	1	3
SEQ ID NO: 3896	attttgatgatgctggccc	10915	10934	SEQ ID NO: 5239	gacatgtttgaagggtgga	14085	14084	1	3
SEQ ID NO: 3897	gaattatcttttaaacatt	10940	10959	SEQ ID NO: 5240	tgggctaaacgtttaattg	12258	12276	1	3
SEQ ID NO: 3898	ttaccaccagtttgaagtt	10987	11006	SEQ ID NO: 5241	tattgaaatatgatttttt	11186	11205	1	3
SEQ ID NO: 3899	ttgcagtgtatcggacaga	10994	11013	SEQ ID NO: 5242	agcttgtttgccagtgggc	13347	13368	1	3
SEQ ID NO: 3900	cattcagaaccagggaaga	10995	11014	SEQ ID NO: 5243	tgaaatcattgaaaattta	13346	13366	1	3
SEQ ID NO: 3901	acaaactgtttatagtccc	11003	11022	SEQ ID NO: 5244	caggaggctttaagttgtc	13622	13641	1	3
SEQ ID NO: 3902	ggatttcaatacatttcac	11016	11037	SEQ ID NO: 5245	agcttgtttgccagtgggc	9820	9299	1	3
SEQ ID NO: 3903	aaagattactttgagaaat	11032	11051	SEQ ID NO: 5246	taaagccattcaggtctct	13166	13185	1	3
SEQ ID NO: 3904	aggaaatagttggattacc	11079	11098	SEQ ID NO: 5247	aattgttgaaagaaaaact	10466	10485	1	3
SEQ ID NO: 3905	attttgatgatgctggccc	11115	11134	SEQ ID NO: 5248	ttaatctttcataagtcaa	7572	7591	1	3
SEQ ID NO: 3906	aggtttaaatggataattt	11199	11216	SEQ ID NO: 5249	taaagccattcaggtctct	12370	12389	1	3
SEQ ID NO: 3907	cagaagctaagcaatgtcc	11239	11258	SEQ ID NO: 5250	aattgttgaaagaaaaact	13148	13187	1	3
SEQ ID NO: 3908	taagattaaatatttactt	11277	11298	SEQ ID NO: 5251	ttaatctttcataagtcaa	8893	8612	1	3
SEQ ID NO: 3909	aaagattactttgagaaat	11332	11351	SEQ ID NO: 5252	ggacaaggcccagaatctg	13334	13363	1	3
SEQ ID NO: 3910	aggaaatagttggattacc	11432	11451	SEQ ID NO: 5253	attcttaacgggattcctt	13110	13129	1	3
SEQ ID NO: 3911	attttgatgatgctggccc	11453	11472	SEQ ID NO: 5254	aattgttgaaagaaaaact	12641	12660	1	3
SEQ ID NO: 3912	gaattatcttttaaacatt	11536	11555	SEQ ID NO: 5255	agcttgtttgccagtgggc	8525	8547	1	3
SEQ ID NO: 3913	ttaccaccagtttgaagtt	11591	11610	SEQ ID NO: 5256	ttaatctttcataagtcaa	12693	12712	1	3
SEQ ID NO: 3914	ttgcagtgtatcggacaga	11608	11627	SEQ ID NO: 5257	agcttgtttgccagtgggc	11833	11652	1	3
SEQ ID NO: 3915	cattcagaaccagggaaga	11639	11685	SEQ ID NO: 5258	tgaaatcattgaaaattta	9231	9550	1	3
SEQ ID NO: 3916	acaaactgtttatagtccc	11691	11710	SEQ ID NO: 5259	caggaggctttaagttgtc	12259	12278	1	3
SEQ ID NO: 3917	ggatttcaatacatttcac	11692	11711	SEQ ID NO: 5260	agcttgtttgccagtgggc	12258	12277	1	3
SEQ ID NO: 3918	ttgtagaaatgaaagtaaa	11703	11722	SEQ ID NO: 5261	taaagccattcaggtctct	13179	13196	1	3
SEQ ID NO: 3919	ctgaacagtgagctgcagt	11807	11826	SEQ ID NO: 5262	cttcatcgttcatttgagt	8504	8523	1	3
SEQ ID NO: 3920	aatccaatctcctctttgc	12004	12023	SEQ ID NO: 5263	cttatgggatttcctaagg	11438	11457	1	3
SEQ ID NO: 3921	atttgattttcaagcaaag	120456	12075	SEQ ID NO: 5264	cttcatcgttcatttgagt	13285	13305	1	3
SEQ ID NO: 3922	ttttgattttcaagcaaat	12060	12079	SEQ ID NO: 5265	ttccatcacaaatcctttg	13980	13979	1	3
SEQ ID NO: 3923	tgatttcaagcaaatgcag	12074	142093	SEQ ID NO: 5266	tctcaaagagttacaacag	13711	13730	1	3
SEQ ID NO: 3924	atgctgtttgggaaattgg	12264	12283	SEQ ID NO: 5267	tattgaaatatgatttttt	11667	11688	1	3
SEQ ID NO: 3925	ttgtagaaatgaaagtaaa	12300	12319	SEQ ID NO: 5268	aattgttgaaagaaaaact	3018	3037	1	3
SEQ ID NO: 3926	attttgatgatgctggccc	12327	12348	SEQ ID NO: 5269	ggacaaggcccagaatctg	6508	6527	1	3
SEQ ID NO: 3927	gaattatcttttaaacatt	12362	12381	SEQ ID NO: 5270	aaagaaacctatggcctta	12763	12782	1	3
SEQ ID NO: 3928	ttaccaccagtttgaagtt	12475	12494	SEQ ID NO: 5271	attcttaacgggattcctt	13115	13124	1	3
SEQ ID NO: 3929	ttgcagtgtatcggacaga	12584	12603	SEQ ID NO: 5272	taaagccattcaggtctct	12640	12669	1	3
SEQ ID NO: 3930	ggatttcaatacatttcac	12736	12755	SEQ ID NO: 5273	gacatgtttgaagggtgga	12865	12884	1	3
SEQ ID NO: 3931	ttgtagaaatgaaagtaaa	12877	12898	SEQ ID NO: 5274	tgggctaaacgtttaattg	13524	13543	1	3
SEQ ID NO: 3932	ctgaacagtgagctgcagt	12974	12993	SEQ ID NO: 5275	tattgaaatatgatttttt	5802	5821	1	3
SEQ ID NO: 3933	aatccaatctcctctttgc	13001	13020	SEQ ID NO: 5276	agcttgtttgccagtgggc	13964	13983	1	3
SEQ ID NO: 3934	atttgattttcaagcaaag	13065	13084	SEQ ID NO: 5277	aaagaaacctatggcctta	13200	13219	1	3
SEQ ID NO: 3935	ttttgattttcaagcaaat	13080	13099	SEQ ID NO: 5278	attcttaacgggattcctt	13615	13834	1	3
SEQ ID NO: 3936	tgatttcaagcaaatgcag	13150	13169	SEQ ID NO: 5279	taaagccattcaggtctct	9566	9585	1	3
SEQ ID NO: 3937	atgctgtttgggaaattgg	13180	13199	SEQ ID NO: 5280	gacatgtttgaagggtgga	11702	11721	1	3
SEQ ID NO: 3938	tgctgttttttggaaatgc	13276	13208	SEQ ID NO: 5281	tgggctaaacgtttaattg	13937	13956	1	3
SEQ ID NO: 3939	aaaaaaatacactggacgt	13311	13330	SEQ ID NO: 5282	tattgaaatatgatttttt	8295	8314	1	3
SEQ ID NO: 3940	ctgaaaaagttagtgaaag	13442	13481	SEQ ID NO: 5283	taaagccattcaggtctct	13834	13853	1	3
SEQ ID NO: 3941	tttttcccagacagtgtca	13509	13628	SEQ ID NO: 5284	attcttaacgggattcctt	13821	13840	1	3
SEQ ID NO: 3942	ttttcccagacagtgtcca	13570	13555	SEQ ID NO: 5285	taaagccattcaggtctct	7999	8018	1	3
SEQ ID NO: 3943	attttgatgatgctggccc	13680	13899	SEQ ID NO: 5286	gacatgtttgaagggtgga	10422	10441	1	3
SEQ ID NO: 3944	gaattatcttttaaacatt	13693	13712	SEQ ID NO: 5287	tgggctaaacgtttaattg	7370	7389	1	3
SEQ ID NO: 3945	ttaccaccagtttgaagtt	13811	13830	SEQ ID NO: 5288	tattgaaatatgatttttt	10382	10401	1	3
SEQ ID NO: 3946	ttgcagtgtatcggacaga	14043	14082	SEQ ID NO: 5289	agcttgtttgccagtgggc	7884	7883	1	3
SEQ ID NO: 3947	aaagattactttgagaaat	14057	14076	SEQ ID NO: 5290	attcttaacgggattcctt	9842	9661	1	3

TABLE 10 — Selected palindromic sequences from human glucose-6-phosphatase

		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 5291	ggccattccagaagggaag	222	241	SEQ ID NO: 5369	cttccgttctgtaatggcc	1340	1359	1	6
SEQ ID NO: 5292	tgccatctcgagagttcca	223	242	SEQ ID NO: 5370	tggaactctctccatggca	1339	1358	1	6
SEQ ID NO: 5293	catgtcaaacactttgtta	417	436	SEQ ID NO: 5371	taacaaattccttgacatg	1492	1511	1	6
SEQ ID NO: 5294	tttgttataaatcttattg	418	437	SEQ ID NO: 5372	caataagatcaatagcaaa	1491	1510	1	6
SEQ ID NO: 5295	tctggaaaaagggtcatga	521	540	SEQ ID NO: 5373	tccatgtcccatttacaga	2945	2964	1	6
SEQ ID NO: 5296	cagctcttgttcaggtcca	1886	1905	SEQ ID NO: 5374	tggacctgcaccaaagctg	271	2750	1	6
SEQ ID NO: 5297	ggaggttccccagctctgc	1956	1975	SEQ ID NO: 5375	gcagccctgggaaaactcc	2983	3002	1	6
SEQ ID NO: 5298	ctgttttgaagactctcca	50	69	SEQ ID NO: 5376	tggagggtagtcataacag	2620	2639	1	5
SEQ ID NO: 5299	agtggctgaaacgtgtgca	51	70	SEQ ID NO: 5377	tgcagagctttctgccact	2619	2638	1	5
SEQ ID NO: 5300	ccaaaatagaagggaatct	487	506	SEQ ID NO: 5378	agattcctttctttttcaa	1295	1314	1	5
SEQ ID NO: 5301	tgaagagaagattgaattt	598	617	SEQ ID NO: 5379	aaattctcttttattttca	2982	3001	1	5
SEQ ID NO: 5302	agtggtggcaacaccagca	651	670	SEQ ID NO: 5380	tgctagtgaggccaacact	773	792	1	5
SEQ ID NO: 5303	aaggctccacaagtcatca	776	795	SEQ ID NO: 5381	tgatgatatctggaacctt	884	903	1	5
SEQ ID NO: 5304	gtccgccaggtttctagca	848	867	SEQ ID NO: 5382	tgctaagaaccttactgac	2107	2126	1	5
SEQ ID NO: 5305	tgatatctggaaccttgga	878	897	SEQ ID NO: 5383	ttcactgttcctgaaatca	2801	2620	1	5
SEQ ID NO: 5306	gtcaagttgagcaatttct	1439	1458	SEQ ID NO: 5384	agaaaaggcacaccttgac	1676	1896	1	5
SEQ ID NO: 5307	atccagatggaaaagggaa	1572	1591	SEQ ID NO: 5385	ttccaatttccctgtggat	2626	2645	1	5
SEQ ID NO: 5308	atttgtttgtcaaagaagt	1573	1592	SEQ ID NO: 5386	acttcagagagaaatacat	2625	2644	4	5
SEQ ID NO: 5309	ctggaaaatgtcagcctgg	1854	1873	SEQ ID NO: 5387	ccagacttcagttaccagc	2683	2702	2	5
SEQ ID NO: 5310	accaggaggttcttcttca	2509	2528	SEQ ID NO: 5388	tgaagtgtgatctcctggt	2996	3015	2	5
SEQ ID NO: 5311	aaagaagttctgaaaagat	0	19	SEQ ID NO: 5389	attccatcacaaaatcctt	612	831	2	4
SEQ ID NO: 5312	gctacagcttatggctcca	12	31	SEQ ID NO: 5390	tggatctaaatgcagtagc	1987	2006	2	4
SEQ ID NO: 5313	atcaatatgatcaatttgt	220	239	SEQ ID NO: 5391	caaagaaatcaagattgat	1440	1459	2	4
SEQ ID NO: 5314	gaattatctttaaaacatt	326	345	SEQ ID NO: 5392	atgtggacaaatataccgg	2426	2444	2	4
SEQ ID NO: 5315	cgaggcccgcgctgctggc	392	411	SEQ ID NO: 5393	gccagaagtgagatcctcg	782	801	1	4
SEQ ID NO: 5316	acaactatgaggctgagag	638	657	SEQ ID NO: 5394	ctctgagcaacaaattttt	1474	1493	1	4
SEQ ID NO: 5317	gctgagagttccagtggag	666	685	SEQ ID NO: 5395	ctccatggcaaatgtcagg	758	777	1	4
SEQ ID NO: 5318	tgaagaaaaaccaagaact	760	779	SEQ ID NO: 5396	gagtcattgaggttcttca	823	842	1	4
SEQ ID NO: 5319	cctacttacatcctgaaca	761	780	SEQ ID NO: 5397	tgttcataagggaggtagg	822	841	1	4
SEQ ID NO: 5320	ctacttacatcctgaacat	762	781	SEQ ID NO: 5398	atgttcataagggaggtag	821	840	1	4
SEQ ID NO: 5321	gagacagaagccaagagcc	767	786	SEQ ID NO: 5399	gcttggttttgcccagtct	2014	2033	1	4
SEQ ID NO: 5322	cactcactttaccgtcaag	862	881	SEQ ID NO: 5400	cttgaacaccaaagtcact	1687	1706	1	4
SEQ ID NO: 5323	ctgatcagcagcagccagt	863	882	SEQ ID NO: 5401	actgggaagtgcttatcag	1696	1705	1	4
SEQ ID NO: 5324	actggacgctaagaggaag	1028	1047	SEQ ID NO: 5402	cttccccaaagagaggagt	1663	1682	1	4
SEQ ID NO: 5325	agaggaagcatgtggcaga	1056	1075	SEQ ID NO: 5403	tctggcatttactttctct	2229	2248	1	4
SEQ ID NO: 5326	tgaagactctccaggaact	1217	1236	SEQ ID NO: 5404	agttgaaggagactattca	1446	1465	1	4
SEQ ID NO: 5327	ctctgagcaaaatatccag	1267	1286	SEQ ID NO: 5405	ctggttactgagctgagag	2311	2330	1	4
SEQ ID NO: 5328	atgaagcagtcacatctct	1598	1617	SEQ ID NO: 5406	agagctgccagtcttcatt	2967	2986	1	4
SEQ ID NO: 5329	ttgccacagctgattgagg	1764	1783	SEQ ID NO: 5407	cctcctacagtggtggcaa	2063	2062	1	4
SEQ ID NO: 5330	agctgattgaggtgtccag	1855	1874	SEQ ID NO: 5408	ctggattccacatgcagct	3003	3022	1	4
SEQ ID NO: 5331	tgctccactcacatcctcc	2215	2234	SEQ ID NO: 5409	ggaggctttaagttcagca	2617	2836	1	4
SEQ ID NO: 5332	tgaaacgtgtgcatgtcaa	2330	2349	SEQ ID NO: 5410	ttgggagagacaagtttca	3007	3026	1	4
SEQ ID NO: 5333	gacattgctaattacctga	2345	2364	SEQ ID NO: 5411	tcagaagctaagcaatgtc	2992	3011	1	4
SEQ ID NO: 5334	ttcttcttcagactttcct	197	216	SEQ ID NO: 5412	aggagagtccaaatttaga	1116	1135	1	3
SEQ ID NO: 5335	ccaatatcttgaaactcag	257	276	SEQ ID NO: 5413	tctgaattcattcaattgg	1446	1465	1	3
SEQ ID NO: 5336	aaagttagtgaaaagaagt	263	282	SEQ ID NO: 5414	aactaccctcactgccttt	423	442	1	3
SEQ ID NO: 5337	aagttagtgaaagaagttc	358	377	SEQ ID NO: 5415	gaacctctggcattttact	2548	2567	1	3
SEQ ID NO: 5338	aaagaagttctgaaagaat	464	483	SEQ ID NO: 5416	attctctggtaactacttt	2705	2724	1	3
SEQ ID NO: 5339	tttggctataccaaagatg	468	487	SEQ ID NO: 5417	catcttaggcactgacaaa	1419	1438	1	3
SEQ ID NO: 5340	tgttgagaagctgattaaa	492	511	SEQ ID NO: 5418	tttagccatcggctcaaca	2828	2647	1	3
SEQ ID NO: 5341	caggaagggctcaaagaat	563	572	SEQ ID NO: 5419	attcctttaacaattcctg	644	663	1	3
SEQ ID NO: 5342	aggaagggctcaaagaatg	564	583	SEQ ID NO: 5420	cattcctttaacaattcct	2036	2057	1	3
SEQ ID NO: 5343	gaagggctcaaagaatgac	572	591	SEQ ID NO: 5421	gtcagtcttcaggctcttc	2779	2798	1	3
SEQ ID NO: 5344	caaagaatgacttttttct	606	825	SEQ ID NO: 5422	agaaggatggcattttttg	2742	2761	1	3
SEQ ID NO: 5345	catggagaatgcctttgaa	645	864	SEQ ID NO: 5423	ttcagagccaaagtccatg	2185	2204	1	3
SEQ ID NO: 5346	ggagccaaggctggagtaa	650	669	SEQ ID NO: 5424	ttactccaacgccagctcc	1818	1837	1	3
SEQ ID NO: 5347	tcattccttccccaaagag	678	697	SEQ ID NO: 5425	ctctctggggcatctatgc	1950	1969	1	3
SEQ ID NO: 5348	acctatgagctccagagag	690	709	SEQ ID NO: 5426	ctctcaagaccacagaagg	2283	2302	1	3
SEQ ID NO: 5349	gggcaaaaacgtcttacag	691	710	SEQ ID NO: 5427	tctgaaagacaacgtgccc	2282	2301	1	3
SEQ ID NO: 5350	accctggaccttcagaaca	744	763	SEQ ID NO: 5428	tgttgctaaggttcagggt	2306	2325	1	3
SEQ ID NO: 5351	atgggcgacctaagttgtg	772	791	SEQ ID NO: 5429	cacaaattagtttcaccat	2713	2732	1	3
SEQ ID NO: 5352	gatgaagagaagattgaat	784	803	SEQ ID NO: 5430	atccagcttccccacactt	2805	2824	1	3
SEQ ID NO: 5354	caatgtgataccaaaaaaa	1004	1023	SEQ ID NO: 5431	tttttggaaatgccattgt	2405	2424	1	3
SEQ ID NO: 5355	gtagataccaaaaaaatga	1351	1370	SEQ ID NO: 5432	tcatgtgatgggtctctac	2847	2865	1	3
SEQ ID NO: 5356	gcttcagttcatttggact	1438	1457	SEQ ID NO: 5433	agtcaagaaggacttaagc	2750	2779	1	3
SEQ ID NO: 5357	tttgtttgtcaaagaagtc	1553	1572	SEQ ID NO: 5434	gacttcagagaaatacaaa	2297	2316	1	3
SEQ ID NO: 5358	ttgtttgtcaaagaagtca	1606	1625	SEQ ID NO: 5435	tgacttcagagaaatacaa	2131	2150	1	3
SEQ ID NO: 5359	tggcaatgggaaactcgct	1785	1804	SEQ ID NO: 5436	agcgagagtcccctgccat	2503	2522	1	3
SEQ ID NO: 5360	aacctctggcatttacttt	1786	1805	SEQ ID NO: 5437	aaaggagatgtcaagggtt	2502	2521	1	3
SEQ ID NO: 5361	catttactttctctcatga	1787	1806	SEQ ID NO: 5438	tcatttgaaagaataaatg	2501	2520	1	3
SEQ ID NO: 5362	aaagtcagtgccctgctta	1788	1807	SEQ ID NO: 5439	taagaaccttactgacttt	2500	2519	1	3
SEQ ID NO: 5363	tcccattttttgagacctt	1982	2001	SEQ ID NO: 5440	aaggacttcaggaatggga	2189	2208	1	3
SEQ ID NO: 5364	catcaatattgatcaattt	2081	2100	SEQ ID NO: 5441	aaattaaaaagtcttgatg	2319	2338	1	3
SEQ ID NO: 5365	taaagatagttatgattta	2086	2105	SEQ ID NO: 5442	taaaccaaaaacttggtta	2669	2908	1	3
SEQ ID NO: 5366	tattgatgaaatcattgaa	2231	2250	SEQ ID NO: 5443	ttcaaagacttaaaaaata	2601	2820	1	3
SEQ ID NO: 5367	atgatctacatttgtttat	2493	2512	SEQ ID NO: 5444	ataaagaaattaaagtcat	2555	2574	1	3
SEQ ID NO: 5368	agagacacatacagaatat	2519	2536	SEQ ID NO: 5445	atatattgtcagtgcctct	2977	2996	1	3
SEQ ID NO: 5369	gacacatacagaatataga	2652	2671	SEQ ID NO: 5446	tctaaattcagttcttgtc	2798	2817	1	3

TABLE 11 — Selected palindromic sequences from human glucose-6-phosphatase

		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 5447	ggccattccagaagggaag	301	320	SEQ ID NO: 5471	cttccgttctgtaatggcc	598	617	1	6
SEQ ID NO: 5448	tgccatctcgagagttcca	831	850	SEQ ID NO: 5472	tggaactctctccatggca	859	878	1	6
SEQ ID NO: 5449	catgtcaaacactttgtta	879	898	SEQ ID NO: 5473	taacaaattccttgacatg	1019	1038	1	6
SEQ ID NO: 5450	tttgttataaatcttattg	376	395	SEQ ID NO: 5474	caataagatcaatagcaaa	1171	1190	1	5
SEQ ID NO: 5451	tctggaaaaagggtcatga	1478	1497	SEQ ID NO: 5475	tccatgtcccatttacaga	2057	2076	1	5
SEQ ID NO: 5452	cagctcttgttcaggtcca	2	21	SEQ ID NO: 5476	tggacctgcaccaaagctg	123	142	1	4
SEQ ID NO: 5453	ggaggttccccagctctgc	13	32	SEQ ID NO: 5477	gcagccctgggaaaactcc	66	85	1	4
SEQ ID NO: 5454	ctgttttgaagactctcca	51	70	SEQ ID NO: 5478	tggagggtagtcataacag	1448	1467	1	4
SEQ ID NO: 5455	agtggctgaaacgtgtgca	108	127	SEQ ID NO: 5479	tgcagagctttctgccact	2018	2037	1	4
SEQ ID NO: 5456	ccaaaatagaagggaatct	155	174	SEQ ID NO: 5480	agattcctttctttttcaa	1076	1095	1	4
SEQ ID NO: 5457	tgaagagaagattgaattt	156	175	SEQ ID NO: 5481	aaattctcttttattttca	1075	1094	1	4
SEQ ID NO: 5458	agtggtggcaacaccagca	177	196	SEQ ID NO: 5482	tgctagtgaggccaacact	1549	1568	1	4
SEQ ID NO: 5459	aaggctccacaagtcatca	325	344	SEQ ID NO: 5483	tgatgatatctggaacctt	1868	1887	1	4
SEQ ID NO: 5460	gtccgccaggtttctagca	1064	1083	SEQ ID NO: 5484	tgctaagaaccttactgac	2034	2053	1	4
SEQ ID NO: 5461	tgatatctggaaccttgga	1111	1130	SEQ ID NO: 5485	ttcactgttcctgaaatca	1659	1678	1	4
SEQ ID NO: 5462	gtcaagttgagcaatttct	1237	1256	SEQ ID NO: 5486	agaaaaggcacaccttgac	2201	2220	1	4
SEQ ID NO: 5463	atccagatggaaaagggaa	1545	1564	SEQ ID NO: 5487	ttccaatttccctgtggat	2121	2140	1	4
SEQ ID NO: 5464	atttgtttgtcaaagaagt	37	56	SEQ ID NO: 5488	acttcagagagaaatacat	724	743	1	3
SEQ ID NO: 5465	ctggaaaatgtcagcctgg	59	78	SEQ ID NO: 5489	ccagacttcagttaccagc	1124	1143	1	3
SEQ ID NO: 5466	accaggaggttcttcttca	132	151	SEQ ID NO: 5490	tgaagtgtgatctcctggt	1911	1930	1	3
SEQ ID NO: 5467	aaagaagttctgaaaagat	148	167	SEQ ID NO: 5491	attccatcacaaaatcctt	1748	1767	1	3
SEQ ID NO: 5468	gctacagcttatggctcca	194	213	SEQ ID NO: 5492	tggatctaaatgcagtagc	357	376	1	3
SEQ ID NO: 5469	atcaatatgatcaatttgt	296	315	SEQ ID NO: 5493	caaagaaatcaagattgat	518	537	1	3
SEQ ID NO: 5470	gaattatctttaaaacatt	966	985	SEQ ID NO: 5494	atgtggacaaatataccgg	1833	1852	1	3
TALBE 12
Selected palindromic sequences from human B-catein
		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 5495	ggccattccagaagggaag	70	89	SEQ ID NO: 5542	cttccgttctgtaatggcc	2152	2171	1	5
SEQ ID NO: 5496	tgccatctcgagagttcca	304	323	SEQ ID NO: 5543	tggaactctctccatggca	2387	2406	1	5
SEQ ID NO: 5497	catgtcaaacactttgtta	328	347	SEQ ID NO: 5544	taacaaattccttgacatg	985	1004	1	5
SEQ ID NO: 5498	tttgttataaatcttattg	334	353	SEQ ID NO: 5545	caataagatcaatagcaaa	791	810	1	5
SEQ ID NO: 5499	tctggaaaaagggtcatga	473	492	SEQ ID NO: 5546	tccatgtcccatttacaga	2037	2056	1	5
SEQ ID NO: 5500	cagctcttgttcaggtcca	677	696	SEQ ID NO: 5547	tggacctgcaccaaagctg	2539	2558	1	5
SEQ ID NO: 5501	ggaggttccccagctctgc	678	697	SEQ ID NO: 5548	gcagccctgggaaaactcc	2538	2557	1	5
SEQ ID NO: 5502	ctgttttgaagactctcca	383	402	SEQ ID NO: 5549	tggagggtagtcataacag	2176	2195	2	4
SEQ ID NO: 5503	agtggctgaaacgtgtgca	1839	1858	SEQ ID NO: 5550	tgcagagctttctgccact	2451	2470	2	4
SEQ ID NO: 5504	ccaaaatagaagggaatct	143	162	SEQ ID NO: 5551	agattcctttctttttcaa	1929	1946	1	4
SEQ ID NO: 5505	tgaagagaagattgaattt	151	170	SEQ ID NO: 5552	aaattctcttttattttca	1680	1699	1	4
SEQ ID NO: 5506	agtggtggcaacaccagca	260	279	SEQ ID NO: 5553	tgctagtgaggccaacact	2494	2513	1	4
SEQ ID NO: 5507	aaggctccacaagtcatca	383	402	SEQ ID NO: 5554	tgatgatatctggaacctt	1652	1671	1	4
SEQ ID NO: 5508	gtccgccaggtttctagca	384	403	SEQ ID NO: 5555	tgctaagaaccttactgac	2175	2194	1	4
SEQ ID NO: 5509	tgatatctggaaccttgga	454	473	SEQ ID NO: 5556	ttcactgttcctgaaatca	2517	2536	1	4
SEQ ID NO: 5510	gtcaagttgagcaatttct	563	582	SEQ ID NO: 5557	agaaaaggcacaccttgac	1652	1671	1	4
SEQ ID NO: 5511	atccagatggaaaagggaa	623	642	SEQ ID NO: 5558	ttccaatttccctgtggat	1820	1839	1	5
SEQ ID NO: 5512	atttgtttgtcaaagaagt	718	737	SEQ ID NO: 5559	acttcagagagaaatacat	1125	1145	1	5
SEQ ID NO: 5513	ctggaaaatgtcagcctgg	915	934	SEQ ID NO: 5560	ccagacttcagttaccagc	2029	2048	1	5
SEQ ID NO: 5514	accaggaggttcttcttca	1291	1310	SEQ ID NO: 5561	tgaagtgtgatctcctggt	2502	2521	1	5
SEQ ID NO: 5515	aaagaagttctgaaaagat	1356	1375	SEQ ID NO: 5562	attccatcacaaaatcctt	2162	2181	1	4
SEQ ID NO: 5516	gctacagcttatggctcca	1549	1568	SEQ ID NO: 5563	tggatctaaatgcagtagc	1605	1624	1	4
SEQ ID NO: 5517	atcaatatgatcaatttgt	2107	2126	SEQ ID NO: 5564	caaagaaatcaagattgat	2134	2153	1	4
SEQ ID NO: 5518	gaattatctttaaaacatt	245	264	SEQ ID NO: 5565	atgtggacaaatataccgg	828	847	2	3
SEQ ID NO: 5519	cgaggcccgcgctgctggc	4	23	SEQ ID NO: 5566	gccagaagtgagatcctcg	2420	2439	1	3
SEQ ID NO: 5520	acaactatgaggctgagag	60	79	SEQ ID NO: 5567	ctctgagcaacaaattttt	359	378	1	3
SEQ ID NO: 5521	gctgagagttccagtggag	174	193	SEQ ID NO: 5568	ctccatggcaaatgtcagg	437	456	1	3
SEQ ID NO: 5522	tgaagaaaaaccaagaact	213	232	SEQ ID NO: 5569	gagtcattgaggttcttca	2500	2519	1	3
SEQ ID NO: 5523	cctacttacatcctgaaca	245	264	SEQ ID NO: 5570	tgttcataagggaggtagg	275	294	1	3
SEQ ID NO: 5524	ctacttacatcctgaacat	323	342	SEQ ID NO: 5571	atgttcataagggaggtag	1579	1598	1	3
SEQ ID NO: 5525	gagacagaagccaagagcc	369	368	SEQ ID NO: 5572	gcttggttttgcccagtct	1972	1991	1	3
SEQ ID NO: 5526	cactcactttaccgtcaag	424	443	SEQ ID NO: 5573	cttgaacaccaaagtcact	2514	2533	1	3
SEQ ID NO: 5527	ctgatcagcagcagccagt	469	488	SEQ ID NO: 5574	actgggaagtgcttatcag	1276	1295	1	3
SEQ ID NO: 5528	actggacgctaagaggaag	516	535	SEQ ID NO: 5575	cttccccaaagagaggagt	1271	1290	1	3
SEQ ID NO: 5529	agaggaagcatgtggcaga	646	664	SEQ ID NO: 5576	tctggcatttactttctct	1430	1449	1	3
SEQ ID NO: 5530	tgaagactctccaggaact	646	665	SEQ ID NO: 5577	agttgaaggagactattca	1429	1448	1	3
SEQ ID NO: 5531	ctctgagcaaaatatccag	846	865	SEQ ID NO: 5578	ctggttactgagctgagag	1743	1762	1	3
SEQ ID NO: 5532	atgaagcagtcacatctct	974	993	SEQ ID NO: 5579	agagctgccagtcttcatt	2542	2561	1	3
SEQ ID NO: 5533	ttgccacagctgattgagg	1222	1241	SEQ ID NO: 5580	cctcctacagtggtggcaa	2037	2056	1	3
SEQ ID NO: 5534	agctgattgaggtgtccag	1347	1366	SEQ ID NO: 5581	ctggattccacatgcagct	1743	1762	1	3
SEQ ID NO: 5535	tgctccactcacatcctcc	1435	1454	SEQ ID NO: 5582	ggaggctttaagttcagca	1562	1581	1	3
SEQ ID NO: 5536	tgaaacgtgtgcatgtcaa	1839	1858	SEQ ID NO: 5583	ttgggagagacaagtttca	2370	2389	1	3
SEQ ID NO: 5537	gacattgctaattacctga	1852	1871	SEQ ID NO: 5584	tcagaagctaagcaatgtc	2053	2072	1	3
SEQ ID NO: 5538	ttcttcttcagactttcct	1915	1934	SEQ ID NO: 5585	aggagagtccaaatttaga	2065	2074	1	3
SEQ ID NO: 5539	ccaatatcttgaaactcag	1962	1981	SEQ ID NO: 5586	tctgaattcattcaattgg	2553	2572	1	3
SEQ ID NO: 5540	aaagttagtgaaaagaagt	2084	2103	SEQ ID NO: 5587	aactaccctcactgccttt	2114	2133	1	3
SEQ ID NO: 5541	aagttagtgaaagaagttc	2247	2256	SEQ ID NO: 5588	gaacctctggcattttact	2421	2440	1	3

TABLE 13 — Selected palindromic sequences from human hepatitis C virus (HCV)

		Start	End			Start	End
	Source	Index	Index		Match	Index	Index	#	B
SEQ ID NO: 5589	ggccattccagaagggaag	5314	5333	SEQ ID NO: 6135	cttccgttctgtaatggcc	6196	6215	1	9
SEQ ID NO: 5590	tgccatctcgagagttcca	1682	1701	SEQ ID NO: 6136	tggaactctctccatggca	8202	8221	1	8
SEQ ID NO: 5591	catgtcaaacactttgtta	1049	1068	SEQ ID NO: 6137	taacaaattccttgacatg	6151	6170	1	7
SEQ ID NO: 5592	ttgttataaatcttaattg	1352	1371	SEQ ID NO: 6138	caataagatcaatagcaaa	6053	6072	1	7
SEQ ID NO: 5593	tctggaaaagggtcatgga	2048	2067	SEQ ID NO: 6139	tccatgtcccatttacaga	6871	6890	1	7
SEQ ID NO: 5594	cagctcttgttcaggtcca	2049	2088	SEQ ID NO: 6140	tggacctgcaccaaagctg	6870	6889	1	7
SEQ ID NO: 5595	ggaggttccccagactcgc	5556	5575	SEQ ID NO: 6141	gcagccctgggaaaactcc	9254	9273	1	7
SEQ ID NO: 5596	ctgttttgaagactctcca	5744	5763	SEQ ID NO: 6142	tggagggtagtcataacag	9291	6310	1	7
SEQ ID NO: 5597	agtggctgaaacgtgtgca	6189	6206	SEQ ID NO: 6143	tgcagagctttctgcccac	5832	5851	1	7
SEQ ID NO: 5598	ccaaaatagaagggaatct	6249	6268	SEQ ID NO: 6144	agattcctttgccttttgg	6830	6649	1	7
SEQ ID NO: 5599	tgaagagaagattgaattt	6250	6269	SEQ ID NO: 6145	aaattctcttttcttttca	6829	6848	1	7
SEQ ID NO: 5600	agtggtgccaataccagca	8216	8235	SEQ ID NO: 6146	tgctagtgaggccaacact	8634	8653	1	7
SEQ ID NO: 5601	aaggctccacaagtcatca	1430	1449	SEQ ID NO: 6147	tgatgatatctggaacctt	9019	9038	2	6
SEQ ID NO: 5602	gtcagccaggtttatagca	370	389	SEQ ID NO: 6148	tgctaagaaccttactgac	4115	4134	1	6
SEQ ID NO: 5603	tgatatctggaaccttgaa	419	436	SEQ ID NO: 6149	ttcactgttcctgaaatca	5734	5753	1	6
SEQ ID NO: 5604	gtcaagttgagcaatttct	584	603	SEQ ID NO: 6150	agaaaaggcacaccttgac	6374	6393	1	6
SEQ ID NO: 5605	atccagatggaaaagggaa	1265	1284	SEQ ID NO: 6151	ttccccatttccctgtggt	8465	8484	1	6
SEQ ID NO: 5606	atttgtttgtcaaagaagt	1508	1527	SEQ ID NO: 6152	acttcagagaaatacaaat	4759	4778	1	6
SEQ ID NO: 5607	ctggaaaatgtcagcctgg	1624	1643	SEQ ID NO: 6153	ccagacttccgtttcccag	2594	2613	1	6
SEQ ID NO: 5608	accaggaggttcttcttca	1897	1916	SEQ ID NO: 6154	tgaagtgtagtctcctggt	4115	4134	1	6
SEQ ID NO: 5609	aaagaagttctgaaagaat	2032	2051	SEQ ID NO: 6155	attccatcacaatcctttt	6537	8556	1	6
SEQ ID NO: 5610	gctacagcttatggctcca	2238	2257	SEQ ID NO: 6156	tggatctaaatgcagtagc	7610	7629	1	6
SEQ ID NO: 5611	atccatcttgatcaatttg	2288	2307	SEQ ID NO: 6157	caaagaagtcaagattgat	9207	9220	1	6
SEQ ID NO: 5612	gaattatcttttaaaacat	2613	2632	SEQ ID NO: 6158	atgtgttaaccaaatattc	6827	6846	1	6
SEQ ID NO: 5613	cgaggcccgcgctgctggc	2817	2836	SEQ ID NO: 6159	gccagaagtgagatcctcg	7763	7762	1	6
SEQ ID NO: 5614	acaactatgaggctgagag	2997	3016	SEQ ID NO: 6160	ctctgagcaacaaatttgt	5454	5473	1	6
SEQ ID NO: 5615	gctgagagttccagtggcg	3014	3033	SEQ ID NO: 6161	ctccatggcaaatgtcagc	5518	5537	1	6
SEQ ID NO: 5616	tgaagaaaaccaagaactc	3314	3333	SEQ ID NO: 6162	gagtcattgcggttcttca	7641	7680	1	6
SEQ ID NO: 5617	cctcttacatcctgaacaa	3324	3343	SEQ ID NO: 6163	tgttcataagggaggtcgg	3915	3934	1	6
SEQ ID NO: 5618	ctacttacatcctgaacat	3874	3893	SEQ ID NO: 6164	atgttcataagggcggtag	7439	7458	1	6
SEQ ID NO: 5619	gagacagaagaagccaagc	3875	3894	SEQ ID NO: 6165	gcttggttttgccagtctc	7438	7457	1	6
SEQ ID NO: 5620	cactcactttaccgtcaag	3876	3895	SEQ ID NO: 6166	cttgaacacaaagtcagtg	7631	7600	1	6
SEQ ID NO: 5621	ctgatcagcagcagccagt	3961	3980	SEQ ID NO: 6167	actgggaagtgcttatcag	7252	7271	1	6
SEQ ID NO: 5622	actggccgctaagaggaag	4174	4193	SEQ ID NO: 6168	cttccccaaagagaccagt	8155	8174	1	6
SEQ ID NO: 5623	agaggaagcatgtggcaga	4245	4264	SEQ ID NO: 6169	tctggcatttactttctct	4853	4872	1	6
SEQ ID NO: 5624	tgaagactctccaggaact	4501	4520	SEQ ID NO: 6170	agttgaaggagactattca	8334	8353	1	6
SEQ ID NO: 5625	ctctgagcaaaatatccag	5517	5536	SEQ ID NO: 6171	ctggttactgagctgagag	3015	3034	1	6
SEQ ID NO: 5626	atgaagcagtcacatctct	7821	7840	SEQ ID NO: 6172	agagctgccagtccttcat	8564	8583	1	6
SEQ ID NO: 5627	ttgccacagctgattgagg	767	788	SEQ ID NO: 6173	cctcctacagtggtggcaa	3674	3693	2	5
SEQ ID NO: 5628	agctgattgaggtgtccag	646	665	SEQ ID NO: 6174	ctggattccacatgcagct	7519	7538	2	5
SEQ ID NO: 5629	tgctccactcacatcctcc	750	789	SEQ ID NO: 6175	ggaggctttaagttcagca	7923	7942	2	5
SEQ ID NO: 5630	tgaagcgtgtgcatgccaa	767	786	SEQ ID NO: 6176	ttgggagagacaagtttca	1363	1402	2	5
SEQ ID NO: 5631	gacattgctaattacctga	1222	1241	SEQ ID NO: 6177	tcagaagctaagcaatgtc	9147	9166	2	5
SEQ ID NO: 5632	ttcttcttcagactttcct	178	197	SEQ ID NO: 6178	aggagagtccaaattagaa	7341	7360	1	5
SEQ ID NO: 5633	ccaatatcttgaactcaga	179	196	SEQ ID NO: 6179	tctgaattcattcaattgg	7340	7359	1	5
SEQ ID NO: 5634	aaagttggtgaaagaagtt	368	367	SEQ ID NO: 6180	aactaccctcactgccttt	9443	9462	1	5
SEQ ID NO: 5635	aagtagtgaaagaaagttc	385	404	SEQ ID NO: 6181	gaacctctggcatttactt	4100	4119	1	5
SEQ ID NO: 5636	aaagaagttctgaaagaat	418	437	SEQ ID NO: 6182	attctctggtaactacttt	4754	4773	1	5
SEQ ID NO: 5637	tttggctataccaaagatg	444	463	SEQ ID NO: 6183	catcttaggcactgacaaa	1267	1285	1	5
SEQ ID NO: 5638	tgttgagaagctgattaaa	450	469	SEQ ID NO: 6184	tttagccatcggctcaaca	3164	3183	1	5
SEQ ID NO: 5639	caggaagggctcaaagaat	460	479	SEQ ID NO: 6185	attcctttaacaggttcag	9296	9315	1	5
SEQ ID NO: 5640	aggaagggctcaaagaatg	657	676	SEQ ID NO: 6186	cattcctttaacaattcct	5783	5802	1	5
SEQ ID NO: 5641	gaagggctcaaagaatgac	658	677	SEQ ID NO: 6187	gtcagtcttcaggctcttc	5782	5801	1	5
SEQ ID NO: 5642	caaagaatgacttttttct	659	678	SEQ ID NO: 6188	agaaggatggcattttttg	5781	5800	1	5
SEQ ID NO: 5643	catggagaatgcctttgaa	715	734	SEQ ID NO: 6189	ttcagagccaaagtccatg	3774	3793	1	5
SEQ ID NO: 5644	ggagccacggatggagtaa	718	737	SEQ ID NO: 6190	ttatccaacgccagctccc	6020	6039	1	5
SEQ ID NO: 5645	tcattccttccccaaagag	751	770	SEQ ID NO: 6191	ctctcggggcatctatgaa	7922	7941	1	5
SEQ ID NO: 5646	acctatgagctccagagag	776	795	SEQ ID NO: 6192	ctctcaagaccacagaggt	6110	5129	1	5
SEQ ID NO: 5647	gggcaaacgtcttacagaa	943	962	SEQ ID NO: 6193	tctgaaagaaacgtgtgca	6498	6517	1	5
SEQ ID NO: 5648	accctggacattcagaaca	1019	1038	SEQ ID NO: 6194	tgttgctaaggttcagggt	1495	1514	1	5
SEQ ID NO: 5649	atgggcgacctaagttgtg	1020	1039	SEQ ID NO: 6195	cacaaattagttcaccatt	1494	1513	1	5
SEQ ID NO: 5650	gatgaagagagagttgagt	1021	1040	SEQ ID NO: 6196	attccagcttccccacatc	1493	1512	1	5
SEQ ID NO: 5651	caatgtagataccaaaaaa	1085	1104	SEQ ID NO: 6197	tttttggaaatgccattgc	4323	4342	1	5
SEQ ID NO: 5652	gtagataccaaaaaaatga	1098	1117	SEQ ID NO: 6198	tcatgtgatgggtctcacc	8092	8111	1	5
SEQ ID NO: 5653	gcttcagttcatttggact	1112	1131	SEQ ID NO: 6199	agtcaagaaggacttaagc	4576	4595	1	5
SEQ ID NO: 5654	tttgtttgttcaaagaagt	1128	1147	SEQ ID NO: 6200	gactcagagaaatacaaac	9164	9183	1	5
SEQ ID NO: 5655	ttgtttgtcaaagaagtca	1215	1234	SEQ ID NO: 6201	tgactcagagaaatacaac	6374	6393	1	5
SEQ ID NO: 5656	tggcaatgggaaactcgct	1265	1265	SEQ ID NO: 6202	agcgagaatcaccctgcca	6340	6359	1	5
SEQ ID NO: 5657	aacctctggcatttacttt	1331	1350	SEQ ID NO: 6203	aaaggagatgtcaagggtt	6048	6065	1	5
SEQ ID NO: 5658	catttactttctctcatga	1558	1577	SEQ ID NO: 6204	tcatttgaaagaataaatg	5838	5857	1	5
SEQ ID NO: 5659	aaagtcagtgccctgctta	1619	1638	SEQ ID NO: 6205	taagaacctactgaacttt	2313	2332	1	5
SEQ ID NO: 5660	tcccattttttgagacctt	1641	1660	SEQ ID NO: 6206	aaggacttcaggaatggga	8609	8628	1	5
SEQ ID NO: 5661	catcaatattgatcaattt	1685	1704	SEQ ID NO: 6207	aatattaaaagtcttgatg	4257	4276	1	5
SEQ ID NO: 5662	taaagatagttatgattta	1738	1757	SEQ ID NO: 6208	taaaccaaaacttggttta	5084	5103	1	5
SEQ ID NO: 5663	tattgatgaaatcattgaa	1894	1913	SEQ ID NO: 6209	ttcaaagacttaaaacaat	6343	6362	1	5
SEQ ID NO: 5664	atgatctacatttgtttat	2026	2045	SEQ ID NO: 6210	ataaagaaattgaatcaat	6919	6938	1	5
SEQ ID NO: 5665	agagacacatacagaatat	2030	2049	SEQ ID NO: 6211	atatattgtcagtgcctct	4202	4221	1	5
SEQ ID NO: 5666	gacacatacagaatataga	2101	2120	SEQ ID NO: 6212	tctaaattcagttcttgtc	3886	3905	1	5
SEQ ID NO: 5667	agcatgtcaaacatttgtc	2115	2134	SEQ ID NO: 6213	acaaagtcagtgccctgct	3137	3156	1	5
SEQ ID NO: 5668	ttttagaggaaaccaaggc	2235	2254	SEQ ID NO: 6214	cctttgtgtacaccccaaa	4011	4030	1	5
SEQ ID NO: 5669	ttttagaggaaaccaaggc	2354	2373	SEQ ID NO: 6215	gcctttgtgtacaccaaaa	3400	3419	1	5
SEQ ID NO: 5670	ggaagatagacttcctgaa	2474	2493	SEQ ID NO: 6216	ttcagaaatactgtttccg	8861	8880	1	5
SEQ ID NO: 5671	cactgtttctgagtcccag	2666	2585	SEQ ID NO: 6217	ctgggacctaccaagagtg	7834	7853	1	5
SEQ ID NO: 5672	cacaaatcctttggctgtg	2698	2717	SEQ ID NO: 6218	cacatttcaggattgtggg	7865	7884	1	5
SEQ ID NO: 5673	ttcctggatacactgttcc	2699	2718	SEQ ID NO: 6219	ggaactgttgactcaggaa	7864	7883	1	5
SEQ ID NO: 5674	gaaatctcaagctttctat	2700	2719	SEQ ID NO: 6220	agagccaggtccgagcttc	7863	7882	1	5
SEQ ID NO: 5675	tttcttcatcttcatctgt	2913	2932	SEQ ID NO: 6221	acagctgaaagagatgaaa	6402	6421	1	5
SEQ ID NO: 5676	tctaccgctaaaggagcag	2928	2947	SEQ ID NO: 6222	ctgcacgcttgaggtagaa	9376	9395	1	5
SEQ ID NO: 5677	ctaccgtcaaaggagcagt	3401	3420	SEQ ID NO: 6223	actgcacgctttgaggtag	8066	8085	1	5
SEQ ID NO: 5678	agggcctctttttcaccaa	3574	3593	SEQ ID NO: 6224	ttggccaggaagtggccct	4158	4177	1	5
SEQ ID NO: 5679	ttctccatccctgtaaaag	3575	3594	SEQ ID NO: 6225	cttttcaccaaacgggaga	4157	4176	1	5
SEQ ID NO: 5680	gaaaaccaaagcagattct	3695	3714	SEQ ID NO: 6226	ataaactgcaagattttct	8543	8562	1	5
SEQ ID NO: 5681	actcactcattgatttcct	3704	3723	SEQ ID NO: 6227	agaaaatcaggatctgagt	6342	8351	1	5
SEQ ID NO: 5682	taaactaatagatgtaatc	3705	3724	SEQ ID NO: 6228	gattaccaccagcggttta	6341	6360	1	5
SEQ ID NO: 5683	caaaagagcttcaggaagc	4325	4344	SEQ ID NO: 6229	cttcgtgaagaatattttg	4979	4998	1	5
SEQ ID NO: 5684	tggaataatgctcagtgtt	4347	4366	SEQ ID NO: 6230	aacacttcattgaattcca	5804	5823	1	5
SEQ ID NO: 5685	gatttgaaatccaaaggag	4361	4380	SEQ ID NO: 6231	cttcagagaatacaaaatc	7443	7462	1	5
SEQ ID NO: 5686	atttgaaatccaaagaagt	4485	4508	SEQ ID NO: 6232	acttcagcgaaatacaaat	5806	5825	1	5
SEQ ID NO: 5687	atcaacagccgcttctttg	5165	5184	SEQ ID NO: 6233	caaagaagtcaagattgat	7225	7244	1	5
SEQ ID NO: 5688	tgttttgaagactctccag	5322	5341	SEQ ID NO: 6234	ctggaaagttaaaacaaca	8765	8784	1	5
SEQ ID NO: 5689	cccttctgatagatgtggt	5390	5409	SEQ ID NO: 6235	accaaagctggcaccaggg	7713	7732	1	5
SEQ ID NO: 5690	tgagcaagtgaagaacttt	5515	5534	SEQ ID NO: 6236	aaagccattcagtctctca	6550	6569	1	5
SEQ ID NO: 5691	atttgaaatccaaagaagt	5592	5611	SEQ ID NO: 6237	acttttctaaacttgaaat	8176	8194	1	5
SEQ ID NO: 5692	atccaaagaagtcccggaa	5603	5622	SEQ ID NO: 6238	ttccggggaaacctgggat	7633	7652	1	5
SEQ ID NO: 5693	agagcctacctccgcatct	5739	5758	SEQ ID NO: 6239	agatggtacgttagcctct	5895	5914	1	5
SEQ ID NO: 5694	aatgcctttgaaactcccc	6306	6325	SEQ ID NO: 6240	tgggaactacaatttcatt	8363	8382	1	5
SEQ ID NO: 5695	gaagtccaaattccggatt	6456	6475	SEQ ID NO: 6241	aatcttcaatttattcttc	4158	4177	1	5
SEQ ID NO: 5696	tgcaagcagaagccagaag	6488	6507	SEQ ID NO: 6242	cttcaggtttccatcgtga	8708	8727	1	5
SEQ ID NO: 5697	gaagagaagattgaatttg	6565	6584	SEQ ID NO: 6243	caaaacctactgtctcctc	8500	8519	1	5
SEQ ID NO: 5698	atgctaaaggcacatatgg	6967	6986	SEQ ID NO: 6244	ccatatgaaagtcaagcat	3814	3833	1	5
SEQ ID NO: 5699	tccctcacctccacctctg	7078	7097	SEQ ID NO: 6245	cagattctcgatgagggat	7278	7297	1	5
SEQ ID NO: 5700	atttacagctctgacaagt	7138	7157	SEQ ID NO: 6246	acttttctaaacttgaatt	9326	9345	1	5
SEQ ID NO: 5701	aggagcctaccaaaattat	7202	7221	SEQ ID NO: 6247	attatgttgaaaacagttc	9150	9169	1	5
SEQ ID NO: 5702	aaagctgaagcacatcaat	7301	7320	SEQ ID NO: 6248	attgttgctcatctccttt	9162	9181	1	5
SEQ ID NO: 5703	ctgctggaaacaacgagaa	7520	7539	SEQ ID NO: 6249	tttctgataccacccacag	645	664	1	5
SEQ ID NO: 5704	ttgaaggaattcttgaaaa	7606	7625	SEQ ID NO: 6250	ttttaaaagaaaatctcaa	4809	4828	1	5
SEQ ID NO: 5705	gaagtaaaagaaatttggg	7626	7645	SEQ ID NO: 6251	caaaaacctactgtctggg	8996	9015	1	5
SEQ ID NO: 5706	tgaagaagatggcaaattt	7627	7646	SEQ ID NO: 6252	aaatgtcagctcttgttca	8995	9014	1	5
SEQ ID NO: 5707	aggatctgagttattttgc	8635	8664	SEQ ID NO: 6253	gcaagtcagcccagttcct	8920	8939	1	5
SEQ ID NO: 5708	gtgcccttctcggttgctg	27	46	SEQ ID NO: 6254	cagccattgacatgagcac	785	804	2	4
SEQ ID NO: 5709	ggcgctgcctgcgctgctg	1206	1225	SEQ ID NO: 6255	cagctccaccgactccgcc	8459	8478	2	4
SEQ ID NO: 5710	ctgcgctgctgctgctgct	1624	1643	SEQ ID NO: 6256	agcagaaggtgcaagcagg	5847	5806	2	4
SEQ ID NO: 5711	gctggtggcgggcgccagg	2238	2257	SEQ ID NO: 6257	cctggattccacatgcagc	7701	7720	2	4
SEQ ID NO: 5712	aagaggaaatgctggaaaa	3322	3341	SEQ ID NO: 6258	ttttcttcactactacttc	5795	5814	2	4
SEQ ID NO: 5713	ctggaaaatgtcagcctgg	3324	3343	SEQ ID NO: 6259	ccagacttccacatcccag	5076	5095	2	4
SEQ ID NO: 5714	tggagtccctgggactgct	8366	3385	SEQ ID NO: 6260	agcatgcctagtttctcca	6545	6564	2	4
SEQ ID NO: 5715	ggagctcccgggacttgtg	3674	3593	SEQ ID NO: 6261	cagtacgcctagtttctcc	7718	7737	2	4
SEQ ID NO: 5716	tgggactgctgattcaaga	6189	6208	SEQ ID NO: 6262	tcttccatcacttggaaac	6382	6401	2	4
SEQ ID NO: 5717	ctgctgattcaagaagtgc	6967	6986	SEQ ID NO: 6263	gcacaccttgacattgaca	9147	9166	2	4
SEQ ID NO: 5718	tgccaccaggatcaactgc	1	20	SEQ ID NO: 6264	gcaggctgaactggtggca	550	569	1	4
SEQ ID NO: 5719	gccaccaggatcaactgca	28	47	SEQ ID NO: 6265	tgcaggctgaactggttgc	784	803	1	4
SEQ ID NO: 5720	tgcaaggttgagctggagg	95	114	SEQ ID NO: 6266	cctccacctctgatctgca	2946	2965	1	4
SEQ ID NO: 5721	cataggttgagctggaggt	104	123	SEQ ID NO: 6267	aaccctacatggaaagctt	6403	6422	1	4
SEQ ID NO: 5722	ctctgcagtccatccctga	105	124	SEQ ID NO: 6268	tcaggaagcttctcaagag	6402	6421	1	4
SEQ ID NO: 5723	cagcttcatcctgaagacc	113	132	SEQ ID NO: 6269	ggtcttgagttaaatgctg	9255	9274	1	4
SEQ ID NO: 5724	gcttcatcctgaatgacag	118	137	SEQ ID NO: 6270	ctggacgctaagaggaagc	6368	6387	1	4
SEQ ID NO: 5725	tcatcctgaagaccagcca	121	140	SEQ ID NO: 6271	tggcatggcattatgatcc	598	617	1	4
SEQ ID NO: 5726	gaaaaccaagaactctgag	243	262	SEQ ID NO: 6272	ctcaaccttaatgattttc	3953	3972	1	4
SEQ ID NO: 5727	agaactctgaggagtttgc	251	270	SEQ ID NO: 6273	gcaagctctacagtattct	3656	3675	1	4
SEQ ID NO: 5728	tctgaggagtttgccgggt	299	318	SEQ ID NO: 6274	ctgcaggggatcccccaga	2139	2158	1	4
SEQ ID NO: 5729	tttgctgcagccatgtcca	306	325	SEQ ID NO: 6275	tggaagtgcagtggcaaat	671	690	1	4
SEQ ID NO: 5730	caagaggggcatcatttct	331	350	SEQ ID NO: 6276	agaataaatgacgttcttg	2437	2456	1	4
SEQ ID NO: 5731	tcactttaccgtcaagacg	412	431	SEQ ID NO: 6277	cgtctacactatagaaatt	997	1016	1	4
SEQ ID NO: 5732	tttaccgtcaagacgaagg	451	470	SEQ ID NO: 6278	tccttgaccatgttgataa	6549	6568	1	4
SEQ ID NO: 5733	cactggacgctaagaaaga	511	530	SEQ ID NO: 6279	ttccagaaagcagccagtg	3891	3910	1	4
SEQ ID NO: 5734	aggaagcatgtggcagaag	512	531	SEQ ID NO: 6280	cttcatacacattatcctt	3890	3909	1	4
SEQ ID NO: 5735	caaggagcaacacctcttc	526	547	SEQ ID NO: 6281	gaagtagtactgcatcttg	553	572	1	4
SEQ ID NO: 5736	acagactttgaaacttgaa	531	550	SEQ ID NO: 6282	ttcaatcttcaatgctgtt	5416	5437	1	4
SEQ ID NO: 5737	tgatgaagcagtcacatct	532	551	SEQ ID NO: 6283	agatttgaggattccatca	5417	5436	1	4
SEQ ID NO: 5738	agcagtcacatctctcttg	577	596	SEQ ID NO: 6284	caaggagaaaactgactgc	8067	8086	1	4
SEQ ID NO: 5739	cagccccatcactttacat	609	628	SEQ ID NO: 6285	tgtagtctcctggtgctgg	706	725	1	4
SEQ ID NO: 5740	ctccactcatccttcagtg	630	649	SEQ ID NO: 6286	caggagcttagtaatggag	8523	8542	1	4
SEQ ID NO: 5741	catgcccaccccctcctgg	661	680	SEQ ID NO: 6287	tcagatgagggaacacatg	4064	4083	1	4
SEQ ID NO: 5742	gagagatcttcaacatggc	662	681	SEQ ID NO: 6288	gccaccctggaaactctct	4063	4082	1	4
SEQ ID NO: 5743	tcaaacatggcgacgcatc	724	743	SEQ ID NO: 6289	tgatcccacctctcattga	6472	6491	1	4
SEQ ID NO: 5744	ccaccttgtatgcgctgag	725	744	SEQ ID NO: 6290	ctcagggatctgaggggtg	64171	6490	1	4
SEQ ID NO: 5745	gtcaaacaactatatcata	764	783	SEQ ID NO: 6291	tcttgagttaaatgctgac	7414	7433	1	4
SEQ ID NO: 5746	tggacattgctaattacct	792	811	SEQ ID NO: 6292	aggtatattcgaaagtcca	5188	5207	1	4
SEQ ID NO: 5747	ggacattgctaattacctg	858	887	SEQ ID NO: 6293	caggtatattcgaaagtcc	5863	5882	1	4
SEQ ID NO: 5748	ttctgcgggtcattggaaa	944	963	SEQ ID NO: 6294	tttcacactgcccaagaaa	6847	6866	1	4
SEQ ID NO: 5749	ccagaactcaagtcttcaa	983	1002	SEQ ID NO: 6295	ttgaagtgtgtctcctggc	5712	5731	1	4
SEQ ID NO: 5750	agtcttcaattcctgaagt	1096	1115	SEQ ID NO: 6296	catttctgattggtggact	4592	4611	1	4
SEQ ID NO: 5751	tgagcaagtgaagaaactt	1207	1226	SEQ ID NO: 6297	aaagtgccactttttacta	8793	8612	1	4
SEQ ID NO: 5752	agcaagtgaaagaacttgt	1222	1241	SEQ ID NO: 6298	acaaagtcagttgcctggt	3814	3833	1	4
SEQ ID NO: 5753	tctgaaaagaatctcaact	1293	1312	SEQ ID NO: 6299	aagtccataatggttcaga	5981	6000	1	4
SEQ ID NO: 5754	actgtcatggacttcagaa	1352	1381	SEQ ID NO: 6300	ttctgaatatattgtcagt	6279	6298	1	4
SEQ ID NO: 5755	acttgacccagcctcagcc	1365	1385	SEQ ID NO: 6301	ggctccccctgagagaagt	5560	5579	1	4
SEQ ID NO: 5756	tccaaataactaccttcct	1381	1400	SEQ ID NO: 6302	aggaagatagaagatggat	5127	5146	1	4
SEQ ID NO: 5757	actaccctcactgcctttg	1387	1406	SEQ ID NO: 6303	caaatttgtggagggtagt	8641	8660	1	4
SEQ ID NO: 5758	ttggatttgcttcagctga	1430	1449	SEQ ID NO: 6304	tcagtataaatacaccaaa	6846	6865	1	4
SEQ ID NO: 5759	ttggaagctcttgggaaat	1454	1473	SEQ ID NO: 6305	tcccgattcacgcttccaa	7665	7684	1	4
SEQ ID NO: 5760	ggaagctctttttgggaag	1500	1519	SEQ ID NO: 6306	cttcagaaagctaccttcc	5530	5549	1	4
SEQ ID NO: 5761	tttttcccagacagtgtca	1512	1531	SEQ ID NO: 6307	tgacctctctaagccaaat	2278	2297	1	4
SEQ ID NO: 5762	agacagtgtcaacaagctt	1564	1583	SEQ ID NO: 6308	agctggttttgcccagttt	8393	8412	1	4
SEQ ID NO: 5763	cttggctataccaaagatt	1586	1605	SEQ ID NO: 6309	atctcgtgtctaggaaaag	2132	2151	1	4
SEQ ID NO: 5764	caaagatgataaaacatgg	1587	1606	SEQ ID NO: 6310	ctcaaggataacgtgtttg	2131	2160	1	4
SEQ ID NO: 5765	gatatggtaaatggaataa	1749	1768	SEQ ID NO: 6311	tttacttagggttaatttg	4176	4195	1	4
SEQ ID NO: 5766	ggaataatgctcagtgttg	1764	1783	SEQ ID NO: 6312	caacacttacttgaattcc	9063	9062	1	4
SEQ ID NO: 5767	tttgaaatccaaagaaatc	1844	1863	SEQ ID NO: 6313	gacttcagagaaatacaat	8807	8836	1	4
SEQ ID NO: 5768	cagatgattggagaggtca	1850	1869	SEQ ID NO: 6314	tccaatttcctgtgggtgg	7949	7968	1	4
SEQ ID NO: 5769	agaatgacttttttcttca	1866	1885	SEQ ID NO: 6315	tgaccacacaaacagtgct	6887	6906	1	4
SEQ ID NO: 5770	agaatgactttttcttcac	1831	1900	SEQ ID NO: 6316	tgaagtccggcttctttct	2295	2314	1	4
SEQ ID NO: 5771	gaactcccactggagctgc	1972	1991	SEQ ID NO: 6317	cagctcaaccgtacagttc	9241	9260	1	4
SEQ ID NO: 5772	atatctaaggtggtccaca	1982	2001	SEQ ID NO: 6318	tgacttcagtgcagaatat	7287	7306	1	4
SEQ ID NO: 5773	gctgaagtttatcattcct	1983	2002	SEQ ID NO: 6319	tggccccgtttaccataga	7286	7305	1	4
SEQ ID NO: 5774	attccttcccccaaagaga	2043	2062	SEQ ID NO: 6320	aggaggctttaagttcagc	9376	9395	1	4
SEQ ID NO: 5775	ctcattgagaaacaggcag	2092	2111	SEQ ID NO: 6321	gtctcttcctccatggaat	5324	5343	1	4
SEQ ID NO: 5776	ctcattgagaacaggcagt	2093	2112	SEQ ID NO: 6322	actgactgcacgctttgag	5323	5342	1	4
SEQ ID NO: 5777	ttgagcagtattctgtcag	2100	2119	SEQ ID NO: 6323	gctgagaaagtgcttcctt	8616	8635	1	4
SEQ ID NO: 5778	accttgtccagtgaagtcc	2124	2143	SEQ ID NO: 6324	ggacggtactgtcccaggt	8201	8220	1	4
SEQ ID NO: 5779	cacgtgaagtccaaattcc	2142	2161	SEQ ID NO: 6325	ggaaggcagagtttactgg	3611	3630	1	4
SEQ ID NO: 5780	acattcagaaaagaaagcg	2287	2306	SEQ ID NO: 6326	atttcctaaagctggattg	8162	8161	1	4
SEQ ID NO: 5781	gaaaaatcaagggtgttat	2314	2333	SEQ ID NO: 6327	ataaactgcaaagattttt	3128	3147	1	4
SEQ ID NO: 5782	aaatcaagggtgttatttc	2317	2336	SEQ ID NO: 6328	gaaaacaatgcattagatt	3837	3858	1	4
SEQ ID NO: 5783	tggcattatgatgaagaga	2323	2342	SEQ ID NO: 6329	tctcccgtgtatatgccaa	6206	6225	1	4
SEQ ID NO: 5784	aagagaagattgaatttga	2383	2402	SEQ ID NO: 6330	tcaaaacctactgtctctt	6286	6305	1	4
SEQ ID NO: 5785	aaatgacttccaatttccc	2391	2410	SEQ ID NO: 6331	gggaactacaatttcattt	5564	5583	1	4
SEQ ID NO: 5786	atgacttccaattttccct	2419	2438	SEQ ID NO: 6332	caggctgattacgagtcat	3939	3948	1	4
SEQ ID NO: 5787	acttccaattttccctgtg	2460	2479	SEQ ID NO: 6333	ccacgaaaaatatggaagt	8875	8694	1	4
SEQ ID NO: 5788	agttgcaatgagctcatgg	2579	2598	SEQ ID NO: 6334	ccatcagttcagataaact	9241	9260	1	4
SEQ ID NO: 5789	tttgcaagaccacctcaat	2582	2601	SEQ ID NO: 6335	attgacctgtccattcaaa	5559	5578	1	4
SEQ ID NO: 5790	gaaggagttcaacctccag	2621	2640	SEQ ID NO: 6336	ctggaaatttgtcattccc	3214	3233	1	4
SEQ ID NO: 5791	acttccacatcccagaaaa	2786	2805	SEQ ID NO: 6337	ttttaacaaaaagtggtgg	7726	7745	1	4
SEQ ID NO: 5792	ctcttcttaaaaagcgatg	2824	2843	SEQ ID NO: 6338	catcactgccaaaggagag	4647	4666	1	4
SEQ ID NO: 5793	aaaagagagggcgggtcaa	2837	2856	SEQ ID NO: 6339	tgactactcagggaaattt	3539	3558	1	4
SEQ ID NO: 5794	ttcctttgccttttggtgg	2838	2857	SEQ ID NO: 6340	ccacaaacaatgaagggaa	3538	3557	1	4
SEQ ID NO: 5795	caagtctcggggattccat	2939	2958	SEQ ID NO: 6341	atggaaaaaaaacagggct	4327	4346	1	4
SEQ ID NO: 5796	aagtccctactttttacat	3201	3220	SEQ ID NO: 6342	atgggaagtataagaactt	8325	8344	1	4
SEQ ID NO: 5797	tgcctctcctgggtgtttc	3217	3236	SEQ ID NO: 6343	agaaaacaaccaaggcaaa	5221	5240	1	4
SEQ ID NO: 5798	accagcacagaccacccca	3233	3252	SEQ ID NO: 6344	tgaagtgtagtctcctggt	9338	9357	1	4
SEQ ID NO: 5799	ccagcacagaccatttcag	260	3279	SEQ ID NO: 6345	ctgaaatacaagcttctgg	7617	7636	1	4
SEQ ID NO: 5800	actatcatgtgatgggtct	3261	3280	SEQ ID NO: 6346	agacacctgatttttatat	7616	7635	1	4
SEQ ID NO: 5801	accacagatgtctgcttca	3297	3316	SEQ ID NO: 6347	tgaaggctgatttcttgtt	4804	4823	1	4
SEQ ID NO: 5802	ccacagatgtctgcttcag	3301	3320	SEQ ID NO: 6348	ctgagcaactttttttgtt	5794	5813	1	4
SEQ ID NO: 5803	tttggactccaaaaaagaa	316	3335	SEQ ID NO: 6349	tttctctcatgatacaaaa	3755	3774	1	4
SEQ ID NO: 5804	tcaaagaagtcaagattga	3378	3397	SEQ ID NO: 6350	tcaaggataacgtgtttga	4761	4780	1	4
SEQ ID NO: 5805	atgagaactacgagctgac	3499	3518	SEQ ID NO: 6351	gtcagatattggtgctcat	4222	4241	1	4
SEQ ID NO: 5806	ttaaatctgacaacattgg	3509	3528	SEQ ID NO: 6352	cattcatttgaaggggaac	7304	7323	1	4
SEQ ID NO: 5807	gaagtataagaactttgcc	3525	3644	SEQ ID NO: 6353	ggcaaatttgaaggacttc	7734	7753	1	4
SEQ ID NO: 5808	aagtataagaactttgcca	3626	3645	SEQ ID NO: 6354	tggcaaatttgaaggactt	7733	7752	1	4
SEQ ID NO: 5809	ttcttcagcctgtttctgg	3659	3678	SEQ ID NO: 6355	cagaatccagatacaagaa	9410	9429	1	4
SEQ ID NO: 5810	ctggatcactaaattccca	3660	3679	SEQ ID NO: 6356	tgggtcttccagaaggccg	9409	9426	1	4
SEQ ID NO: 5811	aaattaatagtggtgctca	3682	3701	SEQ ID NO: 6357	tgagaagccccagaatttt	7938	7957	1	4
SEQ ID NO: 5812	agtgcaacgaccaacttga	3711	3730	SEQ ID NO: 6358	tcaaattcctggattacac	7279	7298	1	4
SEQ ID NO: 5813	ctgggaagtgcttatcagg	3715	3734	SEQ ID NO: 6359	cctgaccttcacataccag	7826	7845	1	4
SEQ ID NO: 5814	gcaaaaacaaatttcaggg	3775	3791	SEQ ID NO: 6360	aagtcaaagaaattttgct	5682	5701	1	4
SEQ ID NO: 5815	aaaaaacatttcaacttca	3792	3811	SEQ ID NO: 6361	tgaagtaaaagaaattttt	6806	6825	1	4
SEQ ID NO: 5816	tcagtcaagaagaacttaa	3793	3812	SEQ ID NO: 6362	ttaaggaacttccattcta	8805	6824	1	4
SEQ ID NO: 5817	tcaaatgacatgatgggct	3822	3841	SEQ ID NO: 6363	agcccatcaatatattgaa	5585	5604	1	4
SEQ ID NO: 5818	cacaaaacagtctgaacaa	3928	3947	SEQ ID NO: 6364	tgttttcaactgccttgta	7717	7736	1	4
SEQ ID NO: 5819	tcttcaaaacttgaccaca	3947	3968	SEQ ID NO: 6365	tgttttcctattttcaaga	4504	4523	1	4
SEQ ID NO: 5820	caagtttttattaggcact	3948	3967	SEQ ID NO: 6366	agttatttgctaaagcttg	4826	4847	1	4
SEQ ID NO: 5821	tggtaactactttaccagg	4032	4051	SEQ ID NO: 6367	ctgttttagaggaaagccg	4993	5012	1	4
SEQ ID NO: 5822	aacagtgacctgaaataca	4136	4157	SEQ ID NO: 6368	tgtatagcaaattcctgtt	4377	4396	1	4
SEQ ID NO: 5823	gggaaactacggctagaac	4139	4158	SEQ ID NO: 6369	gttccttccagatttccgc	4376	4395	1	4
SEQ ID NO: 5824	aacaaacttgccatcctcc	4183	4202	SEQ ID NO: 6370	gagaacagctttcgtttgg	6119	6138	1	4
SEQ ID NO: 5825	tcagcaagctataaagcag	4193	4212	SEQ ID NO: 6371	ctgctaagaaccttactga	5833	5852	1	4
SEQ ID NO: 5826	gcagacactgttgctaagg	4215	4237	SEQ ID NO: 6372	cctttcaagcactgactgc	8668	8688	1	4
SEQ ID NO: 5827	tctggggagaacatactgg	4335	4354	SEQ ID NO: 6373	ccaggttttccacaaccga	9560	9579	1	4
SEQ ID NO: 5828	ttctctcatgattacaaag	4500	4519	SEQ ID NO: 6374	ctttttcaaccaacgggaa	5948	5967	1	4
SEQ ID NO: 5829	ctgagcagacaggcacctg	4526	4545	SEQ ID NO: 6375	caggaggctttaagttgtc	9589	9588	1	4
SEQ ID NO: 5830	caatttaaacaaacaagat	4577	4596	SEQ ID NO: 6376	attccttcctttacaattg	8682	8701	1	4
SEQ ID NO: 5831	tggacgaaactctggctac	4618	4637	SEQ ID NO: 6377	gtcagcccaggttcctcaa	8128	8147	1	4
SEQ ID NO: 5832	ctttttactcagtgaccca	4611	4830	SEQ ID NO: 6378	tgggctaaacgtttaattg	5992	6011	1	4
SEQ ID NO: 5833	tcattgtggctttagaaga	4857	4876	SEQ ID NO: 6379	atcttcataagttcaatga	5863	6882	1	4
SEQ ID NO: 5834	aaaaccaagatgttcactc	4869	4888	SEQ ID NO: 6380	gagtgaaatgctgtttttt	8067	8086	1	4
SEQ ID NO: 5835	aggaatcgacaaccattat	4922	4941	SEQ ID NO: 6381	taatgatttcaatgttcct	8662	8681	1	4
SEQ ID NO: 5836	tagttgtactggaaaaacg	4982	4981	SEQ ID NO: 6382	acgttagcctctatagact	9518	9537	1	4
SEQ ID NO: 5837	ggaaaactgatcagagaag	4983	4982	SEQ ID NO: 6383	cttttacaattcatttccc	9517	9536	1	4
SEQ ID NO: 5838	gaaaacgtacaggaccccc	5082	5101	SEQ ID NO: 6384	gctttctctttcacaaatg	6908	6927	1	4
SEQ ID NO: 5839	aaagctgaagcccatcaat	5140	5159	SEQ ID NO: 6385	atttgattagttggcaagg	7906	7925	1	4
SEQ ID NO: 5840	aagtctaagacctaggaga	5278	5297	SEQ ID NO: 6386	tattgatgttagagtgctt	9596	9615	1	4
SEQ ID NO: 5841	tgaagcacatcaatattga	5293	5312	SEQ ID NO: 6387	tcaaccttaatgattttaa	7885	7904	1	4
SEQ ID NO: 5842	atcaatattgatcaatttg	5294	5313	SEQ ID NO: 6388	caaagccatacatgatggg	7884	7903	1	4
SEQ ID NO: 5843	taatgattatctgaattca	5301	5320	SEQ ID NO: 6389	tgaaatcattgaaaattta	6676	6695	1	4
SEQ ID NO: 5844	gattatcgaattcattcat	5316	5336	SEQ ID NO: 6390	tgaaagtacgctgaagttc	7620	7639	1	4
SEQ ID NO: 5845	aattgggagagaagccttt	5383	5402	SEQ ID NO: 6391	aaacattcctttaccactt	6478	6497	1	4
SEQ ID NO: 5846	aaaatagctattgctaata	5429	5448	SEQ ID NO: 6392	tattgaaatatgatttttt	8383	8402	1	4
SEQ ID NO: 5847	aaaattaacaagtccgtat	5461	5480	SEQ ID NO: 6393	atcataggttgaggttttt	5924	5943	1	4
SEQ ID NO: 5848	ttgaaaaatatttgattta	5598	5617	SEQ ID NO: 6394	ttaatctttcataagtcaa	7240	7259	1	4
SEQ ID NO: 5849	agacatccagcacctagct	5645	5664	SEQ ID NO: 6395	tgggctaaacgtttaattg	8541	8580	1	4
SEQ ID NO: 5850	caattcatttgaaagaaat	5868	5687	SEQ ID NO: 6396	tattgaaatatgatttttt	5913	5932	1	4
SEQ ID NO: 5851	aggtttaaatggataattt	5736	5756	SEQ ID NO: 6397	aattgttgaaagaaaaact	5832	5851	1	4
SEQ ID NO: 5852	cagaagctaagcaatgtcc	5831	5850	SEQ ID NO: 6398	ggacaaggcccagaatctg	7948	7967	1	4
SEQ ID NO: 5853	taagattaaatatttactt	5877	5896	SEQ ID NO: 6399	aaagaaacctatggcctta	8142	8161	1	4
SEQ ID NO: 5854	aaagattactttgagaaat	5944	5963	SEQ ID NO: 6400	attcttaacgggattcctt	7463	7472	1	4
SEQ ID NO: 5855	aggaaatagttggattacc	6056	6075	SEQ ID NO: 6401	taaagccattcaggtctct	6969	6989	1	4
SEQ ID NO: 5856	attttgatgatgctggccc	6057	6076	SEQ ID NO: 6402	gacatgtttgaagggtgga	6968	6987	1	4
SEQ ID NO: 5857	gaattatcttttaaacatt	6074	6093	SEQ ID NO: 6403	tgggctaaacgtttaattg	7505	7524	1	4
SEQ ID NO: 5858	ttaccaccagtttgaagtt	6104	6123	SEQ ID NO: 6404	tattgaaatatgatttttt	2751	2770	1	4
SEQ ID NO: 5859	ttgcagtgtatcggacaga	6117	6136	SEQ ID NO: 6405	agcttgtttgccagtgggc	7531	7550	1	4
SEQ ID NO: 5860	cattcagaaccagggaaga	6199	6218	SEQ ID NO: 6406	tgaaatcattgaaaattta	7130	7149	1	4
SEQ ID NO: 5861	acaaactgtttatagtccc	6240	6259	SEQ ID NO: 6407	caggaggctttaagttgtc	7444	7463	1	4
SEQ ID NO: 5862	ggatttcaatacatttcac	6314	6333	SEQ ID NO: 6408	agcttgtttgccagtgggc	8541	8560	1	4
SEQ ID NO: 5863	ttgtagaaatgaaagtaaa	6338	6357	SEQ ID NO: 6409	taaagccattcaggtctct	7804	7823	1	4
SEQ ID NO: 5864	ctgaacagtgagctgcagt	6538	6557	SEQ ID NO: 6410	aattgttgaaagaaaaact	6613	6632	1	4
SEQ ID NO: 5865	aatccaatctcctctttgc	6616	6635	SEQ ID NO: 6411	taaagccattcaggtctct	7317	7336	1	4
SEQ ID NO: 5866	atttgattttcaagcaaag	6617	6636	SEQ ID NO: 6412	ttaatctttcataagtcaa	7455	7474	1	4
SEQ ID NO: 5867	ttttgattttcaagcaaat	6682	6701	SEQ ID NO: 6413	ggacaaggcccagaatctg	7059	7078	1	4
SEQ ID NO: 5868	tgatttcaagcaaatgcag	7152	7171	SEQ ID NO: 6414	attcttaacgggattcctt	8185	8184	1	4
SEQ ID NO: 5869	atgctgtttgggaaattgg	7239	7258	SEQ ID NO: 6415	aattgttgaaagaaaaact	5599	6618	1	4
SEQ ID NO: 5870	tgctgttttttggaaatgc	7295	7314	SEQ ID NO: 6416	agcttgtttgccagtgggc	8409	8428	1	4
SEQ ID NO: 5871	aaaaaaatacactggacgt	7327	7346	SEQ ID NO: 6417	ttaatctttcataagtcaa	9502	9521	1	4
SEQ ID NO: 5872	actggagcttagtaagggc	7328	7347	SEQ ID NO: 6418	agcttgtttgccagtgggc	9504	9523	1	4
SEQ ID NO: 5873	cttctggaaagagggtcat	7330	7349	SEQ ID NO: 6419	agcttgtttgccagtgggc	7770	7789	1	4
SEQ ID NO: 5874	ggaaaagggtcatggaaat	7351	7370	SEQ ID NO: 6420	attccttccttacaattgg	7390	7409	1	4
SEQ ID NO: 5875	gggcctgccccagatttct	7460	7479	SEQ ID NO: 6421	gagaacatttatggaggcc	7617	7638	1	4
SEQ ID NO: 5876	ttctcagataggggaacac	7667	7686	SEQ ID NO: 6422	gtgtcctcaagctgagggg	7686	7705	1	4
SEQ ID NO: 5877	gatgaaagaattaagggga	7792	7811	SEQ ID NO: 6423	attccagcttcccaactac	9238	9257	1	4
SEQ ID NO: 5878	cttggactgtcaaataagt	8028	8047	SEQ ID NO: 6424	cttatgggatttcctaagg	8931	8950	1	4
SEQ ID NO: 5879	gcatccacaaacaatggag	8029	8048	SEQ ID NO: 6425	cttcatcgttcatttgagt	8930	8949	1	4
SEQ ID NO: 5880	cacaaacaatgaagggaat	8204	8223	SEQ ID NO: 6426	cttatgggatttcctaagg	7860	7679	1	4
SEQ ID NO: 5881	ccaaaatttctctcggctt	8470	8489	SEQ ID NO: 6427	cttcatcgttcatttgagt	9091	9110	1	4
SEQ ID NO: 5882	caaattttctctgctaaaa	8766	8785	SEQ ID NO: 6428	ttccatcacaaatcctttg	9089	9108	1	4
SEQ ID NO: 5883	tctgctggaaacaacaacg	8876	8895	SEQ ID NO: 6429	tctcaaagagttacaacag	2459	2478	1	4
SEQ ID NO: 5884	cagaagctaagcaatgtcc	8976	8995	SEQ ID NO: 6430	tattgaaatatgatttttt	9052	9071	1	4
SEQ ID NO: 5885	taagattaaatatttactt	9090	9109	SEQ ID NO: 6431	aattgttgaaagaaaaact	8765	8784	1	4
SEQ ID NO: 5886	aaagattactttgagaaat	1624	1643	SEQ ID NO: 6432	ggacaaggcccagaatctg	5897	5916	3	3
SEQ ID NO: 5887	aggaaatagttggattacc	1	20	SEQ ID NO: 6433	aaagaaacctatggcctta	1391	1410	2	3
SEQ ID NO: 5888	attttgatgatgctggccc	520	539	SEQ ID NO: 6434	attcttaacgggattcctt	6918	6937	2	3
SEQ ID NO: 5889	gaattatcttttaaacatt	662	881	SEQ ID NO: 6435	taaagccattcaggtctct	4553	4572	2	3
SEQ ID NO: 5890	ttaccaccagtttgaagtt	983	1002	SEQ ID NO: 6436	gacatgtttgaagggtgga	8697	8716	2	3
SEQ ID NO: 5891	ttgcagtgtatcggacaga	1249	1266	SEQ ID NO: 6437	tgggctaaacgtttaattg	5330	5349	2	3
SEQ ID NO: 5892	cattcagaaccagggaaga	1637	1656	SEQ ID NO: 6438	tattgaaatatgatttttt	5599	5618	2	3
SEQ ID NO: 5893	acaaactgtttatagtccc	1830	1849	SEQ ID NO: 6439	agcttgtttgccagtgggc	8989	9008	2	3
SEQ ID NO: 5894	ggatttcaatacatttcac	2026	2045	SEQ ID NO: 6440	tgaaatcattgaaaattta	9156	9175	2	3
SEQ ID NO: 5895	ttgtagaaatgaaagtaaa	2264	2283	SEQ ID NO: 6441	caggaggctttaagttgtc	9405	9424	2	3
SEQ ID NO: 5896	ttaccaccagtttgaagtt	2383	2402	SEQ ID NO: 6442	agcttgtttgccagtgggc	7287	7288	2	3
SEQ ID NO: 5897	ttgcagtgtatcggacaga	2838	2857	SEQ ID NO: 6443	taaagccattcaggtctct	5654	5573	2	3
SEQ ID NO: 5898	cattcagaaccagggaaga	4489	4508	SEQ ID NO: 6444	aattgttgaaagaaaaact	8097	6116	2	3
SEQ ID NO: 5899	acaaactgtttatagtccc	5736	5755	SEQ ID NO: 6445	taaagccattcaggtctct	6382	6401	2	3
SEQ ID NO: 5900	ggatttcaatacatttcac	8635	8654	SEQ ID NO: 6446	ttaatctttcataagtcaa	9284	9303	2	3
SEQ ID NO: 5901	ttgtagaaatgaaagtaaa	24	43	SEQ ID NO: 6447	ggacaaggcccagaatctg	9111	9130	1	3
SEQ ID NO: 5902	ctgaacagtgagctgcagt	25	44	SEQ ID NO: 6448	attcttaacgggattcctt	5451	5470	1	3
SEQ ID NO: 5903	aatccaatctcctctttgc	27	48	SEQ ID NO: 6449	aattgttgaaagaaaaact	458	477	1	3
SEQ ID NO: 5904	atttgattttcaagcaaag	36	55	SEQ ID NO: 6450	agcttgtttgccagtgggc	809	826	1	3
SEQ ID NO: 5905	ttttgattttcaagcaaat	88	107	SEQ ID NO: 6451	ttaatctttcataagtcaa	5191	5210	1	3
SEQ ID NO: 5906	acaaactgtttatagtccc	100	119	SEQ ID NO: 6452	agcttgtttgccagtgggc	1879	1898	1	3
SEQ ID NO: 5907	ggatttcaatacatttcac	119	136	SEQ ID NO: 6453	agcttgtttgccagtgggc	645	664	1	3
SEQ ID NO: 5908	ttgtagaaatgaaagtaaa	131	150	SEQ ID NO: 6454	attccttccttacaattgg	8993	9012	1	3
SEQ ID NO: 5909	ttaccaccagtttgaagtt	134	153	SEQ ID NO: 6455	tgggctaaacgtttaattg	1046	1067	1	3
SEQ ID NO: 5910	ttgcagtgtatcggacaga	142	161	SEQ ID NO: 6456	tattgaaatatgatttttt	5038	5057	1	3
SEQ ID NO: 5911	cattcagaaccagggaaga	161	180	SEQ ID NO: 6457	agcttgtttgccagtgggc	1406	1425	1	3
SEQ ID NO: 5912	acaaactgtttatagtccc	188	207	SEQ ID NO: 6458	tgaaatcattgaaaattta	6304	6323	1	3
SEQ ID NO: 5913	ggatttcaatacatttcac	194	213	SEQ ID NO: 6459	caggaggctttaagttgtc	7134	7153	1	3
SEQ ID NO: 5914	ttgtagaaatgaaagtaaa	210	229	SEQ ID NO: 6460	agcttgtttgccagtgggc	7486	7505	1	3
SEQ ID NO: 5915	ccaaaatttctctcggctt	215	234	SEQ ID NO: 6461	taaagccattcaggtctct	1967	1986	1	3
SEQ ID NO: 5916	caaattttctctgctaaaa	216	235	SEQ ID NO: 6462	ggacaaggcccagaatctg	1966	1985	1	3
SEQ ID NO: 5917	tctgctggaaacaacaacg	221	240	SEQ ID NO: 6463	attcttaacgggattcctt	1891	1910	1	3
SEQ ID NO: 5918	cagaagctaagcaatgtcc	273	292	SEQ ID NO: 6464	aattgttgaaagaaaaact	7756	7775	1	3
SEQ ID NO: 5919	taagattaaatatttactt	274	293	SEQ ID NO: 6465	agcttgtttgccagtgggc	7755	7774	1	3
SEQ ID NO: 5920	aaagattactttgagaaat	282	301	SEQ ID NO: 6466	ttaatctttcataagtcaa	2385	2404	1	3
SEQ ID NO: 5921	aggaaatagttggattacc	291	310	SEQ ID NO: 6467	agcttgtttgccagtgggc	544	563	1	3
SEQ ID NO: 5922	attttgatgatgctggccc	307	326	SEQ ID NO: 6468	agcttgtttgccagtgggc	4780	4799	1	3
SEQ ID NO: 5923	gaattatcttttaaacatt	312	331	SEQ ID NO: 6469	attccttccttacaattgg	4122	4141	1	3
SEQ ID NO: 5924	ttaccaccagtttgaagtt	442	451	SEQ ID NO: 6470	agcttgtttgccagtgggc	1023	1042	1	3
SEQ ID NO: 5925	taagattaaatatttactt	443	462	SEQ ID NO: 6471	agcttgtttgccagtgggc	1022	1041	1	3
SEQ ID NO: 5926	aaagattactttgagaaat	445	464	SEQ ID NO: 6472	attccttccttacaattgg	8348	8367	1	3
SEQ ID NO: 5927	aggaaatagttggattacc	446	465	SEQ ID NO: 6473	tgggctaaacgtttaattg	8347	8366	1	3
SEQ ID NO: 5928	attttgatgatgctggccc	497	516	SEQ ID NO: 6474	tattgaaatatgatttttt	7922	7941	1	3
SEQ ID NO: 5929	gaattatcttttaaacatt	504	523	SEQ ID NO: 6475	agcttgtttgccagtgggc	3268	3287	1	3
SEQ ID NO: 5930	ttaccaccagtttgaagtt	505	524	SEQ ID NO: 6476	tgaaatcattgaaaattta	4749	4768	1	3
SEQ ID NO: 5931	ctgaaaaagttagtgaaag	520	539	SEQ ID NO: 6477	caggaggctttaagttgtc	774	793	1	3
SEQ ID NO: 5932	tttttcccagacagtgtca	527	546	SEQ ID NO: 6478	agcttgtttgccagtgggc	2252	2271	1	3
SEQ ID NO: 5933	ttttcccagacagtgtcca	554	573	SEQ ID NO: 6479	taaagccattcaggtctct	5020	5039	1	3
SEQ ID NO: 5934	cattcagaaccagggaaga	559	578	SEQ ID NO: 6480	cttcatcgttcatttgagt	4324	4343	1	3
SEQ ID NO: 5935	acaaactgtttatagtccc	561	580	SEQ ID NO: 6481	cttatgggatttcctaagg	4064	4083	1	3
SEQ ID NO: 5936	ggatttcaatacatttcac	564	583	SEQ ID NO: 6482	cttcatcgttcatttgagt	4162	4181	1	3
SEQ ID NO: 5937	ttgtagaaatgaaagtaaa	590	609	SEQ ID NO: 6483	ttccatcacaaatcctttg	727	746	1	3
SEQ ID NO: 5938	ccaaaatttctctcggctt	605	624	SEQ ID NO: 6484	tctcaaagagttacaacag	1180	1199	1	3
SEQ ID NO: 5939	caaattttctctgctaaaa	606	625	SEQ ID NO: 6485	tattgaaatatgatttttt	2370	2389	1	3
SEQ ID NO: 5940	tctgctggaaacaacaacg	622	641	SEQ ID NO: 6486	aattgttgaaagaaaaact	2459	2478	1	3
SEQ ID NO: 5941	cagaagctaagcaatgtcc	624	643	SEQ ID NO: 6487	ggacaaggcccagaatctg	4261	4280	1	3
SEQ ID NO: 5942	taagattaaatatttactt	633	652	SEQ ID NO: 6488	aaagaaacctatggcctta	3688	3707	1	3
SEQ ID NO: 5943	aaagattactttgagaaat	634	653	SEQ ID NO: 6489	attcttaacgggattcctt	3687	3706	1	3
SEQ ID NO: 5944	aggaaatagttggattacc	642	861	SEQ ID NO: 6490	taaagccattcaggtctct	2935	2954	1	3
SEQ ID NO: 5945	attttgatgatgctggccc	646	665	SEQ ID NO: 6491	gacatgtttgaagggtgga	4439	4458	1	3
SEQ ID NO: 5946	gaattatcttttaaacatt	711	730	SEQ ID NO: 6492	tgggctaaacgtttaattg	8839	8858	1	3
SEQ ID NO: 5947	ttaccaccagtttgaagtt	750	769	SEQ ID NO: 6493	tattgaaatatgatttttt	5625	5644	1	3
SEQ ID NO: 5948	taagattaaatatttactt	767	786	SEQ ID NO: 6494	agcttgtttgccagtgggc	898	817	1	3
SEQ ID NO: 5949	aaagattactttgagaaat	832	851	SEQ ID NO: 6495	tgaaatcattgaaaattta	3092	3111	1	3
SEQ ID NO: 5950	aggaaatagttggattacc	922	941	SEQ ID NO: 6496	caggaggctttaagttgtc	2751	2770	1	3
SEQ ID NO: 5951	attttgatgatgctggccc	968	987	SEQ ID NO: 6497	agcttgtttgccagtgggc	7512	7531	1	3
SEQ ID NO: 5952	aggtttaaatggataattt	1070	1080	SEQ ID NO: 6498	taaagccattcaggtctct	5763	5782	1	3
SEQ ID NO: 5953	cagaagctaagcaatgtcc	1080	1099	SEQ ID NO: 6499	aattgttgaaagaaaaact	5279	5298	1	3
SEQ ID NO: 5954	taagattaaatatttactt	1106	1125	SEQ ID NO: 6500	taaagccattcaggtctct	1888	1907	1	3
SEQ ID NO: 5955	aaagattactttgagaaat	1140	1159	SEQ ID NO: 6501	ttaatctttcataagtcaa	8921	8940	1	3
SEQ ID NO: 5956	aggaaatagttggattacc	1168	1187	SEQ ID NO: 6502	ggacaaggcccagaatctg	2615	2634	1	3
SEQ ID NO: 5957	attttgatgatgctggccc	1206	1225	SEQ ID NO: 6503	attcttaacgggattcctt	3779	3798	1	3
SEQ ID NO: 5958	gaattatcttttaaacatt	1208	1227	SEQ ID NO: 6504	aattgttgaaagaaaaact	9282	9301	1	3
SEQ ID NO: 5959	ttaccaccagtttgaagtt	1249	1268	SEQ ID NO: 6505	agcttgtttgccagtgggc	4809	44828	1	3
SEQ ID NO: 5960	ttgcagtgtatcggacaga	1254	1273	SEQ ID NO: 6506	ttaatctttcataagtcaa	3615	3634	1	3
SEQ ID NO: 5961	cattcagaaccagggaaga	1262	1281	SEQ ID NO: 6507	agcttgtttgccagtgggc	2766	2785	1	3
SEQ ID NO: 5962	acaaactgtttatagtccc	1270	1289	SEQ ID NO: 6508	tgaaatcattgaaaattta	1460	1479	1	3
SEQ ID NO: 5963	ggatttcaatacatttcac	1288	1307	SEQ ID NO: 6509	caggaggctttaagttgtc	3943	3962	1	3
SEQ ID NO: 5964	ttgtagaaatgaaagtaaa	1344	1363	SEQ ID NO: 6510	agcttgtttgccagtgggc	8593	8612	1	3
SEQ ID NO: 5965	ctgaacagtgagctgcagt	1360	1379	SEQ ID NO: 6511	taaagccattcaggtctct	5306	5325	1	3
SEQ ID NO: 5966	aatccaatctcctctttgc	1396	1415	SEQ ID NO: 6512	cttcatcgttcatttgagt	1695	1714	1	3
SEQ ID NO: 5967	atttgattttcaagcaaag	1408	1427	SEQ ID NO: 6513	cttatgggatttcctaagg	7865	7884	1	3
SEQ ID NO: 5968	ttttgattttcaagcaaat	1472	1491	SEQ ID NO: 6514	cttcatcgttcatttgagt	7886	7907	1	3
SEQ ID NO: 5969	tgatttcaagcaaatgcag	1492	1511	SEQ ID NO: 6515	ttccatcacaaatcctttg	4420	4439	1	3
SEQ ID NO: 5970	atgctgtttgggaaattgg	1504	1523	SEQ ID NO: 6516	tctcaaagagttacaacag	7440	7459	1	3
SEQ ID NO: 5971	tgctgttttttggaaatgc	1507	1526	SEQ ID NO: 6517	tattgaaatatgatttttt	3789	3808	1	3
SEQ ID NO: 5972	aaaaaaatacactggacgt	1517	1536	SEQ ID NO: 6518	aattgttgaaagaaaaact	1493	1512	1	3
SEQ ID NO: 5973	actggagcttagtaagggc	1571	1590	SEQ ID NO: 6519	ggacaaggcccagaatctg	5382	5401	1	3
SEQ ID NO: 5974	cttctggaaagagggtcat	1637	1656	SEQ ID NO: 6520	aaagaaacctatggcctta	4346	4365	1	3
SEQ ID NO: 5975	ggaaaagggtcatggaaat	1696	1715	SEQ ID NO: 6521	attcttaacgggattcctt	3859	3888	1	3
SEQ ID NO: 5976	gggcctgccccagatttct	1714	1733	SEQ ID NO: 6522	taaagccattcaggtctct	2667	2686	1	3
SEQ ID NO: 5977	ttctcagataggggaacac	1730	1749	SEQ ID NO: 6523	gacatgtttgaagggtgga	8673	8692	1	3
SEQ ID NO: 5978	gatgaaagaattaagggga	1746	1765	SEQ ID NO: 6524	tgggctaaacgtttaattg	2914	2933	1	3
SEQ ID NO: 5979	cttggactgtcaaataagt	1786	1805	SEQ ID NO: 6525	tattgaaatatgatttttt	3658	3677	1	3
SEQ ID NO: 5980	ggaaaagggtcatggaaat	1810	1829	SEQ ID NO: 6526	agcttgtttgccagtgggc	3264	3303	1	3
SEQ ID NO: 5981	gggcctgccccagatttct	1830	1849	SEQ ID NO: 6527	tgaaatcattgaaaattta	4748	4767	1	3
SEQ ID NO: 5982	cttggactgtcaaataagt	1837	1856	SEQ ID NO: 6528	tgaaatcattgaaaattta	6123	6142	1	3
SEQ ID NO: 5983	ggatttcaatacatttcac	1890	1909	SEQ ID NO: 6529	aaagaaacctatggcctta	4761	4780	1	3
SEQ ID NO: 5984	ttgtagaaatgaaagtaaa	1924	1943	SEQ ID NO: 6530	attcttaacgggattcctt	2320	2339	1	3
SEQ ID NO: 5985	ccaaaatttctctcggctt	1974	1993	SEQ ID NO: 6531	taaagccattcaggtctct	8596	8615	1	3
SEQ ID NO: 5986	caaattttctctgctaaaa	1975	1994	SEQ ID NO: 6532	gacatgtttgaagggtgga	8595	8614	1	3
SEQ ID NO: 5987	tctgctggaaacaacaacg	1996	2015	SEQ ID NO: 6533	tgggctaaacgtttaattg	8366	8365	1	3
SEQ ID NO: 5988	cagaagctaagcaatgtcc	1997	2016	SEQ ID NO: 6534	tattgaaatatgatttttt	7607	7626	1	3
SEQ ID NO: 5989	taagattaaatatttactt	2020	2039	SEQ ID NO: 6535	agcttgtttgccagtgggc	6740	5759	1	3
SEQ ID NO: 5990	aaagattactttgagaaat	2028	2047	SEQ ID NO: 6536	tgaaatcattgaaaattta	3512	3531	1	3
SEQ ID NO: 5991	aggaaatagttggattacc	2042	2061	SEQ ID NO: 6537	caggaggctttaagttgtc	9376	9396	1	3
SEQ ID NO: 5992	attttgatgatgctggccc	2071	2090	SEQ ID NO: 6538	agcttgtttgccagtgggc	8581	8580	1	3
SEQ ID NO: 5993	gaattatcttttaaacatt	2184	2203	SEQ ID NO: 6539	taaagccattcaggtctct	8600	8619	1	3
SEQ ID NO: 5994	ttaccaccagtttgaagtt	2201	2220	SEQ ID NO: 6540	aattgttgaaagaaaaact	6717	6736	1	3
SEQ ID NO: 5995	taagattaaatatttactt	2257	2276	SEQ ID NO: 6541	taaagccattcaggtctct	6411	6430	1	3
SEQ ID NO: 5996	aaagattactttgagaaat	2264	2283	SEQ ID NO: 6542	ttaatctttcataagtcaa	9009	9028	1	3
SEQ ID NO: 5997	aggaaatagttggattacc	2268	2287	SEQ ID NO: 6543	ggacaaggcccagaatctg	3236	3255	1	3
SEQ ID NO: 5998	attttgatgatgctggccc	2275	2294	SEQ ID NO: 6544	attcttaacgggattcctt	6761	6780	1	3
SEQ ID NO: 5999	gaattatcttttaaacatt	2309	2328	SEQ ID NO: 6545	aattgttgaaagaaaaact	3799	3818	1	3
SEQ ID NO: 6000	ttaccaccagtttgaagtt	2315	2334	SEQ ID NO: 6546	agcttgtttgccagtgggc	4357	4376	1	3
SEQ ID NO: 6001	ctgaaaaagttagtgaaag	2316	2335	SEQ ID NO: 6547	ttaatctttcataagtcaa	5201	5220	1	3
SEQ ID NO: 6002	tttttcccagacagtgtca	2402	2421	SEQ ID NO: 6548	agcttgtttgccagtgggc	6068	6087	1	3
SEQ ID NO: 6003	ttttcccagacagtgtcca	2405	2424	SEQ ID NO: 6549	tgaaatcattgaaaattta	2551	2570	1	3
SEQ ID NO: 6004	cattcagaaccagggaaga	2461	2480	SEQ ID NO: 6550	caggaggctttaagttgtc	7344	7363	1	3
SEQ ID NO: 6005	acaaactgtttatagtccc	2492	2511	SEQ ID NO: 6551	agcttgtttgccagtgggc	9202	9221	1	3
SEQ ID NO: 6006	ggatttcaatacatttcac	2547	2566	SEQ ID NO: 6552	taaagccattcaggtctct	5707	5723	1	3
SEQ ID NO: 6007	ttgtagaaatgaaagtaaa	2574	2593	SEQ ID NO: 6553	cttcatcgttcatttgagt	3015	3034	1	3
SEQ ID NO: 6008	ccaaaatttctctcggctt	2589	2809	SEQ ID NO: 6554	cttatgggatttcctaagg	3000	3019	1	3
SEQ ID NO: 6009	caaattttctctgctaaaa	2672	2691	SEQ ID NO: 6555	cttcatcgttcatttgagt	3637	3656	1	3
SEQ ID NO: 6010	tctgctggaaacaacaacg	2677	2696	SEQ ID NO: 6556	ttccatcacaaatcctttg	9355	9374	1	3
SEQ ID NO: 6011	cagaagctaagcaatgtcc	2720	2739	SEQ ID NO: 6557	tctcaaagagttacaacag	6848	6867	1	3
SEQ ID NO: 6012	taagattaaatatttactt	2721	2740	SEQ ID NO: 6558	tattgaaatatgatttttt	6847	6866	1	3
SEQ ID NO: 6013	aaagattactttgagaaat	2725	2744	SEQ ID NO: 6559	aattgttgaaagaaaaact	9160	9179	1	3
SEQ ID NO: 6014	aggaaatagttggattacc	2779	2498	SEQ ID NO: 6560	ggacaaggcccagaatctg	7443	7462	1	3
SEQ ID NO: 6015	attttgatgatgctggccc	2783	2602	SEQ ID NO: 6561	aaagaaacctatggcctta	9404	9423	1	3
SEQ ID NO: 6016	gaattatcttttaaacatt	2801	2826	SEQ ID NO: 6562	attcttaacgggattcctt	4192	4211	1	3
SEQ ID NO: 6017	ttaccaccagtttgaagtt	2687	2906	SEQ ID NO: 6563	taaagccattcaggtctct	3482	3501	1	3
SEQ ID NO: 6018	taagattaaatatttactt	2918	2937	SEQ ID NO: 6564	gacatgtttgaagggtgga	5242	5261	1	3
SEQ ID NO: 6019	aaagattactttgagaaat	2926	2945	SEQ ID NO: 6565	tgggctaaacgtttaattg	6261	6280	1	3
SEQ ID NO: 6020	aggaaatagttggattacc	2933	2952	SEQ ID NO: 6566	tattgaaatatgatttttt	8959	8978	1	3
SEQ ID NO: 6021	attttgatgatgctggccc	3006	3027	SEQ ID NO: 6567	agcttgtttgccagtgggc	8130	8149	1	3
SEQ ID NO: 6022	aggtttaaatggataattt	3010	3029	SEQ ID NO: 6568	aaagaaacctatggcctta	8267	8286	1	3
SEQ ID NO: 6023	cagaagctaagcaatgtcc	3032	3051	SEQ ID NO: 6569	attcttaacgggattcctt	7171	7190	1	3
SEQ ID NO: 6024	taagattaaatatttactt	3033	3052	SEQ ID NO: 6570	taaagccattcaggtctct	3945	3964	1	3
SEQ ID NO: 6025	aaagattactttgagaaat	3106	3125	SEQ ID NO: 6571	gacatgtttgaagggtgga	9348	9367	1	3
SEQ ID NO: 6026	aggaaatagttggattacc	3139	3158	SEQ ID NO: 6572	tgggctaaacgtttaattg	8354	8973	1	3
SEQ ID NO: 6027	attttgatgatgctggccc	3145	3164	SEQ ID NO: 6573	tattgaaatatgatttttt	7552	7571	1	3
SEQ ID NO: 6028	gaattatcttttaaacatt	353	3172	SEQ ID NO: 6574	agcttgtttgccagtgggc	5927	5946	1	3
SEQ ID NO: 6029	ttaccaccagtttgaagtt	3241	3260	SEQ ID NO: 6575	ggacaaggcccagaatctg	3349	3368	1	3
SEQ ID NO: 6030	ttgcagtgtatcggacaga	3262	3281	SEQ ID NO: 6576	aaagaaacctatggcctta	7717	7736	1	3
SEQ ID NO: 6031	cattcagaaccagggaaga	3299	3318	SEQ ID NO: 6577	attcttaacgggattcctt	8083	8102	1	3
SEQ ID NO: 6032	acaaactgtttatagtccc	3300	3319	SEQ ID NO: 6578	taaagccattcaggtctct	5792	5811	1	3
SEQ ID NO: 6033	ttaccaccagtttgaagtt	3321	3340	SEQ ID NO: 6579	aattgttgaaagaaaaact	4020	4039	1	3
SEQ ID NO: 6034	taagattaaatatttactt	3322	3341	SEQ ID NO: 6580	ggacaaggcccagaatctg	4019	4038	1	3
SEQ ID NO: 6035	aaagattactttgagaaat	3323	3342	SEQ ID NO: 6581	aaagaaacctatggcctta	6913	5932	1	3
SEQ ID NO: 6036	aggaaatagttggattacc	3343	3362	SEQ ID NO: 6582	attcttaacgggattcctt	7770	7789	1	3
SEQ ID NO: 6037	attttgatgatgctggccc	3346	3365	SEQ ID NO: 6583	taaagccattcaggtctct	3962	3981	1	3
SEQ ID NO: 6038	aggtttaaatggataattt	3365	3385	SEQ ID NO: 6584	gacatgtttgaagggtgga	4018	4037	1	3
SEQ ID NO: 6039	cagaagctaagcaatgtcc	3439	3458	SEQ ID NO: 6585	tgggctaaacgtttaattg	7519	7538	1	3
SEQ ID NO: 6040	taagattaaatatttactt	3530	3549	SEQ ID NO: 6586	tattgaaatatgatttttt	6215	6234	1	3
SEQ ID NO: 6041	aaagattactttgagaaat	3540	3559	SEQ ID NO: 6587	agcttgtttgccagtgggc	6020	6039	1	3
SEQ ID NO: 6042	aggaaatagttggattacc	3671	3690	SEQ ID NO: 6588	tgaaatcattgaaaattta	5542	5561	1	3
SEQ ID NO: 6043	attttgatgatgctggccc	3685	3704	SEQ ID NO: 6589	caggaggctttaagttgtc	4840	4859	1	3
SEQ ID NO: 6044	gaattatcttttaaacatt	3698	3717	SEQ ID NO: 6590	agcttgtttgccagtgggc	6818	6637	1	3
SEQ ID NO: 6045	ttaccaccagtttgaagtt	3762	3761	SEQ ID NO: 6591	taaagccattcaggtctct	3931	3950	1	3
SEQ ID NO: 6046	ttgcagtgtatcggacaga	3802	3821	SEQ ID NO: 6592	aattgttgaaagaaaaact	9111	9130	1	3
SEQ ID NO: 6047	cattcagaaccagggaaga	3904	3923	SEQ ID NO: 6593	taaagccattcaggtctct	5680	5699	1	3
SEQ ID NO: 6048	acaaactgtttatagtccc	3926	3945	SEQ ID NO: 6594	ttaatctttcataagtcaa	5813	5632	1	3
SEQ ID NO: 6049	ggatttcaatacatttcac	3996	4015	SEQ ID NO: 6595	ggacaaggcccagaatctg	8940	8959	1	3
SEQ ID NO: 6050	ttgtagaaatgaaagtaaa	4013	4032	SEQ ID NO: 6596	attcttaacgggattcctt	5385	5408	1	3
SEQ ID NO: 6051	ctgaacagtgagctgcagt	4028	4047	SEQ ID NO: 6597	aattgttgaaagaaaaact	5815	5834	1	3
SEQ ID NO: 6052	aatccaatctcctctttgc	4068	4067	SEQ ID NO: 6598	agcttgtttgccagtgggc	5937	5956	1	3
SEQ ID NO: 6053	atttgattttcaagcaaag	4140	4159	SEQ ID NO: 6599	ttaatctttcataagtcaa	5163	5182	1	3
SEQ ID NO: 6054	ttttgattttcaagcaaat	4141	4160	SEQ ID NO: 6600	agcttgtttgccagtgggc	5162	5181	1	3
SEQ ID NO: 6055	tgatttcaagcaaatgcag	4191	4210	SEQ ID NO: 6601	tgaaatcattgaaaattta	6167	5186	1	3
SEQ ID NO: 6056	atgctgtttgggaaattgg	4209	4228	SEQ ID NO: 6602	caggaggctttaagttgtc	5059	6076	1	3
SEQ ID NO: 6057	tgctgttttttggaaatgc	4236	4255	SEQ ID NO: 6603	agcttgtttgccagtgggc	7839	7858	1	3
SEQ ID NO: 6058	aaaaaaatacactggacgt	4352	4371	SEQ ID NO: 6604	taaagccattcaggtctct	7637	7556	1	3
SEQ ID NO: 6059	actggagcttagtaagggc	4384	4403	SEQ ID NO: 6605	cttcatcgttcatttgagt	7619	7538	1	3
SEQ ID NO: 6060	cttctggaaagagggtcat	4453	4472	SEQ ID NO: 6606	cttatgggatttcctaagg	6845	6864	1	3
SEQ ID NO: 6061	ggaaaagggtcatggaaat	4477	4496	SEQ ID NO: 6607	cttcatcgttcatttgagt	9257	9276	1	3
SEQ ID NO: 6062	gggcctgccccagatttct	4482	4501	SEQ ID NO: 6608	ttccatcacaaatcctttg	7527	7546	1	3
SEQ ID NO: 6063	ttctcagataggggaacac	4483	4502	SEQ ID NO: 6609	tctcaaagagttacaacag	7526	7545	1	3
SEQ ID NO: 6064	gatgaaagaattaagggga	4586	4587	SEQ ID NO: 6610	tattgaaatatgatttttt	7414	7433	1	3
SEQ ID NO: 6065	cttggactgtcaaataagt	4612	4631	SEQ ID NO: 6611	aattgttgaaagaaaaact	6217	6290	1	3
SEQ ID NO: 6066	ggaaaagggtcatggaaat	4616	4635	SEQ ID NO: 6612	ggacaaggcccagaatctg	6968	8957	1	3
SEQ ID NO: 6067	gggcctgccccagatttct	4668	4687	SEQ ID NO: 6613	aaagaaacctatggcctta	7184	7203	1	3
SEQ ID NO: 6068	cttggactgtcaaataagt	4684	4703	SEQ ID NO: 6614	attcttaacgggattcctt	8213	8232	1	3
SEQ ID NO: 6069	ggatttcaatacatttcac	4724	4743	SEQ ID NO: 6615	taaagccattcaggtctct	8947	8966	1	3
SEQ ID NO: 6070	ttgtagaaatgaaagtaaa	4736	4757	SEQ ID NO: 6616	gacatgtttgaagggtgga	6301	6320	1	3
SEQ ID NO: 6071	ccaaaatttctctcggctt	4760	4779	SEQ ID NO: 6617	tgggctaaacgtttaattg	1891	1910	1	3
SEQ ID NO: 6072	caaattttctctgctaaaa	4805	4825	SEQ ID NO: 6618	tattgaaatatgatttttt	9657	8676	1	3
SEQ ID NO: 6073	tctgctggaaacaacaacg	4831	4850	SEQ ID NO: 6619	agcttgtttgccagtgggc	7622	7641	1	3
SEQ ID NO: 6074	cagaagctaagcaatgtcc	4855	4874	SEQ ID NO: 6620	tgaaatcattgaaaattta	6546	6565	1	3
SEQ ID NO: 6075	taagattaaatatttactt	4906	4925	SEQ ID NO: 6621	tgaaatcattgaaaattta	8009	8028	1	3
SEQ ID NO: 6076	aaagattactttgagaaat	4907	4926	SEQ ID NO: 6622	aaagaaacctatggcctta	6008	8027	1	3
SEQ ID NO: 6077	aggaaatagttggattacc	4918	4937	SEQ ID NO: 6623	attcttaacgggattcctt	5849	588	1	3
SEQ ID NO: 6078	attttgatgatgctggccc	5050	5069	SEQ ID NO: 6624	taaagccattcaggtctct	3164	3183	1	3
SEQ ID NO: 6079	gaattatcttttaaacatt	5101	5120	SEQ ID NO: 6625	gacatgtttgaagggtgga	8002	8021	1	3
SEQ ID NO: 6080	ttaccaccagtttgaagtt	5130	5149	SEQ ID NO: 6626	tgggctaaacgtttaattg	8065	8085	1	3
SEQ ID NO: 6081	taagattaaatatttactt	5147	5165	SEQ ID NO: 6627	tattgaaatatgatttttt	5892	5911	1	3
SEQ ID NO: 6082	aaagattactttgagaaat	5175	5194	SEQ ID NO: 6628	agcttgtttgccagtgggc	5933	5952	1	3
SEQ ID NO: 6083	aggaaatagttggattacc	5214	5233	SEQ ID NO: 6629	tgaaatcattgaaaattta	6832	6851	1	3
SEQ ID NO: 6084	attttgatgatgctggccc	5268	5287	SEQ ID NO: 6630	caggaggctttaagttgtc	9072	9091	1	3
SEQ ID NO: 6085	gaattatcttttaaacatt	5336	5355	SEQ ID NO: 6631	agcttgtttgccagtgggc	6956	6974	1	3
SEQ ID NO: 6086	ttaccaccagtttgaagtt	5364	5383	SEQ ID NO: 6632	taaagccattcaggtctct	9401	9420	1	3
SEQ ID NO: 6087	ctgaaaaagttagtgaaag	5557	5576	SEQ ID NO: 6633	aattgttgaaagaaaaact	8820	8839	1	3
SEQ ID NO: 6088	tttttcccagacagtgtca	5635	5654	SEQ ID NO: 6634	taaagccattcaggtctct	7702	7721	1	3
SEQ ID NO: 6089	ttttcccagacagtgtcca	5728	5747	SEQ ID NO: 6635	ttaatctttcataagtcaa	9565	9564	1	3
SEQ ID NO: 6090	cattcagaaccagggaaga	5751	5770	SEQ ID NO: 6636	ggacaaggcccagaatctg	5771	5790	1	3
SEQ ID NO: 6091	acaaactgtttatagtccc	5762	5781	SEQ ID NO: 6637	attcttaacgggattcctt	9378	9397	1	3
SEQ ID NO: 6092	ggatttcaatacatttcac	5777	5796	SEQ ID NO: 6638	aattgttgaaagaaaaact	6564	6583	1	3
SEQ ID NO: 6093	ttgtagaaatgaaagtaaa	5818	5937	SEQ ID NO: 6639	agcttgtttgccagtgggc	6832	6851	1	3
SEQ ID NO: 6094	ccaaaatttctctcggctt	5829	5848	SEQ ID NO: 6640	ggacaaggcccagaatctg	8453	8472	1	3
SEQ ID NO: 6095	caaattttctctgctaaaa	5845	5864	SEQ ID NO: 6641	aaagaaacctatggcctta	7727	7746	1	3
SEQ ID NO: 6096	tctgctggaaacaacaacg	5853	5872	SEQ ID NO: 6642	attcttaacgggattcctt	7111	7130	1	3
SEQ ID NO: 6097	cagaagctaagcaatgtcc	5909	5928	SEQ ID NO: 6643	taaagccattcaggtctct	5794	5813	1	3
SEQ ID NO: 6098	taagattaaatatttactt	5922	5941	SEQ ID NO: 6644	gacatgtttgaagggtgga	8063	8082	1	3
SEQ ID NO: 6099	aaagattactttgagaaat	5923	5942	SEQ ID NO: 6645	tgggctaaacgtttaattg	8062	8081	1	3
SEQ ID NO: 6100	aggaaatagttggattacc	5950	5969	SEQ ID NO: 6646	tattgaaatatgatttttt	7596	7615	1	3
SEQ ID NO: 6101	attttgatgatgctggccc	6060	8079	SEQ ID NO: 6647	agcttgtttgccagtgggc	4019	4038	1	3
SEQ ID NO: 6102	gaattatcttttaaacatt	6095	5114	SEQ ID NO: 6648	tgaaatcattgaaaattta	5664	5583	1	3
SEQ ID NO: 6103	ttaccaccagtttgaagtt	6146	6165	SEQ ID NO: 6649	tgaaatcattgaaaattta	7428	7447	1	3
SEQ ID NO: 6104	taagattaaatatttactt	6237	6256	SEQ ID NO: 6650	aaagaaacctatggcctta	6399	6418	1	3
SEQ ID NO: 6105	aaagattactttgagaaat	6331	6350	SEQ ID NO: 6651	attcttaacgggattcctt	7969	7988	1	3
SEQ ID NO: 6106	aggaaatagttggattacc	6442	6461	SEQ ID NO: 6652	taaagccattcaggtctct	9287	9306	1	3
SEQ ID NO: 6107	attttgatgatgctggccc	6506	6525	SEQ ID NO: 6653	gacatgtttgaagggtgga	7257	7276	1	3
SEQ ID NO: 6108	aggtttaaatggataattt	6544	6563	SEQ ID NO: 6654	tgggctaaacgtttaattg	7860	7679	1	3
SEQ ID NO: 6109	ggatttcaatacatttcac	6675	6694	SEQ ID NO: 6655	tattgaaatatgatttttt	8803	8622	1	3
SEQ ID NO: 6110	ttgtagaaatgaaagtaaa	6677	6696	SEQ ID NO: 6656	agcttgtttgccagtgggc	3164	3183	1	3
SEQ ID NO: 6111	ctgaacagtgagctgcagt	6693	6712	SEQ ID NO: 6657	tgaaatcattgaaaattta	7593	7612	1	3
SEQ ID NO: 6112	aatccaatctcctctttgc	6708	6727	SEQ ID NO: 6658	caggaggctttaagttgtc	8163	8172	1	3
SEQ ID NO: 6113	atttgattttcaagcaaag	6709	6726	SEQ ID NO: 6659	agcttgtttgccagtgggc	8152	7171	1	3
SEQ ID NO: 6114	ttttgattttcaagcaaat	6872	6891	SEQ ID NO: 6660	taaagccattcaggtctct	8276	7297	1	3
SEQ ID NO: 6115	acaaactgtttatagtccc	6911	6930	SEQ ID NO: 6661	aattgttgaaagaaaaact	5782	5801	1	3
SEQ ID NO: 6116	ggatttcaatacatttcac	6972	6991	SEQ ID NO: 6662	taaagccattcaggtctct	7811	7830	1	3
SEQ ID NO: 6117	ttgtagaaatgaaagtaaa	7060	7079	SEQ ID NO: 6663	ttaatctttcataagtcaa	8887	8906	1	3
SEQ ID NO: 6118	ttaccaccagtttgaagtt	7061	7080	SEQ ID NO: 6664	ggacaaggcccagaatctg	8885	8905	1	3
SEQ ID NO: 6119	ttgcagtgtatcggacaga	7112	7131	SEQ ID NO: 6665	attcttaacgggattcctt	8457	8476	1	3
SEQ ID NO: 6120	cattcagaaccagggaaga	7113	7132	SEQ ID NO: 6666	aattgttgaaagaaaaact	7556	7575	1	3
SEQ ID NO: 6121	acaaactgtttatagtccc	7149	7168	SEQ ID NO: 6667	agcttgtttgccagtgggc	8030	8049	1	3
SEQ ID NO: 6122	ggatttcaatacatttcac	7164	7183	SEQ ID NO: 6668	ttaatctttcataagtcaa	8730	8749	1	3
SEQ ID NO: 6123	ttgtagaaatgaaagtaaa	7580	7599	SEQ ID NO: 6669	agcttgtttgccagtgggc	9305	9324	1	3
SEQ ID NO: 6124	ccaaaatttctctcggctt	7594	7613	SEQ ID NO: 6670	tgaaatcattgaaaattta	9197	9216	1	3
SEQ ID NO: 6125	caaattttctctgctaaaa	7601	7620	SEQ ID NO: 6671	caggaggctttaagttgtc	7716	7735	1	3
SEQ ID NO: 6126	tctgctggaaacaacaacg	7704	7723	SEQ ID NO: 6672	agcttgtttgccagtgggc	6123	6142	1	3
SEQ ID NO: 6127	cagaagctaagcaatgtcc	7757	7776	SEQ ID NO: 6673	taaagccattcaggtctct	8031	8050	1	3
SEQ ID NO: 6128	taagattaaatatttactt	7789	7808	SEQ ID NO: 6674	cttcatcgttcatttgagt	7936	7955	1	3
SEQ ID NO: 6129	aaagattactttgagaaat	7807	7826	SEQ ID NO: 6675	cttatgggatttcctaagg	9116	9135	1	3
SEQ ID NO: 6130	aggaaatagttggattacc	8032	8051	SEQ ID NO: 6676	cttcatcgttcatttgagt	7756	7775	1	3
SEQ ID NO: 6131	attttgatgatgctggccc	8110	8129	SEQ ID NO: 6677	ttccatcacaaatcctttg	9069	9088	1	3
SEQ ID NO: 6132	gaattatcttttaaacatt	8365	8384	SEQ ID NO: 6678	tctcaaagagttacaacag	9551	9570	1	3
SEQ ID NO: 6133	ttaccaccagtttgaagtt	8900	8919	SEQ ID NO: 6679	tattgaaatatgatttttt	7134	7153	1	3
SEQ ID NO: 6134	taagattaaatatttactt	9303	9322	SEQ ID NO: 6680	aattgttgaaagaaaaact	3688	3707	1	3
		Start	End		Match	Start	End	Match
	Source	Index	Index			Index	Index	#
SEQ ID	tttttttttttttttttttt	9446	9465	SEQ ID	tttttttttttttttttttt	9466	9485	2
NO: 4088				NO: 4661
SEQ ID	tttttttttttttttttttt	9446	9465	SEQ ID	tttttttttttttttttttt	9465	9484	1
NO: 4347				NO: 5229
SEQ ID	tttttttttttttttttttt	9447	9466	SEQ ID	tttttttttttttttttttt	9466	9485	1
NO: 4348				NO: 5230

TABLE 16 — Solutions adminstered to mice

Group	n	Injection Mix
1	7	Buffer (PBS [pH 7.4])
2	8	Cholesterol conjugated siRNA
		(ALN-3001)

TABLE 17 — Test iRNA agents targeting Luciferase 2 cholesterol is conjugated to the antisense strand via the linker: U-pyrroline carrier-C(O)-(CH2)5-NHC(O)-cholesterol (via cholesterol C-3 hydroxyl).

siRNA	Sequence
ALN-1070	5′-GAA CUG UGU GUG AGA GGU CCU-3′
	(SEQ ID NO: 6700)
	3′-CG CUU GAC ACA CAC UCU CCA GGA-5′
	(SEQ ID NO: 6701)
ALN-1000	5′-GAA CUG UGU GUG AGA GGU CCU-GS-3′
	(SEQ ID NO: 6702)
	3′-CG CUU GAC ACA CAC UCU CCA GGA-5′
	(SEQ ID NO: 6703)
ALN-3000	5′-GAA CUG UGU GUG AGA GGU CCU-3′
	(SEQ ID NO: 6704)
	3′-Cs 1 Gs 1 CUU GAC ACA CAC UCU CCA GGA-5′
	(SEQ ID NO: 6705)
ALN-3001	5′-GAA CUG UGU GUG AGA GGU CCU-chol. 2 -3′
	(SEQ ID NO: 6706)
	3′-Cs 1 Gs 1 CUU GAC ACA CAC UCU CCA GGA-5′
	(SEQ ID NO: 6707)
1 2′ O-Me group is attached to the nucleotide and the nucleotides have phosphorothioate linkages (indicated by “s”)

TABLE 18 — Test iRNA agents targeting glucose-6-phosphatase

siRNA	Description
U/U	No cholesterol; dinucleotide 3′ overhangs
	on sense and antisense strands
3′C/U	dinucleotide 3′ overhangs on sense and
	antisense strands; cholesterol conjugated
	to 3′ end of sense strand (mono-conjugate)
3′C/3′C	dinucleotide 3′ overhangs on sense and
	antisense strands; cholesterol conjugated
	to 3′ end of both sense and antisense
	strands (bis-conjugate)

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Claims as granted

20 claims

Classifications

12 codes

IPC · International Patent Classification

Section A — Human necessities

A61K31/713
A61K48/00
A61K47/54
A01K67/027
A61K/

Section C — Chemistry; metallurgy

C12Q1/68
C12N15/113
C07K14/775
C07H21/02
C07H21/04
C12N15/00
C12N15/11

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Tracy Vivlemore

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