Antisense modulation of Apaf-1 expression

The present invention provides compositions and methods for modulating the expression of Apaf-1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Apaf-1. Such compounds have been shown to modulate the expression of Apaf-1.

Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process goes awry becoming overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGFβ), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa, Mol. Pathol ., 2000, 53, 55-63).

Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa, Mol. Pathol ., 2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.

Apoptotic protease activating factor 1 (also known as Apaf-1) is the human homolog of the C. elegans gene, ced-4, originally purified and cloned from HeLa cell cytosol (Zou et al., Cell , 1997, 90, 405-413). Disclosed in the PCT Publication WO 98/55615 are the protein and nucleic acid sequences of Apaf-1 as are antibodies to the protein, host cells which express a vector encoding the nucleic acid sequence of Apaf-1, transgenic and knock-out animals and methods to detect Apaf-1 modulating compounds. Generally disclosed is an isolated nucleic acid comprising a nucleotide which would hybridize to Apaf-1 under stringent conditions (Zou et al., 1998).

Characterization of the protein has revealed that Apaf-1 contains a domain structure with defined regions of homology to existing proteins including WD repeats, and caspase-related domains (Zou et al., Cell , 1997, 90, 405-413). Tissue localization studies have demonstrated that Apaf-1 can be found in most tissues (Zou et al., Cell , 1997, 90, 405-413), however there is debate on the presence of Apaf-1 in skeletal muscle (Burgess et al., Cell Death and Differentiation , 1999, 6, 256-261). Within the cell, Apaf-1 is localized to the cytosol (Hausmann et al., Journal of Cell Biology , 2000, 149, 623-634) and at least three variants of the protein have been identified to date. Hahn et al. describe three novel forms of Apaf-1 isolated from six lymphoma cell lines, three non-lymphoid tumor cell lines, peripheral blood lymphocytes, and in tissues from heart, kidney and liver (Hahn et al., Biochemical and Biophysical Research Communications , 1999, 261, 746-749).

Others have also identified alternate forms of Apaf-1 resulting from differential splicing events (Benedict et al., Journal of Biological Chemistry , 2000, 275, 8461-8468; Hu et al., The Embo Journal , 1999, 18, 3586-3595). Characterization of these variants demonstrated that longer forms of the protein containing an extra WD repeat were required to activate the apoptotic cascade orchestrated by caspases (Benedict et al., Journal of Biological Chemistry , 2000, 275, 8461-8468). Disclosed in the PCT publication WO 99/65937 are the nucleic acid sequence of Apaf-1 including truncated variants of Apaf-1 which may oligomerize with members of the caspase family as are host cells which express a vector encoding the nucleic acid sequence of Apaf-1, and methods to detect inhibitors of apoptosis and Apaf-1 modulating compounds. Generally disclosed is an isolated nucleic acid comprising a nucleotide which would hybridize to Apaf-1 under stringent conditions (Alnemri, 1999).

Since its isolation, Apaf-1 has been shown to play a critical role in the regulation of apoptosis through multiple signaling pathways reviewed in (Cecconi, Cell Death and Differentiation , 1999, 6, 1087-1098). Primarily Apaf-1 acts through cytochrome c-mediated caspase activation (Saleh et al., Journal of Biological Chemistry , 1999, 274, 17941-17945). Upon release of cytochrome c from the mitochondria and in the presence of DATP (deoxy ATP), Apaf-1 oligomerizes and forms a complex with procaspase-9 (Cain et al., Journal of Biological Chemistry , 2000, 275, 6067-6070; Zou et al., Journal of Biological Chemistry , 1999, 274, 11549-11556). In turn, procaspase-9 is cleaved into its active form, leading to the activation of caspase-3, the major caspase in the apoptotic pathway. Overexpression of Apaf-1 has been shown to increase sensitivity of human myeloid leukemia HL-60 cells to the apoptosis-inducing agents paclitaxel and etoposide (Perkins et al., Cancer Research , 1998, 58, 4561-4566).

Although Apaf-1 has been localized to chromosome 12q23, a region recurrently deleted in male germ cell tumors, genetic analysis has shown that Apaf-1 is not a tumor suppressor gene candidate (Bala et al., Genes, Chromosomes & Cancer , 2000, 28, 258-268; Kim et al., Cytogenet. Cell Genet ., 1999, 87, 252-253). Expression of Apaf-1, nonetheless has been associated with several types of cancers. Frameshift mutations in the Apaf-1 gene have been suggested to provide a survival advantage to some tumor types (Yamamoto et al., Cell Death and Differentiation , 2000, 7, 238-239) and mouse embryonic cells lacking Apaf-1 and caspase-9 were shown to be resistant to apoptotic stimuli (Soengas et al., Science , 1999, 284, 156-159). The majority of homozygous knockout mice lacking Apaf-1 die early in embryogenesis and show reduced apoptosis leading to many morphological abnormalities. In the mice reaching maturity, the adult heterozygote males exhibit infertility (Cecconi et al., Cell , 1998, 94, 727-737; Honarpour et al., Cevelopmental Biology , 2000, 218, 248-258).

Collectively, these data suggest that modulation of Apaf-1 would render opportunity to treat patients with various cancers and deregulated apoptotic pathologic conditions.

Strategies aimed at modulating Apaf-1 function have involved the use of antibodies and gene knockouts in mice. However, these strategies are untested as therapeutic protocols and consequently there remains a long felt need for agents capable of effectively inhibiting Apaf-1 function.

Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of Apaf-1.

The present invention provides compositions and methods for modulating Apaf-1 expression, including modulation of the truncated forms, including splice variants of Apaf-1.

The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Apaf-1, and which modulate the expression of Apaf-1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Apaf-1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Apaf-1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Apaf-1, ultimately modulating the amount of Apaf-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Apaf-1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Apaf-1” encompass DNA encoding Apaf-1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Apaf-1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Apaf-1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Apaf-1, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense 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, and in the case of in vitro assays, under conditions in which the assays are performed.

Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3 1 -most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.

Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science , 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—O—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 — [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 — [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1 to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl. Particularly preferred are O [(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1 to C 10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′ -methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta , 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples hereinbelow.

A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH 2 —) n group bridging the 2′ oxygen atom and the 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases 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, those disclosed by Englisch et al., Angewandte Chemie , International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15 , Antisense Research and Applications , pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications , CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA , 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let ., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci ., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let ., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res ., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J ., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett ., 1990, 259, 327-330; Svinarchuk et al., Biochimie , 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett ., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res ., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides , 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett ., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta , 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther ., 1996, 277, 923-937. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules. The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci ., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Apaf-1 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Apaf-1, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Apaf-1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Apaf-1 in a sample may also be prepared.

The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. 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 and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

Pharmaceutical compositions and 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. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no hermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases posess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems , Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research , 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol ., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research , 1994, 11, 1385; Ho et al., J. Pharm. Sci ., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, p. 92). Each of these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

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. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

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 drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., 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.

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 liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that 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 a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun ., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is 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 , 1992, 19, 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.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting , 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research , 1992, 18, 259-265).

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 cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci ., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G M1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters , 1987, 223, 42; Wu et al., Cancer Research , 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. ( Ann. N.Y. Acad. Sci ., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. ( Proc. Natl. Acad. Sci. U.S.A ., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. ( Bull. Chem. Soc. Jpn ., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Illum et al. ( FEBS Lett ., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. ( FEBS Lett ., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. ( Biochimica et Biophysica Acta , 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms , Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic 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).

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol ., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems , 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics , 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences , 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems , 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther ., 1992, 263, 25; Yamashita et al., J. Pharm. Sci ., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr ., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems , 1990, 7, 1-33; Buur et al., J. Control Rel ., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems , 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems , 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol ., 1987, 39, 621-626)

Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev ., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev ., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy , 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy , 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state 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 the 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 oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

Artificial Sequence

Antisense Oligonucleotide

1

tccgtcatcg ctcctcaggg 20

2

20

Artificial Sequence

Antisense Oligonucleotide

2

atgcattctg cccccaagga 20

3

324

Homo sapiens

3

ggaggcgcct gtgaggcccg gacctgcccc ggggcgaagg gtatgtggcg agacagagcc 60

ctgcacccct aattcccggt ggaaaactcc tgttgccgtt tccctccacc ggcctggagt 120

ctcccagtct tgtcccggca gtgccgccct ccccactaag acctaggcgc aaaggcttgg 180

gtaagttgac ctcctcgctt ttctccccga gccaggttct ttggaggctt ggtagctgat 240

ttcagggact gagaatttga gacatggaat atttgataac cacctgctcc tgtcccagga 300

agaaaattct gttatgagga aatt 324

4

20

Artificial Sequence

PCR Primer

4

agagccctgc acccctaatt 20

5

21

Artificial Sequence

PCR Primer

5

tttgcgccta ggtcttagtg g 21

6

24

Artificial Sequence

PCR Probe

6

tctcccagtc ttgtcccggc agtg 24

7

21

Artificial Sequence

PCR Primer

7

caacggattt ggtcgtattg g 21

8

26

Artificial Sequence

PCR Primer

8

ggcaacaata tccactttac cagagt 26

9

21

Artificial Sequence

PCR Probe

9

cgcctggtca ccagggctgc t 21

10

cggcttgagg cagagaccag gaggcagcta gaggagcaga cgtctcactc cgctcgcgga 60

agggtgtgag aggggtgtgt gggggtcggc agcgaggggt gtgtgccatc agccaccggc 120

gacgatctga gacagtcgca gcggctttcc gagcggcgtc cgcttcccgc ccgggcagct 180

cccgccagag gggtgaagcg gcgactggag tggccgtgct tttgtgccct gggtcccggt 240

accctcccct ggtgcggccc gaggcaagcc caccgaggtg accacccctc gacgccgctt 300

ggagatcccg ggcatccacc ctgcgccccg agcagctgat acccagggag gtgtcaggac 360

ctgcccgggg cgcggggtcg ccggaagcca ggcgggagcc ccggctgctt tctggcaatc 420

tagtctcata agtgaccctc cctgggctgc tttctttcga ttatcatcag tgaccctacc 480

ccggctgctc ttcccagcac aactccggtg caaaggcttg ggcatcctgg tgctttgcct 540

ctagcccatg ctccacagcg aggagagaga aaaccctgag gcaca atg gat gca aag 597

Met Asp Ala Lys

1

gcc cgc aat tgt ttg ctt caa cat aga gaa gct ttg gaa aag gac atc 645

Ala Arg Asn Cys Leu Leu Gln His Arg Glu Ala Leu Glu Lys Asp Ile

5 10 15 20

aaa aca tcc tac atc atg gat cac atg atc agt aat ggc gtc ttg tca 693

Lys Thr Ser Tyr Ile Met Asp His Met Ile Ser Asn Gly Val Leu Ser

25 30 35

gtg ata gag gag gag aag gtc aaa agt cag gcc act caa tat caa cga 741

Val Ile Glu Glu Glu Lys Val Lys Ser Gln Ala Thr Gln Tyr Gln Arg

40 45 50

gca gcc gct tta att aaa atg ata ctt aat aaa gac aac tgt gcc tac 789

Ala Ala Ala Leu Ile Lys Met Ile Leu Asn Lys Asp Asn Cys Ala Tyr

55 60 65

att tca ttc tac aac gct ctg cta cac gag ggc tat aag gac ctt gct 837

Ile Ser Phe Tyr Asn Ala Leu Leu His Glu Gly Tyr Lys Asp Leu Ala

70 75 80

gcg ctt ctg cag agt ggc ctt cct ctt gtg tcg tct tcc agt gca agg 885

Ala Leu Leu Gln Ser Gly Leu Pro Leu Val Ser Ser Ser Ser Ala Arg

85 90 95 100

aca gtg ctg tgt gaa ggt gga gta ccc cag agg ccg gtt att ttc gtt 933

Thr Val Leu Cys Glu Gly Gly Val Pro Gln Arg Pro Val Ile Phe Val

105 110 115

act aga aag aag ctg gtt cat gcg att cag cag aag ctc tgg aaa ctg 981

Thr Arg Lys Lys Leu Val His Ala Ile Gln Gln Lys Leu Trp Lys Leu

120 125 130

aat gga gaa cca ggg tgg gtc acc atc tat ggg atg gca ggc tgc ggc 1029

Asn Gly Glu Pro Gly Trp Val Thr Ile Tyr Gly Met Ala Gly Cys Gly

135 140 145

aag tct gtg tta gct gcg gaa gcc gtt cga gat cac tcc ctc tta gaa 1077

Lys Ser Val Leu Ala Ala Glu Ala Val Arg Asp His Ser Leu Leu Glu

150 155 160

ggt tgc ttt tca ggg ggt gta cac tgg gtt tcc att gga aaa caa gac 1125

Gly Cys Phe Ser Gly Gly Val His Trp Val Ser Ile Gly Lys Gln Asp

165 170 175 180

aaa tct ggg ctt ctc atg aaa ctg cag aat ctg tgc atg cgc ttg gac 1173

Lys Ser Gly Leu Leu Met Lys Leu Gln Asn Leu Cys Met Arg Leu Asp

185 190 195

caa gat gag agt ttc tct cag agg ctt cca ctt aat att gag gag gcc 1221

Gln Asp Glu Ser Phe Ser Gln Arg Leu Pro Leu Asn Ile Glu Glu Ala

200 205 210

aaa gac cgc ctc cgt gtt ctg atg ctg cgc aaa cac cca agg tct ctg 1269

Lys Asp Arg Leu Arg Val Leu Met Leu Arg Lys His Pro Arg Ser Leu

215 220 225

ttg atc ttg gat gat gtt tgg gat cct tgg gtg tta aaa gct ttt gac 1317

Leu Ile Leu Asp Asp Val Trp Asp Pro Trp Val Leu Lys Ala Phe Asp

230 235 240

aat cag tgt cag att ctt ctt acg acc aga gat aag agt gtt aca gat 1365

Asn Gln Cys Gln Ile Leu Leu Thr Thr Arg Asp Lys Ser Val Thr Asp

245 250 255 260

tca gta atg ggt cct aag cat gtt gtc cct gtg gag agt ggt cta ggg 1413

Ser Val Met Gly Pro Lys His Val Val Pro Val Glu Ser Gly Leu Gly

265 270 275

aga gag aaa gga ctt gag atc ttg tca ctt ttt gtt aat atg aag aaa 1461

Arg Glu Lys Gly Leu Glu Ile Leu Ser Leu Phe Val Asn Met Lys Lys

280 285 290

gaa gat ctg cca gcg gag gct cac agt att ata aag gaa tgc aaa ggt 1509

Glu Asp Leu Pro Ala Glu Ala His Ser Ile Ile Lys Glu Cys Lys Gly

295 300 305

tct cct ctt gta gtg tct tta att ggt gca ctt tta cgt gat ttt ccc 1557

Ser Pro Leu Val Val Ser Leu Ile Gly Ala Leu Leu Arg Asp Phe Pro

310 315 320

aat cgc tgg gcg tac tac ctc aga cag ctt cag aat aag cag ttt aag 1605

Asn Arg Trp Ala Tyr Tyr Leu Arg Gln Leu Gln Asn Lys Gln Phe Lys

325 330 335 340

aga ata agg aag tct tca tct tat gat tat gag gct cta gat gaa gcc 1653

Arg Ile Arg Lys Ser Ser Ser Tyr Asp Tyr Glu Ala Leu Asp Glu Ala

345 350 355

atg tcg ata agt gtt gaa atg ctc aga gaa gac atc aaa gac tat tac 1701

Met Ser Ile Ser Val Glu Met Leu Arg Glu Asp Ile Lys Asp Tyr Tyr

360 365 370

aca gac ctt tcc atc ctt cag aag gac gtc aag gta cct aca aag gtg 1749

Thr Asp Leu Ser Ile Leu Gln Lys Asp Val Lys Val Pro Thr Lys Val

375 380 385

ttg tgc gtt ctc tgg gac ttg gaa acg gaa gaa gtt gaa gac atc ctg 1797

Leu Cys Val Leu Trp Asp Leu Glu Thr Glu Glu Val Glu Asp Ile Leu

390 395 400

cag gag ttc gtt aat aag tct ctc tta ttc tgt aat cgg aat gga aag 1845

Gln Glu Phe Val Asn Lys Ser Leu Leu Phe Cys Asn Arg Asn Gly Lys

405 410 415 420

tca ttt tgt tat tat tta cac gat ctt caa gta gat ttt ctt aca gag 1893

Ser Phe Cys Tyr Tyr Leu His Asp Leu Gln Val Asp Phe Leu Thr Glu

425 430 435

aag aat cgc agt cag ctt cag gat ctg cac agg aag atg gtc act cag 1941

Lys Asn Arg Ser Gln Leu Gln Asp Leu His Arg Lys Met Val Thr Gln

440 445 450

ttt cag agg tat tac cag ccc cac acg ctg tct cca gac cag gag gac 1989

Phe Gln Arg Tyr Tyr Gln Pro His Thr Leu Ser Pro Asp Gln Glu Asp

455 460 465

tgc atg tat tgg tac aac ttc cta gcc tat cac atg gct agt gcc aat 2037

Cys Met Tyr Trp Tyr Asn Phe Leu Ala Tyr His Met Ala Ser Ala Asn

470 475 480

atg cac aaa gaa ctt tgt gct tta atg ttt tcc ctg gac tgg att aaa 2085

Met His Lys Glu Leu Cys Ala Leu Met Phe Ser Leu Asp Trp Ile Lys

485 490 495 500

gca aaa aca gaa ctt gtc ggc cct gcc cat ctg att cac gag ttc gtg 2133

Ala Lys Thr Glu Leu Val Gly Pro Ala His Leu Ile His Glu Phe Val

505 510 515

gca tat agg cat ata ttg gat gaa aag gat tgt gca gtc tgt gag aat 2181

Ala Tyr Arg His Ile Leu Asp Glu Lys Asp Cys Ala Val Cys Glu Asn

520 525 530

ttt caa gag ttt tta tct tta aat gga cac ctc ctt gga cga cag cca 2229

Phe Gln Glu Phe Leu Ser Leu Asn Gly His Leu Leu Gly Arg Gln Pro

535 540 545

ttt cct aat att gta cag ctg ggc ctc tgt gaa cca gaa act tcc gaa 2277

Phe Pro Asn Ile Val Gln Leu Gly Leu Cys Glu Pro Glu Thr Ser Glu

550 555 560

gtt tat cga caa gca aag ctg cag gcc aag cag gag ggg gat act ggg 2325

Val Tyr Arg Gln Ala Lys Leu Gln Ala Lys Gln Glu Gly Asp Thr Gly

565 570 575 580

ccg ctt tac ctg gaa tgg ata aac aaa aaa act atc aag aat ctg tcc 2373

Pro Leu Tyr Leu Glu Trp Ile Asn Lys Lys Thr Ile Lys Asn Leu Ser

585 590 595

cgc tta gtc gtc cgc ccc cac aca gat gct gtt tac cac gcg tgt ttt 2421

Arg Leu Val Val Arg Pro His Thr Asp Ala Val Tyr His Ala Cys Phe

600 605 610

tct cag gat ggc cag aga ata gct tct tgt ggg gct gat aaa acc tta 2469

Ser Gln Asp Gly Gln Arg Ile Ala Ser Cys Gly Ala Asp Lys Thr Leu

615 620 625

cag gtg ttc aaa gcc gag aca gga gag aaa ctt ctt gac att aaa gct 2517

Gln Val Phe Lys Ala Glu Thr Gly Glu Lys Leu Leu Asp Ile Lys Ala

630 635 640

cat gaa gat gag gtg ctc tgc tgc gcg ttc tcc tca gac gac agt tac 2565

His Glu Asp Glu Val Leu Cys Cys Ala Phe Ser Ser Asp Asp Ser Tyr

645 650 655 660

ata gcg acc tgc tca gcg gat aag aag gtt aag att tgg gat tct gcg 2613

Ile Ala Thr Cys Ser Ala Asp Lys Lys Val Lys Ile Trp Asp Ser Ala

665 670 675

act ggg aag ctt gtg cac acc tac gac gag cac tcg gag caa gtc aat 2661

Thr Gly Lys Leu Val His Thr Tyr Asp Glu His Ser Glu Gln Val Asn

680 685 690

tgc tgc cat ttc acc aac agt agt aac cac ctt ctc ttg gcc acc ggg 2709

Cys Cys His Phe Thr Asn Ser Ser Asn His Leu Leu Leu Ala Thr Gly

695 700 705

tca aat gat ttc ttc ctc aag ctc tgg gat ttg aat caa aaa gaa tgt 2757

Ser Asn Asp Phe Phe Leu Lys Leu Trp Asp Leu Asn Gln Lys Glu Cys

710 715 720

cga aat acc atg ttt ggt cac acg aac tca gtc aac cac tgc agg ttc 2805

Arg Asn Thr Met Phe Gly His Thr Asn Ser Val Asn His Cys Arg Phe

725 730 735 740

tca cca gac gat gag ctc ttg gct agc tgc tca gct gac ggg act tta 2853

Ser Pro Asp Asp Glu Leu Leu Ala Ser Cys Ser Ala Asp Gly Thr Leu

745 750 755

agg ctt tgg gat gtg aga tca gca aac gag agg aaa agc att aat gtg 2901

Arg Leu Trp Asp Val Arg Ser Ala Asn Glu Arg Lys Ser Ile Asn Val

760 765 770

aag cgc ttc ttc ctg agt tca gaa gac cct cca gag gat gtg gag gtg 2949

Lys Arg Phe Phe Leu Ser Ser Glu Asp Pro Pro Glu Asp Val Glu Val

775 780 785

atc gtg aag tgt tgt tcc tgg tct gca gat ggt gac aaa ata ata gtg 2997

Ile Val Lys Cys Cys Ser Trp Ser Ala Asp Gly Asp Lys Ile Ile Val

790 795 800

gca gca aaa aac aaa gtc ctc ctt ttt gat att cat act agt ggc cta 3045

Ala Ala Lys Asn Lys Val Leu Leu Phe Asp Ile His Thr Ser Gly Leu

805 810 815 820

ttg gca gag atc cac aca ggc cat cac agc acc atc cag tac tgt gac 3093

Leu Ala Glu Ile His Thr Gly His His Ser Thr Ile Gln Tyr Cys Asp

825 830 835

ttc tcc ccc tat gac cat ttg gct gtg att gcc ctg tct cag tac tgt 3141

Phe Ser Pro Tyr Asp His Leu Ala Val Ile Ala Leu Ser Gln Tyr Cys

840 845 850

gtg gag ttg tgg aac ata gac tcc cgc cta aag gtg gcc gac tgc aga 3189

Val Glu Leu Trp Asn Ile Asp Ser Arg Leu Lys Val Ala Asp Cys Arg

855 860 865

gga cat ttg agt tgg gtt cac ggt gtg atg ttt tct ccc gat ggc tcc 3237

Gly His Leu Ser Trp Val His Gly Val Met Phe Ser Pro Asp Gly Ser

870 875 880

tca ttt ttg aca gct tct gat gac caa aca ata agg gtc tgg gag aca 3285

Ser Phe Leu Thr Ala Ser Asp Asp Gln Thr Ile Arg Val Trp Glu Thr

885 890 895 900

aaa aag gta tgc aag aac tct gct atc gtg cta aag cag gaa ata gac 3333

Lys Lys Val Cys Lys Asn Ser Ala Ile Val Leu Lys Gln Glu Ile Asp

905 910 915

gtc gtg ttt caa gag aac gaa acg atg gtc ctt gca gtt gac aac ata 3381

Val Val Phe Gln Glu Asn Glu Thr Met Val Leu Ala Val Asp Asn Ile

920 925 930

aga ggc ctg caa ctc att gct gga aaa aca ggc cag att gat tac ctg 3429

Arg Gly Leu Gln Leu Ile Ala Gly Lys Thr Gly Gln Ile Asp Tyr Leu

935 940 945

cct gaa gcc caa gtg agt tgc tgc tgc ctc agt cca cac ctt gag tac 3477

Pro Glu Ala Gln Val Ser Cys Cys Cys Leu Ser Pro His Leu Glu Tyr

950 955 960

gtg gca ttc gga gat gaa gat gga gcc att aag att ata gaa ctt cca 3525

Val Ala Phe Gly Asp Glu Asp Gly Ala Ile Lys Ile Ile Glu Leu Pro

965 970 975 980

aac aac aga gtc ttc agt tct ggg gtt ggg cac aag aaa gct gtg cgg 3573

Asn Asn Arg Val Phe Ser Ser Gly Val Gly His Lys Lys Ala Val Arg

985 990 995

cac atc cag ttc aca gct gat ggg aag aca ctg att tca agt tct gaa 3621

His Ile Gln Phe Thr Ala Asp Gly Lys Thr Leu Ile Ser Ser Ser Glu

1000 1005 1010

gat tct gtg att cag gta tgg aat tgg cag aca ggg gac tat gta ttt 3669

Asp Ser Val Ile Gln Val Trp Asn Trp Gln Thr Gly Asp Tyr Val Phe

1015 1020 1025

ttg caa gcc cac cag gaa acg gta aag gac ttc agg ctc ctc caa gat 3717

Leu Gln Ala His Gln Glu Thr Val Lys Asp Phe Arg Leu Leu Gln Asp

1030 1035 1040

tca aga ttg ctt tct tgg tca ttt gat gga acg gtg aag gtg tgg aat 3765

Ser Arg Leu Leu Ser Trp Ser Phe Asp Gly Thr Val Lys Val Trp Asn

1045 1050 1055 1060

gtc att acc gga aga ata gaa aga gac ttt act tgt cat cag ggc aca 3813

Val Ile Thr Gly Arg Ile Glu Arg Asp Phe Thr Cys His Gln Gly Thr

1065 1070 1075

gtg ctt tcc tgt gct atc tct tct gat gcg acc aag ttt tcc tct acc 3861

Val Leu Ser Cys Ala Ile Ser Ser Asp Ala Thr Lys Phe Ser Ser Thr

1080 1085 1090

tct gct gat aag act gcc aag atc tgg agt ttt gac ctc ctt tcc cct 3909

Ser Ala Asp Lys Thr Ala Lys Ile Trp Ser Phe Asp Leu Leu Ser Pro

1095 1100 1105

ctt cat gag ctg aag ggc cat aat ggc tgt gtc cgc tgc tct gcc ttc 3957

Leu His Glu Leu Lys Gly His Asn Gly Cys Val Arg Cys Ser Ala Phe

1110 1115 1120

tcg ctg gat ggc atc ctg ttg gca act gga gat gac aat gga gaa atc 4005

Ser Leu Asp Gly Ile Leu Leu Ala Thr Gly Asp Asp Asn Gly Glu Ile

1125 1130 1135 1140

cgg ata tgg aat gtc tca gat ggc cag ctt ctt cat tcg tgt gct ccg 4053

Arg Ile Trp Asn Val Ser Asp Gly Gln Leu Leu His Ser Cys Ala Pro

1145 1150 1155

atc tcg gta gag gaa gga act gct acc cac ggc ggc tgg gta act gat 4101

Ile Ser Val Glu Glu Gly Thr Ala Thr His Gly Gly Trp Val Thr Asp

1160 1165 1170

gtg tgc ttc tct ccc gac agt aaa acg ctt gtc tct gct gga gga tat 4149

Val Cys Phe Ser Pro Asp Ser Lys Thr Leu Val Ser Ala Gly Gly Tyr

1175 1180 1185

ctc aag tgg tgg aat gtt gcc act ggg gac tcc tca cag acc ttc tac 4197

Leu Lys Trp Trp Asn Val Ala Thr Gly Asp Ser Ser Gln Thr Phe Tyr

1190 1195 1200

aca aat gga aca aac ctc aag aaa atc cac gtg tcc cct gac ttc aga 4245

Thr Asn Gly Thr Asn Leu Lys Lys Ile His Val Ser Pro Asp Phe Arg

1205 1210 1215 1220

acc tat gtg act gtc gat aat ctc ggt att tta tat att tta cag gtt 4293

Thr Tyr Val Thr Val Asp Asn Leu Gly Ile Leu Tyr Ile Leu Gln Val

1225 1230 1235

tta gag taa aatggctaag cgttaatgta gttcagcttc cttaagtttg aattggaaaa 4352

Leu Glu

aatctaaacc ctctatatca acttttataa agctgtgaat tgtactgcag tattgcaaca 4412

tttacaaagg tggtttaaca gctgggccag tgctctgtat agttgggtga atcatagtat 4472

atctccacaa aggaacatac ttttactcta tttttttaaa tatagtcatc attgttatta 4532

acagtttgtc cttaacatgc aaatgaaatg gtaaatatgt accttgttat gctattggta 4592

aattctttga tgcattcaaa ttggttggca aaattagtga gaatcatttg aaggccttct 4652

ataggttgat gctgtcatta tgtaggctat gtcccaaggt aatggtggtc agttttctga 4712

accacactta ctcccagggg gtatggtttt ccaaataaat catgcttatt tgcactcttt 4772

aaatttgctt taagatattg tgtcatggtg caagcacagt ctgcaacatt tctcttgtta 4832

actcagtgag tattggtttt caacagttct tcctatgagc tagtcatgtg ttagagagat 4892

ttctctctgt agctctgtgt ttagctggac tctggctgtc agtgtctgtg aattgggcac 4952

caaccacctt gcactctttt agttgtttcg tttataaaga cagggtttat gtagtcctgg 5012

ctctcttggc actcagcatg tacaccaggt tagccttgaa ctcagagctc agcctgcctc 5072

tctgcctcct gagttctgga attgaaggcg tgtgccaaca gcacctctct ctctcagtta 5132

tactcgactg agcttgaccc 5152

11

23

Artificial Sequence

PCR Primer

11

aagcaggaaa tagacgtcgt gtt 23

12

23

Artificial Sequence

PCR Primer

12

caatgagttg caggcctctt atg 23

13

26

Artificial Sequence

PCR Probe

13

tgtcaactgc aaggaccatc gtttcg 26

14

20

Artificial Sequence

PCR Primer

14

ggcaaattca acggcacagt 20

15

20

Artificial Sequence

PCR Primer

15

gggtctcgct cctggaagct 20

16

27

Artificial Sequence

PCR Probe

16

aaggccgaga atgggaagct tgtcatc 27

17

atg gat gca aaa gct cga aat tgt ttg ctt caa cat aga gaa gct ctg 48

Met Asp Ala Lys Ala Arg Asn Cys Leu Leu Gln His Arg Glu Ala Leu

1 5 10 15

gaa aag gac atc aag aca tcc tac atc atg gat cac atg att agt gat 96

Glu Lys Asp Ile Lys Thr Ser Tyr Ile Met Asp His Met Ile Ser Asp

20 25 30

gga ttt tta aca ata tca gaa gag gaa aaa gta aga aat gag ccc act 144

Gly Phe Leu Thr Ile Ser Glu Glu Glu Lys Val Arg Asn Glu Pro Thr

35 40 45

caa cag caa aga gca gct atg ctg att aaa atg ata ctt aaa aaa gat 192

Gln Gln Gln Arg Ala Ala Met Leu Ile Lys Met Ile Leu Lys Lys Asp

50 55 60

aat gat tcc tac gta tca ttc tac aat gct cta cta cat gaa gga tat 240

Asn Asp Ser Tyr Val Ser Phe Tyr Asn Ala Leu Leu His Glu Gly Tyr

65 70 75 80

aaa gat ctt gct gcc ctt ctc cat gat ggc att cct gtt gtc tct tct 288

Lys Asp Leu Ala Ala Leu Leu His Asp Gly Ile Pro Val Val Ser Ser

85 90 95

tcc agt ggt aaa gat tca gtt agt gga ata act tcg tat gta agg aca 336

Ser Ser Gly Lys Asp Ser Val Ser Gly Ile Thr Ser Tyr Val Arg Thr

100 105 110

gtc ctg tgt gaa ggt gga gta cca cag agg cca gtt gtt ttt gtc aca 384

Val Leu Cys Glu Gly Gly Val Pro Gln Arg Pro Val Val Phe Val Thr

115 120 125

agg aag aag ctg gtg aat gca att cag cag aag ctc tcc aaa ttg aaa 432

Arg Lys Lys Leu Val Asn Ala Ile Gln Gln Lys Leu Ser Lys Leu Lys

130 135 140

ggt gaa cca gga tgg gtc acc ata cat gga atg gca ggc tgt ggg aag 480

Gly Glu Pro Gly Trp Val Thr Ile His Gly Met Ala Gly Cys Gly Lys

145 150 155 160

tct gta tta gct gca gaa gct gtt aga gat cat tcc ctt tta gaa ggt 528

Ser Val Leu Ala Ala Glu Ala Val Arg Asp His Ser Leu Leu Glu Gly

165 170 175

tgt ttc cca ggg gga gtg cat tgg gtt tca gtt ggg aaa caa gac aaa 576

Cys Phe Pro Gly Gly Val His Trp Val Ser Val Gly Lys Gln Asp Lys

180 185 190

tct ggg ctt ctg atg aaa ctg cag aat ctt tgc aca cgg ttg gat cag 624

Ser Gly Leu Leu Met Lys Leu Gln Asn Leu Cys Thr Arg Leu Asp Gln

195 200 205

gat gag agt ttt tcc cag agg ctt cca ctt aat att gaa gag gct aaa 672

Asp Glu Ser Phe Ser Gln Arg Leu Pro Leu Asn Ile Glu Glu Ala Lys

210 215 220

gac cgt ctc cgc att ctg atg ctt cgc aaa cac cca agg tct ctc ttg 720

Asp Arg Leu Arg Ile Leu Met Leu Arg Lys His Pro Arg Ser Leu Leu

225 230 235 240

atc ttg gat gat gtt tgg gac tct tgg gtg ttg aaa gct ttt gac agt 768

Ile Leu Asp Asp Val Trp Asp Ser Trp Val Leu Lys Ala Phe Asp Ser

245 250 255

cag tgt cag att ctt ctt aca acc aga gac aag agt gtt aca gat tca 816

Gln Cys Gln Ile Leu Leu Thr Thr Arg Asp Lys Ser Val Thr Asp Ser

260 265 270

gta atg ggt cct aaa tat gta gtc cct gtg gag agt tcc tta gga aag 864

Val Met Gly Pro Lys Tyr Val Val Pro Val Glu Ser Ser Leu Gly Lys

275 280 285

gaa aaa gga ctt gaa att tta tcc ctt ttt gtt aat atg aag aag gca 912

Glu Lys Gly Leu Glu Ile Leu Ser Leu Phe Val Asn Met Lys Lys Ala

290 295 300

gat ttg cca gaa caa gct cat agt att ata aaa gaa tgt aaa ggc tct 960

Asp Leu Pro Glu Gln Ala His Ser Ile Ile Lys Glu Cys Lys Gly Ser

305 310 315 320

ccc ctt gta gta tct tta att ggt gca ctt tta cgt gat ttt ccc aat 1008

Pro Leu Val Val Ser Leu Ile Gly Ala Leu Leu Arg Asp Phe Pro Asn

325 330 335

cgc tgg gag tac tac ctc aaa cag ctt cag aat aag cag ttt aag aga 1056

Arg Trp Glu Tyr Tyr Leu Lys Gln Leu Gln Asn Lys Gln Phe Lys Arg

340 345 350

ata agg aaa tct tcg tct tat gat tat gag gct cta gat gaa gcc atg 1104

Ile Arg Lys Ser Ser Ser Tyr Asp Tyr Glu Ala Leu Asp Glu Ala Met

355 360 365

tct ata agt gtt gaa atg ctc aga gaa gac atc aaa gat tat tac aca 1152

Ser Ile Ser Val Glu Met Leu Arg Glu Asp Ile Lys Asp Tyr Tyr Thr

370 375 380

gat ctt tcc atc ctt cag aag gac gtt aag gtg cct aca aag gtg tta 1200

Asp Leu Ser Ile Leu Gln Lys Asp Val Lys Val Pro Thr Lys Val Leu

385 390 395 400

tgt att ctc tgg gac atg gaa act gaa gaa gtt gaa gac ata ctg cag 1248

Cys Ile Leu Trp Asp Met Glu Thr Glu Glu Val Glu Asp Ile Leu Gln

405 410 415

gag ttt gta aat aag tct ctt tta ttc tgt gat cgg aat gga aag tcg 1296

Glu Phe Val Asn Lys Ser Leu Leu Phe Cys Asp Arg Asn Gly Lys Ser

420 425 430

ttt cgt tat tat tta cat gat ctt caa gta gat ttt ctt aca gag aag 1344

Phe Arg Tyr Tyr Leu His Asp Leu Gln Val Asp Phe Leu Thr Glu Lys

435 440 445

aat tgc agc cag ctt cag gat cta cat aag aag ata atc act cag ttt 1392

Asn Cys Ser Gln Leu Gln Asp Leu His Lys Lys Ile Ile Thr Gln Phe

450 455 460

cag aga tat cac cag ccg cat act ctt tca cca gat cag gaa gac tgt 1440

Gln Arg Tyr His Gln Pro His Thr Leu Ser Pro Asp Gln Glu Asp Cys

465 470 475 480

atg tat tgg tac aac ttt ctg gcc tat cac atg gcc agt gcc aag atg 1488

Met Tyr Trp Tyr Asn Phe Leu Ala Tyr His Met Ala Ser Ala Lys Met

485 490 495

cac aag gaa ctt tgt gct tta atg ttt tcc ctg gat tgg att aaa gca 1536

His Lys Glu Leu Cys Ala Leu Met Phe Ser Leu Asp Trp Ile Lys Ala

500 505 510

aaa aca gaa ctt gta ggc cct gct cat ctg att cat gaa ttt gtg gaa 1584

Lys Thr Glu Leu Val Gly Pro Ala His Leu Ile His Glu Phe Val Glu

515 520 525

tac aga cat ata cta gat gaa aag gat tgt gca gtc agt gag aat ttt 1632

Tyr Arg His Ile Leu Asp Glu Lys Asp Cys Ala Val Ser Glu Asn Phe

530 535 540

cag gag ttt tta tct tta aat gga cac ctt ctt gga cga cag cca ttt 1680

Gln Glu Phe Leu Ser Leu Asn Gly His Leu Leu Gly Arg Gln Pro Phe

545 550 555 560

cct aat att gta caa ctg ggt ctc tgt gag ccg gaa act tca gaa gtt 1728

Pro Asn Ile Val Gln Leu Gly Leu Cys Glu Pro Glu Thr Ser Glu Val

565 570 575

tat cag caa gct aag ctg cag gcc aag cag gag gtc gat aat gga atg 1776

Tyr Gln Gln Ala Lys Leu Gln Ala Lys Gln Glu Val Asp Asn Gly Met

580 585 590

ctt tac ctg gaa tgg ata aac aaa aaa aac atc acg aat ctt tcc cgc 1824

Leu Tyr Leu Glu Trp Ile Asn Lys Lys Asn Ile Thr Asn Leu Ser Arg

595 600 605

tta gtt gtc cgc ccc cac aca gat gct gtt tac cat gcc tgc ttt tct 1872

Leu Val Val Arg Pro His Thr Asp Ala Val Tyr His Ala Cys Phe Ser

610 615 620

gag gat ggt cag aga ata gct tct tgt gga gct gat aaa acc tta cag 1920

Glu Asp Gly Gln Arg Ile Ala Ser Cys Gly Ala Asp Lys Thr Leu Gln

625 630 635 640

gtg ttc aaa gct gaa aca gga gag aaa ctt cta gaa atc aag gct cat 1968

Val Phe Lys Ala Glu Thr Gly Glu Lys Leu Leu Glu Ile Lys Ala His

645 650 655

gag gat gaa gtg ctt tgt tgt gca ttc tct aca gat gac aga ttt ata 2016

Glu Asp Glu Val Leu Cys Cys Ala Phe Ser Thr Asp Asp Arg Phe Ile

660 665 670

gca acc tgc tca gtg gat aaa aaa gtg aag att tgg aat tct atg act 2064

Ala Thr Cys Ser Val Asp Lys Lys Val Lys Ile Trp Asn Ser Met Thr

675 680 685

ggg gaa cta gta cac acc tat gat gag cac tca gag caa gtc aat tgc 2112

Gly Glu Leu Val His Thr Tyr Asp Glu His Ser Glu Gln Val Asn Cys

690 695 700

tgc cat ttc acc aac agt agt cat cat ctt ctc tta gcc act ggg tca 2160

Cys His Phe Thr Asn Ser Ser His His Leu Leu Leu Ala Thr Gly Ser

705 710 715 720

agt gac tgc ttc ctc aaa ctt tgg gat ttg aat caa aaa gaa tgt cga 2208

Ser Asp Cys Phe Leu Lys Leu Trp Asp Leu Asn Gln Lys Glu Cys Arg

725 730 735

aat acc atg ttt ggt cat aca aat tca gtc aat cac tgc aga ttt tca 2256

Asn Thr Met Phe Gly His Thr Asn Ser Val Asn His Cys Arg Phe Ser

740 745 750

cca gat gat aag ctt ttg gct agt tgt tca gct gat gga acc tta aag 2304

Pro Asp Asp Lys Leu Leu Ala Ser Cys Ser Ala Asp Gly Thr Leu Lys

755 760 765

ctt tgg gat gcg aca tca gca aat gag agg aaa agc att aat gtg aaa 2352

Leu Trp Asp Ala Thr Ser Ala Asn Glu Arg Lys Ser Ile Asn Val Lys

770 775 780

cag ttc ttc cta aat ttg gag gac cct caa gag gat atg gaa gtg ata 2400

Gln Phe Phe Leu Asn Leu Glu Asp Pro Gln Glu Asp Met Glu Val Ile

785 790 795 800

gtg aag tgt tgt tcg tgg tct gct gat ggt gca agg ata atg gtg gca 2448

Val Lys Cys Cys Ser Trp Ser Ala Asp Gly Ala Arg Ile Met Val Ala

805 810 815

gca aaa aat aaa atc ttt ctt ttt gac att cat act agt ggc cta ttg 2496

Ala Lys Asn Lys Ile Phe Leu Phe Asp Ile His Thr Ser Gly Leu Leu

820 825 830

gga gaa atc cac acg ggc cat cac agc acc atc cag tac tgt gac ttc 2544

Gly Glu Ile His Thr Gly His His Ser Thr Ile Gln Tyr Cys Asp Phe

835 840 845

tcc cca caa aac cat ttg gca gtg gtt gct ttg tcc cag tac tgt gta 2592

Ser Pro Gln Asn His Leu Ala Val Val Ala Leu Ser Gln Tyr Cys Val

850 855 860

gag ttg tgg aat aca gac tca cgt tca aag gtg gct gat tgc aga gga 2640

Glu Leu Trp Asn Thr Asp Ser Arg Ser Lys Val Ala Asp Cys Arg Gly

865 870 875 880

cat tta agt tgg gtt cat ggt gtg atg ttt tct cct gat gga tca tca 2688

His Leu Ser Trp Val His Gly Val Met Phe Ser Pro Asp Gly Ser Ser

885 890 895

ttt ttg aca tct tct gat gac cag aca atc agg ctc tgg gag aca aag 2736

Phe Leu Thr Ser Ser Asp Asp Gln Thr Ile Arg Leu Trp Glu Thr Lys

900 905 910

aaa gta tgt aag aac tct gct gta atg tta aag caa gaa gta gat gtt 2784

Lys Val Cys Lys Asn Ser Ala Val Met Leu Lys Gln Glu Val Asp Val

915 920 925

gtg ttt caa gaa aat gaa gtg atg gtc ctt gca gtt gac cat ata aga 2832

Val Phe Gln Glu Asn Glu Val Met Val Leu Ala Val Asp His Ile Arg

930 935 940

cgt ctg caa ctc att aat gga aga aca ggt cag att gat tat ctg act 2880

Arg Leu Gln Leu Ile Asn Gly Arg Thr Gly Gln Ile Asp Tyr Leu Thr

945 950 955 960

gaa gct caa gtt agc tgc tgt tgc tta agt cca cat ctt cag tac att 2928

Glu Ala Gln Val Ser Cys Cys Cys Leu Ser Pro His Leu Gln Tyr Ile

965 970 975

gca ttt gga gat gaa aat gga gcc att gag att tta gaa ctt gta aac 2976

Ala Phe Gly Asp Glu Asn Gly Ala Ile Glu Ile Leu Glu Leu Val Asn

980 985 990

aat aga atc ttc cag tcc agg ttt cag cac aag aaa act gta tgg cac 3024

Asn Arg Ile Phe Gln Ser Arg Phe Gln His Lys Lys Thr Val Trp His

995 1000 1005

atc cag ttc aca gcc gat gag aag act ctt att tca agt tct gat gat 3072

Ile Gln Phe Thr Ala Asp Glu Lys Thr Leu Ile Ser Ser Ser Asp Asp

1010 1015 1020

gct gaa att cag gta tgg aat tgg caa ttg gac aaa tgt atc ttt cta 3120

Ala Glu Ile Gln Val Trp Asn Trp Gln Leu Asp Lys Cys Ile Phe Leu

1025 1030 1035 1040

cga ggc cat cag gaa aca gtg aaa gac ttt aga ctc ttg aaa aat tca 3168

Arg Gly His Gln Glu Thr Val Lys Asp Phe Arg Leu Leu Lys Asn Ser

1045 1050 1055

aga ctg ctt tct tgg tca ttt gat gga aca gtg aag gta tgg aat att 3216

Arg Leu Leu Ser Trp Ser Phe Asp Gly Thr Val Lys Val Trp Asn Ile

1060 1065 1070

att act gga aat aaa gaa aaa gac ttt gtc tgt cac cag ggt aca gta 3264

Ile Thr Gly Asn Lys Glu Lys Asp Phe Val Cys His Gln Gly Thr Val

1075 1080 1085

ctt tct tgt gac att tct cac gat gct acc aag ttt tca tct acc tct 3312

Leu Ser Cys Asp Ile Ser His Asp Ala Thr Lys Phe Ser Ser Thr Ser

1090 1095 1100

gct gac aag act gca aag atc tgg agt ttt gat ctc ctt ttg cca ctt 3360

Ala Asp Lys Thr Ala Lys Ile Trp Ser Phe Asp Leu Leu Leu Pro Leu

1105 1110 1115 1120

cat gaa ttg agg ggc cac aac ggc tgt gtg cgc tgc tct gcc ttc tct 3408

His Glu Leu Arg Gly His Asn Gly Cys Val Arg Cys Ser Ala Phe Ser

1125 1130 1135

gtg gac agt acc ctg ctg gca acg gga gat gac aat gga gaa atc agg 3456

Val Asp Ser Thr Leu Leu Ala Thr Gly Asp Asp Asn Gly Glu Ile Arg

1140 1145 1150

ata tgg aat gtc tca aac ggt gag ctt ctt cat ttg tgt gct ccg ctt 3504

Ile Trp Asn Val Ser Asn Gly Glu Leu Leu His Leu Cys Ala Pro Leu

1155 1160 1165

tca gaa gaa gga gct gct acc cat gga ggc tgg gtg act gac ctt tgc 3552

Ser Glu Glu Gly Ala Ala Thr His Gly Gly Trp Val Thr Asp Leu Cys

1170 1175 1180

ttt tct cca gat ggc aaa atg ctt atc tct gct gga gga tat att aag 3600

Phe Ser Pro Asp Gly Lys Met Leu Ile Ser Ala Gly Gly Tyr Ile Lys

1185 1190 1195 1200

tgg tgg aac gtt gtc act ggg gaa tcc tca cag acc ttc tac aca aat 3648

Trp Trp Asn Val Val Thr Gly Glu Ser Ser Gln Thr Phe Tyr Thr Asn

1205 1210 1215

gga acc aat ctt aag aaa ata cac gtg tcc cct gac ttc aaa aca tat 3696

Gly Thr Asn Leu Lys Lys Ile His Val Ser Pro Asp Phe Lys Thr Tyr

1220 1225 1230

gtg act gtg gat aat ctt ggt att tta tat att tta cag act tta gaa 3744

Val Thr Val Asp Asn Leu Gly Ile Leu Tyr Ile Leu Gln Thr Leu Glu

1235 1240 1245

taa 3747

18

454

Homo sapiens

18

aaaaacagaa cttgtaggcc ctgctcatct gattcatgaa tttgtggaat acagacatat 60

actagatgaa aaggtatata tattaacatg aaaaattagt gctaaaaagg aatctcattt 120

ttttttacat ttaattctag atttaatgat gggaatgagc agaatagaag gggtgaagat 180

aatttccctc ctttgatttt tggttattct aattaatttg cttcctaccc tactgggaaa 240

gtaggtagga ttgaagttta aaagctagga aaataagatg ttcantggtt tttttaaaaa 300

aagaatgtat taagaaatgt gtttgataat gagattttta ccctatgttt aaaagtccga 360

agaagattac tatatatttg ttacttaatg gttggctgtt caattttagt tcatataaag 420

aatatttcat ttaagctttt aaagtggcca gatc 454

19

605

Homo sapiens

19

aagtttatcg gcaagctaag ctgcaggcca agcaggaggt cgataatgga atgctttacc 60

tggaatggat gtaagtaggt taggagagaa accaaaggga gtggtgcgct aactatatca 120

ttatttttca ggtggtgaat acgatcactc caggaggatt taactacttt cgaaagggct 180

ggaactttta ataagcattc ttacttattg aaaagttcta gagaagtaag cataggagaa 240

aatgattgta ttttcaaaga atccatataa atagatgcct gtaaggcatt tggacaaatt 300

atcctaagta ttcagtacta agtgcttgac ttactgtaat ttagtatagt ggtgaggagc 360

atggactttg gagccacaga actgggtttg agcctcagtt ctaaaaccta ataattctgt 420

gactttgaac aagttacagg tttttctctc atatgtaaaa tggagataac aacctttgaa 480

ataagtgctg tttccatgaa gatttattta ttatttattt atttattttg agacagatct 540

cctatgttgc cangntggat gcattggtgt gatcttggtc actgcacctc tgctcccgga 600

tcaac 605

20

491

Homo sapiens

20

aagtccacat cttcagtaca ttgcatttgg agatgaaaat ggagccattg aggtattcag 60

tgctagtctt cagaatcttt ctgtacagaa ttaaataaaa ctaattatat gtctggcatt 120

gtgcacttct atttttattt atttttattt ttgaaatggg gtcttgctgt gtcacccacg 180

ctgagtacag tgacacgatc acagccgact gcagccttga ccttctgagc tcaagtgatt 240

ttcccactta agcctcctga gtagctggga ctaaaggtgt acgtcaccat gcccgactaa 300

tttttttttt tttgtagaat gggtccctgt gttgcccagg ctggtctcaa actcctgggt 360

ttaagcgatt gtccggcctc agcctcccaa agtgtgggga ttataggcat gggaactttt 420

gtgcctggct attgtttacc tttttttttt tgaatcaagg atccaccttg tttgcacagg 480

ctggaatgca a 491

21

aagaagaggt agcgagtgga cgtgactgct ctatcccggg caaaagggat agaaccagag 60

gtggggagtc tgggcagtcg gcgacccgcg aagacttgag gtgccgcagc ggcatccgga 120

gtagcgccgg gctccctccg gggtgcagcc gccgtcgggg gaagggcgcc acaggccggg 180

aagacctcct ccctttgtgt ccagtagtgg ggtccaccgg agggcggccc gtgggccggg 240

cctcaccgcg gcgctccggg actgtggggt caggctgcgt tgggtggacg cccacctcgc 300

caaccttcgg aggtccctgg gggtcttcgt gcgccccggg gctgcagaga tccaggggag 360

gcgcctgtga ggcccggacc tgccccgggg cgaagggtat gtggcgagac agagccctgc 420

acccctaatt cccggtggaa aactcctgtt gccgtttccc tccaccggcc tggagtctcc 480

cagtcttgtc ccggcagtgc cgccctcccc actaagacct aggcgcaaag gcttggctca 540

tggttgacag ctcagagaga gaaagatctg agggaag atg gat gca aaa gct cga 595

Met Asp Ala Lys Ala Arg

1 5

aat tgt ttg ctt caa cat aga gaa gct ctg gaa aag gac atc aag aca 643

Asn Cys Leu Leu Gln His Arg Glu Ala Leu Glu Lys Asp Ile Lys Thr

10 15 20

tcc tac atc atg gat cac atg att agt gat gga ttt tta aca ata tca 691

Ser Tyr Ile Met Asp His Met Ile Ser Asp Gly Phe Leu Thr Ile Ser

25 30 35

gaa gag gaa aaa gta aga aat gag ccc act caa cag caa aga gca gct 739

Glu Glu Glu Lys Val Arg Asn Glu Pro Thr Gln Gln Gln Arg Ala Ala

40 45 50

atg ctg att aaa atg ata ctt aaa aaa gat aat gat tcc tac gta tca 787

Met Leu Ile Lys Met Ile Leu Lys Lys Asp Asn Asp Ser Tyr Val Ser

55 60 65 70

ttc tac aat gct cta cta cat gaa gga tat aaa gat ctt gct gcc ctt 835

Phe Tyr Asn Ala Leu Leu His Glu Gly Tyr Lys Asp Leu Ala Ala Leu

75 80 85

ctc cat gat ggc att cct gtt gtc tct tct tcc agt gta agg aca gtc 883

Leu His Asp Gly Ile Pro Val Val Ser Ser Ser Ser Val Arg Thr Val

90 95 100

ctg tgt gaa ggt gga gta cca cag agg cca gtt gtt ttt gtc aca agg 931

Leu Cys Glu Gly Gly Val Pro Gln Arg Pro Val Val Phe Val Thr Arg

105 110 115

aag aag ctg gtg aat gca att cag cag aag ctc tcc aaa ttg aaa ggt 979

Lys Lys Leu Val Asn Ala Ile Gln Gln Lys Leu Ser Lys Leu Lys Gly

120 125 130

gaa cca gga tgg gtc acc ata cat gga atg gca ggc tgt ggg aag tct 1027

Glu Pro Gly Trp Val Thr Ile His Gly Met Ala Gly Cys Gly Lys Ser

135 140 145 150

gta tta gct gca gaa gct gtt aga gat cat tcc ctt tta gaa ggt tgt 1075

Val Leu Ala Ala Glu Ala Val Arg Asp His Ser Leu Leu Glu Gly Cys

155 160 165

ttc cca ggg gga gtg cat tgg gtt tca gtt ggg aaa caa gac aaa tct 1123

Phe Pro Gly Gly Val His Trp Val Ser Val Gly Lys Gln Asp Lys Ser

170 175 180

ggg ctt ctg atg aaa ctg cag aat ctt tgc aca cgg ttg gat cag gat 1171

Gly Leu Leu Met Lys Leu Gln Asn Leu Cys Thr Arg Leu Asp Gln Asp

185 190 195

gag agt ttt tcc cag agg ctt cca ctt aat att gaa gag gct aaa gac 1219

Glu Ser Phe Ser Gln Arg Leu Pro Leu Asn Ile Glu Glu Ala Lys Asp

200 205 210

cgt ctc cgc att ctg atg ctt cgc aaa cac cca agg tct ctc ttg atc 1267

Arg Leu Arg Ile Leu Met Leu Arg Lys His Pro Arg Ser Leu Leu Ile

215 220 225 230

ttg gat gat gtt tgg gac tct tgg gtg ttg aaa gct ttt gac agt cag 1315

Leu Asp Asp Val Trp Asp Ser Trp Val Leu Lys Ala Phe Asp Ser Gln

235 240 245

tgt cag att ctt ctt aca acc aga gac aag agt gtt aca gat tca gta 1363

Cys Gln Ile Leu Leu Thr Thr Arg Asp Lys Ser Val Thr Asp Ser Val

250 255 260

atg ggt cct aaa tat gta gtc cct gtg gag agt tcc tta gga aag gaa 1411

Met Gly Pro Lys Tyr Val Val Pro Val Glu Ser Ser Leu Gly Lys Glu

265 270 275

aaa gga ctt gaa att tta tcc ctt ttt gtt aat atg aag aag gca gat 1459

Lys Gly Leu Glu Ile Leu Ser Leu Phe Val Asn Met Lys Lys Ala Asp

280 285 290

ttg cca gaa caa gct cat agt att ata aaa gaa tgt aaa ggc tct ccc 1507

Leu Pro Glu Gln Ala His Ser Ile Ile Lys Glu Cys Lys Gly Ser Pro

295 300 305 310

ctt gta gta tct tta att ggt gca ctt tta cgt gat ttt ccc aat cgc 1555

Leu Val Val Ser Leu Ile Gly Ala Leu Leu Arg Asp Phe Pro Asn Arg

315 320 325

tgg gag tac tac ctc aaa cag ctt cag aat aag cag ttt aag aga ata 1603

Trp Glu Tyr Tyr Leu Lys Gln Leu Gln Asn Lys Gln Phe Lys Arg Ile

330 335 340

agg aaa tct tcg tct tat gat tat gag gct cta gat gaa gcc atg tct 1651

Arg Lys Ser Ser Ser Tyr Asp Tyr Glu Ala Leu Asp Glu Ala Met Ser

345 350 355

ata agt gtt gaa atg ctc aga gaa gac atc aaa gat tat tac aca gat 1699

Ile Ser Val Glu Met Leu Arg Glu Asp Ile Lys Asp Tyr Tyr Thr Asp

360 365 370

ctt tcc atc ctt cag aag gac gtt aag gtg cct aca aag gtg tta tgt 1747

Leu Ser Ile Leu Gln Lys Asp Val Lys Val Pro Thr Lys Val Leu Cys

375 380 385 390

att ctc tgg gac atg gaa act gaa gaa gtt gaa gac ata ctg cag gag 1795

Ile Leu Trp Asp Met Glu Thr Glu Glu Val Glu Asp Ile Leu Gln Glu

395 400 405

ttt gta aat aag tct ctt tta ttc tgt gat cgg aat gga aag tcg ttt 1843

Phe Val Asn Lys Ser Leu Leu Phe Cys Asp Arg Asn Gly Lys Ser Phe

410 415 420

cgt tat tat tta cat gat ctt caa gta gat ttt ctt aca gag aag aat 1891

Arg Tyr Tyr Leu His Asp Leu Gln Val Asp Phe Leu Thr Glu Lys Asn

425 430 435

tgc agc cag ctt cag gat cta cat aag aag ata atc act cag ttt cag 1939

Cys Ser Gln Leu Gln Asp Leu His Lys Lys Ile Ile Thr Gln Phe Gln

440 445 450

aga tat cac cag ccg cat act ctt tca cca gat cag gaa gac tgt atg 1987

Arg Tyr His Gln Pro His Thr Leu Ser Pro Asp Gln Glu Asp Cys Met

455 460 465 470

tat tgg tac aac ttt ctg gcc tat cac atg gcc agt gcc aag atg cac 2035

Tyr Trp Tyr Asn Phe Leu Ala Tyr His Met Ala Ser Ala Lys Met His

475 480 485

aag gaa ctt tgt gct tta atg ttt tcc ctg gat tgg att aaa gca aaa 2083

Lys Glu Leu Cys Ala Leu Met Phe Ser Leu Asp Trp Ile Lys Ala Lys

490 495 500

aca gaa ctt gta ggc cct gct cat ctg att cat gaa ttt gtg gaa tac 2131

Thr Glu Leu Val Gly Pro Ala His Leu Ile His Glu Phe Val Glu Tyr

505 510 515

aga cat ata cta gat gaa aag gat tgt gca gtc agt gag aat ttt cag 2179

Arg His Ile Leu Asp Glu Lys Asp Cys Ala Val Ser Glu Asn Phe Gln

520 525 530

gag ttt tta tct tta aat gga cac ctt ctt gga cga cag cca ttt cct 2227

Glu Phe Leu Ser Leu Asn Gly His Leu Leu Gly Arg Gln Pro Phe Pro

535 540 545 550

aat att gta caa ctg ggt ctc tgt gag ccg gaa act tca gaa gtt tat 2275

Asn Ile Val Gln Leu Gly Leu Cys Glu Pro Glu Thr Ser Glu Val Tyr

555 560 565

cag caa gct aag ctg cag gcc aag cag gag gtc gat aat gga atg ctt 2323

Gln Gln Ala Lys Leu Gln Ala Lys Gln Glu Val Asp Asn Gly Met Leu

570 575 580

tac ctg gaa tgg ata aac aaa aaa aac atc acg aat ctt tcc cgc tta 2371

Tyr Leu Glu Trp Ile Asn Lys Lys Asn Ile Thr Asn Leu Ser Arg Leu

585 590 595

gtt gtc cgc ccc cac aca gat gct gtt tac cat gcc tgc ttt tct gag 2419

Val Val Arg Pro His Thr Asp Ala Val Tyr His Ala Cys Phe Ser Glu

600 605 610

gat ggt cag aga ata gct tct tgt gga gct gat aaa acc tta cag gtg 2467

Asp Gly Gln Arg Ile Ala Ser Cys Gly Ala Asp Lys Thr Leu Gln Val

615 620 625 630

ttc aaa gct gaa aca gga gag aaa ctt cta gaa atc aag gct cat gag 2515

Phe Lys Ala Glu Thr Gly Glu Lys Leu Leu Glu Ile Lys Ala His Glu

635 640 645

gat gaa gtg ctt tgt tgt gca ttc tct aca gat gac aga ttt ata gca 2563

Asp Glu Val Leu Cys Cys Ala Phe Ser Thr Asp Asp Arg Phe Ile Ala

650 655 660

acc tgc tca gtg gat aaa aaa gtg aag att tgg aat tct atg act ggg 2611

Thr Cys Ser Val Asp Lys Lys Val Lys Ile Trp Asn Ser Met Thr Gly

665 670 675

gaa cta gta cac acc tat gat gag cac tca gag caa gtc aat tgc tgc 2659

Glu Leu Val His Thr Tyr Asp Glu His Ser Glu Gln Val Asn Cys Cys

680 685 690

cat ttc acc aac agt agt cat cat ctt ctc tta gcc act ggg tca agt 2707

His Phe Thr Asn Ser Ser His His Leu Leu Leu Ala Thr Gly Ser Ser

695 700 705 710

gac tgc ttc ctc aaa ctt tgg gat ttg aat caa aaa gaa tgt cga aat 2755

Asp Cys Phe Leu Lys Leu Trp Asp Leu Asn Gln Lys Glu Cys Arg Asn

715 720 725

acc atg ttt ggt cat aca aat tca gtc aat cac tgc aga ttt tca cca 2803

Thr Met Phe Gly His Thr Asn Ser Val Asn His Cys Arg Phe Ser Pro

730 735 740

gat gat aag ctt ttg gct agt tgt tca gct gat gga acc tta aag ctt 2851

Asp Asp Lys Leu Leu Ala Ser Cys Ser Ala Asp Gly Thr Leu Lys Leu

745 750 755

tgg gat gcg aca tca gca aat gag agg aaa agc att aat gtg aaa cag 2899

Trp Asp Ala Thr Ser Ala Asn Glu Arg Lys Ser Ile Asn Val Lys Gln

760 765 770

ttc ttc cta aat ttg gag gac cct caa gag gat atg gaa gtg ata gtg 2947

Phe Phe Leu Asn Leu Glu Asp Pro Gln Glu Asp Met Glu Val Ile Val

775 780 785 790

aag tgt tgt tcg tgg tct gct gat ggt gca agg ata atg gtg gca gca 2995

Lys Cys Cys Ser Trp Ser Ala Asp Gly Ala Arg Ile Met Val Ala Ala

795 800 805

aaa aat aaa atc ttt ttg tgg aat aca gac tca cgt tca aag gtg gct 3043

Lys Asn Lys Ile Phe Leu Trp Asn Thr Asp Ser Arg Ser Lys Val Ala

810 815 820

gat tgc aga gga cat tta agt tgg gtt cat ggt gtg atg ttt tct cct 3091

Asp Cys Arg Gly His Leu Ser Trp Val His Gly Val Met Phe Ser Pro

825 830 835

gat gga tca tca ttt ttg aca tct tct gat gac cag aca atc agg ctc 3139

Asp Gly Ser Ser Phe Leu Thr Ser Ser Asp Asp Gln Thr Ile Arg Leu

840 845 850

tgg gag aca aag aaa gta tgt aag aac tct gct gta atg tta aag caa 3187

Trp Glu Thr Lys Lys Val Cys Lys Asn Ser Ala Val Met Leu Lys Gln

855 860 865 870

gaa gta gat gtt gtg ttt caa gaa aat gaa gtg atg gtc ctt gca gtt 3235

Glu Val Asp Val Val Phe Gln Glu Asn Glu Val Met Val Leu Ala Val

875 880 885

gac cat ata aga cgt ctg caa ctc att aat gga aga aca ggt cag att 3283

Asp His Ile Arg Arg Leu Gln Leu Ile Asn Gly Arg Thr Gly Gln Ile

890 895 900

gat tat ctg act gaa gct caa gtt agc tgc tgt tgc tta agt cca cat 3331

Asp Tyr Leu Thr Glu Ala Gln Val Ser Cys Cys Cys Leu Ser Pro His

905 910 915

ctt cag tac att gca ttt gga gat gaa aat gga gcc att gag att tta 3379

Leu Gln Tyr Ile Ala Phe Gly Asp Glu Asn Gly Ala Ile Glu Ile Leu

920 925 930

gaa ctt gta aac aat aga atc ttc cag tcc agg ttt cag cac aag aaa 3427

Glu Leu Val Asn Asn Arg Ile Phe Gln Ser Arg Phe Gln His Lys Lys

935 940 945 950

act gta tgg cac atc cag ttc aca gcc gat gag aag act ctt att tca 3475

Thr Val Trp His Ile Gln Phe Thr Ala Asp Glu Lys Thr Leu Ile Ser

955 960 965

agt tct gat gat gct gaa att cag gta tgg aat tgg caa ttg gac aaa 3523

Ser Ser Asp Asp Ala Glu Ile Gln Val Trp Asn Trp Gln Leu Asp Lys

970 975 980

tgt atc ttt cta cga ggc cat cag gaa aca gtg aaa gac ttt aga ctc 3571

Cys Ile Phe Leu Arg Gly His Gln Glu Thr Val Lys Asp Phe Arg Leu

985 990 995

ttg aaa aat tca aga ctg ctt tct tgg tca ttt gat gga aca gtg aag 3619

Leu Lys Asn Ser Arg Leu Leu Ser Trp Ser Phe Asp Gly Thr Val Lys

1000 1005 1010

gta tgg aat att att act gga aat aaa gaa aaa gac ttt gtc tgt cac 3667

Val Trp Asn Ile Ile Thr Gly Asn Lys Glu Lys Asp Phe Val Cys His

1015 1020 1025 1030

cag ggt aca gta ctt tct tgt gac att tct cac gat gct acc aag ttt 3715

Gln Gly Thr Val Leu Ser Cys Asp Ile Ser His Asp Ala Thr Lys Phe

1035 1040 1045

tca tct acc tct gct gac aag act gca aag atc tgg agt ttt gat ctc 3763

Ser Ser Thr Ser Ala Asp Lys Thr Ala Lys Ile Trp Ser Phe Asp Leu

1050 1055 1060

ctt ttg cca ctt cat gaa ttg agg ggc cac aac ggc tgt gtg cgc tgc 3811

Leu Leu Pro Leu His Glu Leu Arg Gly His Asn Gly Cys Val Arg Cys

1065 1070 1075

tct gcc ttc tct gtg gac agt acc ctg ctg gca acg gga gat gac aat 3859

Ser Ala Phe Ser Val Asp Ser Thr Leu Leu Ala Thr Gly Asp Asp Asn

1080 1085 1090

gga gaa atc agg ata tgg aat gtc tca aac ggt gag ctt ctt cat ttg 3907

Gly Glu Ile Arg Ile Trp Asn Val Ser Asn Gly Glu Leu Leu His Leu

1095 1100 1105 1110

tgt gct ccg ctt tca gaa gaa gga gct gct acc cat gga ggc tgg gtg 3955

Cys Ala Pro Leu Ser Glu Glu Gly Ala Ala Thr His Gly Gly Trp Val

1115 1120 1125

act gac ctt tgc ttt tct cca gat ggc aaa atg ctt atc tct gct gga 4003

Thr Asp Leu Cys Phe Ser Pro Asp Gly Lys Met Leu Ile Ser Ala Gly

1130 1135 1140

gga tat att aag tgg tgg aac gtt gtc act ggg gaa tcc tca cag acc 4051

Gly Tyr Ile Lys Trp Trp Asn Val Val Thr Gly Glu Ser Ser Gln Thr

1145 1150 1155

ttc tac aca aat gga acc aat ctt aag aaa ata cac gtg tcc cct gac 4099

Phe Tyr Thr Asn Gly Thr Asn Leu Lys Lys Ile His Val Ser Pro Asp

1160 1165 1170

ttc aaa aca tat gtg act gtg gat aat ctt ggt att tta tat att tta 4147

Phe Lys Thr Tyr Val Thr Val Asp Asn Leu Gly Ile Leu Tyr Ile Leu

1175 1180 1185 1190

cag act tta gaa taa aatagttaag cattaatgta gttgaacttt ttaaattttt 4202

Gln Thr Leu Glu

1195

gaattggaaa aaaattctaa tgaaaccctg atatcaactt tttataaagc tcttaattgt 4262

tgtgcagtat tgcattcatt acaaaagtgt ttgtggttgg atgaataata ttaatgtagc 4322

tttttcccaa atgaacatac ctttaatctt gtttttcatg atcatcatta acagtttgtc 4382

cttaggatgc aaatgaaaat gtgaatacat accttgttgt actgttggta aaattctgtc 4442

ttgatgcatt caaaatggtt gacataatta atgagaagaa tttggaagaa attggtattt 4502

taatactgtc tgtatttatt actgttatgc aggctgtgcc tcagggtagc agtggcctgc 4562

tttttgaacc acacttaccc caagggggtt ttgttctcct aaatacaatc ttagaggttt 4622

tttgcactct ttaaatttgc tttaaaaata ttgtgtctgt gtgcatagtc tgcagcattt 4682

cctttaattg actcaataag tgagtcttgg atttagcagg cccccccacc tttttttttt 4742

gtttttggag acagagtctt gctttgttgc caggctggag tgcagtggcg cgatctcggc 4802

tcaccacaat cgctgcctcc tgggttcaag caattctcct gcctcagcct cccgagtagc 4862

tgggactaca ggtgtgcgca catgccaggc taatttttgt atttttagta gagacggggt 4922

ttcaccatgt tggccgggat ggtctcgatc tcttgacctc atgatctacc cgccttggcc 4982

tcccaaagtg ctgagattac aggcgtgagc caccgtgcct ggccaggccc cttctctttt 5042

aatggagaca gggtcttgca ctatcaccca ggctggagtg cagtggcata atcatacctc 5102

attgcagcct cagactcctg ggttcaagca atcctcctgc ctcagcctcc caagtagctg 5162

agactgcagg cacgagccac cacacccagc taatttttaa gttttcttgt agagacaggg 5222

tctcactatg ttgtctaggc tggtcttgaa ctcttggcct caagtaatcc tcctgcctca 5282

gcctcccaaa gtgttgggat tgcagatatg agccactggc ctggccttca gcagttcttt 5342

ttgtgaagta aaacttgtat gttggaaaga gtagatttta ttggtctacc cttttctcac 5402

tgtagctgct ggcagccctg tgccatatct ggactctagt tgtcagtatc tgagttggac 5462

actattcctg ctccctcttg tttcttacat atcagacttc ttacttgaat gaaacctgat 5522

ctttcctaat cctcactttt ttctttttta aaaagcagtt tctccactgc taaatgttag 5582

tcattgaggt ggggccaatt ttaatcataa gccttaataa gatttttcta agaaatgtga 5642

aatagaacaa ttttcatcta attccattta cttttagatg aatggcattg tgaatgccat 5702

tcttttaatg aatttcaaga gaattctctg gttttctgtg taattccaga tgagtcactg 5762

taactctaga agattaacct tccagccaac ctattttcct ttcccttgtc tctctcatcc 5822

tcttttcctt ccttctttcc tttctcttct tttatctcca aggttaatca ggaaaaatag 5882

cttttgacag gggaaaaaac tcaataacta gctatttttg acctcctgat caggaacttt 5942

agttgaagcg taaatctaaa gaaacatttt ctctgaaata tattattaag ggcaatggag 6002

ataaattaat agtagatgtg gttcccagaa aatataatca aaattcaaag attttttttg 6062

tttctgtaac tggaactaaa tcaaatgatt actagtgtta atagtagata acttgttttt 6122

attgttggtg catattagta taactgtggg gtaggtcggg gagagggtaa gggaatagat 6182

cactcagatg tattttagat aagctattta gcctttgatg gaatcataaa tacagtgaat 6242

acaatccttt gcattgttaa ggaggttttt tgtttttaaa tggtgggtca aggagctagt 6302

ttacaggctt actgtgattt aagcaaatgt gaaaagtgaa accttaattt tatcaaaaga 6362

aatttctgta aatggtatgt ctccttagaa tacccaaatc ataattttat ttgtacacac 6422

tgttaggggc tcatctcatg taggcagagt ataaagtatt accttttgga attaaaagcc 6482

actgactgtt ataaagtata acaacacaca tcaggtttta aaaagccttg aatggccctt 6542

gtcttaaaaa gaaattagga gccaggtgcg gtggcacgtg cctgtagtcc cagctccttg 6602

ggaggctgag acaggaggat tccttgagcc ctggagtttg agtccagcct gggtgacata 6662

gcaagaccct gtcttaaaag aaaaatggga agaaagacaa ggtaacatga agaaagaaga 6722

gatacctagt atgatggagc tgcaaatttc atggcagttc atgcagtcgg tcaagaggag 6782

gattttgttt tgtagtttgc agatgagcat ttctaaagca ttttcccttg ctgtattttt 6842

ttgtattata aattacattg gacttcatat atataatttt tttttacatt atatgtctct 6902

tgtatgtttt gaaactcttg tatttatgat atagcttata tgattttttt gccttggtat 6962

acattttaaa atatgaattt aaaaaatttt tgtaaaaata aaattcacaa aattgttttg 7022

aaaaacaaaa aaaaaaaaaa 7042

22

359

Homo sapiens

22

cattatatgt ctcttgtatg ttttgaaact cttgtattta tgatatagct tatatgattt 60

ttttgccttg gtatacattt taaaatatga atttaaaaaa tttttgtaaa aataaaattc 120

acaaaattgt tttgaaaaac atttttggat tgtttcattc tttgcttgtc atttatctgt 180

tgattagacc actaaagtga aggattcaag ctaaatacat caacctttct atttaggctt 240

tatcagctat atgtaaattc aattctatca aaattttctg agtgcctcct cagtgtgtct 300

ctctgatggt tcctgcccgg tatggctggc atgaagaaga tcctgtaaaa aagagaatt 359

23

20

Artificial Sequence

Antisense Oligonucleotide

23

caggactgtc cttacatacg 20

24

20

Artificial Sequence

Antisense Oligonucleotide

24

caataggcca ctagtatgaa 20

25

20

Artificial Sequence

Antisense Oligonucleotide

25

ctggatggtg ctgtgatggc 20

26

20

Artificial Sequence

Antisense Oligonucleotide

26

acagtactgg atggtgctgt 20

27

20

Artificial Sequence

Antisense Oligonucleotide

27

gggagaagtc acagtactgg 20

28

20

Artificial Sequence

Antisense Oligonucleotide

28

cccaagcctt tgcgcctagg 20

29

20

Artificial Sequence

Antisense Oligonucleotide

29

cataacagaa ttttcttcct 20

30

20

Artificial Sequence

Antisense Oligonucleotide

30

aatgagattc ctttttagca 20

31

20

Artificial Sequence

Antisense Oligonucleotide

31

tgaacatctt attttcctag 20

32

20

Artificial Sequence

Antisense Oligonucleotide

32

tatagtaatc ttcttcggac 20

33

20

Artificial Sequence

Antisense Oligonucleotide

33

aatcctcctg gagtgatcgt 20

34

20

Artificial Sequence

Antisense Oligonucleotide

34

atacctcaat ggctccattt 20

35

20

Artificial Sequence

Antisense Oligonucleotide

35

gctgaggccg gacaatcgct 20

36

20

Artificial Sequence

Antisense Oligonucleotide

36

ccccagggac ctccgaaggt 20

37

20

Artificial Sequence

Antisense Oligonucleotide

37

caaccatgag ccaagccttt 20

38

20

Artificial Sequence

Antisense Oligonucleotide

38

tttgcatcca tcttccctca 20

39

20

Artificial Sequence

Antisense Oligonucleotide

39

ctatgttgaa gcaaacaatt 20

40

20

Artificial Sequence

Antisense Oligonucleotide

40

gcttctctat gttgaagcaa 20

41

20

Artificial Sequence

Antisense Oligonucleotide

41

tcatgtgatc catgatgtag 20

42

20

Artificial Sequence

Antisense Oligonucleotide

42

ggtactccac cttcacacag 20

43

20

Artificial Sequence

Antisense Oligonucleotide

43

agcccagatt tgtcttgttt 20

44

20

Artificial Sequence

Antisense Oligonucleotide

44

aatattaagt ggaagcctct 20

45

20

Artificial Sequence

Antisense Oligonucleotide

45

agagaccttg ggtgtttgcg 20

46

20

Artificial Sequence

Antisense Oligonucleotide

46

cccaaacatc atccaagatc 20

47

20

Artificial Sequence

Antisense Oligonucleotide

47

gtaagaagaa tctgacactg 20

48

20

Artificial Sequence

Antisense Oligonucleotide

48

actgaatctg taacactctt 20

49

20

Artificial Sequence

Antisense Oligonucleotide

49

cccattactg aatctgtaac 20

50

20

Artificial Sequence

Antisense Oligonucleotide

50

ttaggaccca ttactgaatc 20

51

20

Artificial Sequence

Antisense Oligonucleotide

51

tatttaggac ccattactga 20

52

20

Artificial Sequence

Antisense Oligonucleotide

52

ttcttcatat taacaaaaag 20

53

20

Artificial Sequence

Antisense Oligonucleotide

53

cgtaaaagtg caccaattaa 20

54

20

Artificial Sequence

Antisense Oligonucleotide

54

ttgggaaaat cacgtaaaag 20

55

20

Artificial Sequence

Antisense Oligonucleotide

55

cagcgattgg gaaaatcacg 20

56

20

Artificial Sequence

Antisense Oligonucleotide

56

tcttaaactg cttattctga 20

57

20

Artificial Sequence

Antisense Oligonucleotide

57

ccttattctc ttaaactgct 20

58

20

Artificial Sequence

Antisense Oligonucleotide

58

tagagcctca taatcataag 20

59

20

Artificial Sequence

Antisense Oligonucleotide

59

ttcatctaga gcctcataat 20

60

20

Artificial Sequence

Antisense Oligonucleotide

60

catggcttca tctagagcct 20

61

20

Artificial Sequence

Antisense Oligonucleotide

61

tctgagcatt tcaacactta 20

62

20

Artificial Sequence

Antisense Oligonucleotide

62

ttgatgtctt ctctgagcat 20

63

20

Artificial Sequence

Antisense Oligonucleotide

63

gtccttctga aggatggaaa 20

64

20

Artificial Sequence

Antisense Oligonucleotide

64

tacataacac ctttgtaggc 20

65

20

Artificial Sequence

Antisense Oligonucleotide

65

ctgtaagaaa atctacttga 20

66

20

Artificial Sequence

Antisense Oligonucleotide

66

tcttctctgt aagaaaatct 20

67

20

Artificial Sequence

Antisense Oligonucleotide

67

tccagggaaa acattaaagc 20

68

20

Artificial Sequence

Antisense Oligonucleotide

68

ctgcacaatc cttttcatct 20

69

20

Artificial Sequence

Antisense Oligonucleotide

69

actgcacaat ccttttcatc 20

70

20

Artificial Sequence

Antisense Oligonucleotide

70

gtgtccattt aaagataaaa 20

71

20

Artificial Sequence

Antisense Oligonucleotide

71

ggaaatggct gtcgtccaag 20

72

20

Artificial Sequence

Antisense Oligonucleotide

72

tgtacaatat taggaaatgg 20

73

20

Artificial Sequence

Antisense Oligonucleotide

73

tcctgcttgg cctgcagctt 20

74

20

Artificial Sequence

Antisense Oligonucleotide

74

tttatccatt ccaggtaaag 20

75

20

Artificial Sequence

Antisense Oligonucleotide

75

gtaaacagca tctgtgtggg 20

76

20

Artificial Sequence

Antisense Oligonucleotide

76

ccacaagaag ctattctctg 20

77

20

Artificial Sequence

Antisense Oligonucleotide

77

acctgtaagg ttttatcagc 20

78

20

Artificial Sequence

Antisense Oligonucleotide

78

ttgaacacct gtaaggtttt 20

79

20

Artificial Sequence

Antisense Oligonucleotide

79

ctttgaacac ctgtaaggtt 20

80

20

Artificial Sequence

Antisense Oligonucleotide

80

atggcagcaa ttgacttgct 20

81

20

Artificial Sequence

Antisense Oligonucleotide

81

actgttggtg aaatggcagc 20

82

20

Artificial Sequence

Antisense Oligonucleotide

82

gacattcttt ttgattcaaa 20

83

20

Artificial Sequence

Antisense Oligonucleotide

83

catggtattt cgacattctt 20

84

20

Artificial Sequence

Antisense Oligonucleotide

84

accaaacatg gtatttcgac 20

85

20

Artificial Sequence

Antisense Oligonucleotide

85

catcccaaag ctttaaggtt 20

86

20

Artificial Sequence

Antisense Oligonucleotide

86

acattaatgc ttttcctctc 20

87

20

Artificial Sequence

Antisense Oligonucleotide

87

tctcccagag cctgattgtc 20

88

20

Artificial Sequence

Antisense Oligonucleotide

88

gttctaaaat ctcaatggct 20

89

20

Artificial Sequence

Antisense Oligonucleotide

89

tgccaattcc atacctgaat 20

90

20

Artificial Sequence

Antisense Oligonucleotide

90

ttccatcaaa tgaccaagaa 20

91

20

Artificial Sequence

Antisense Oligonucleotide

91

aactccagat ctttgcagtc 20

92

20

Artificial Sequence

Antisense Oligonucleotide

92

atttctccat tgtcatctcc 20

93

20

Artificial Sequence

Antisense Oligonucleotide

93

cgttccacca cttaatatat 20

94

20

Artificial Sequence

Antisense Oligonucleotide

94

gtgtagaagg tctgtgagga 20

95

20

Artificial Sequence

Antisense Oligonucleotide

95

ccatttgtgt agaaggtctg 20

96

20

Artificial Sequence

Antisense Oligonucleotide

96

aactatttta ttctaaagtc 20

97

20

Artificial Sequence

Antisense Oligonucleotide

97

gccaggccag tggctcatat 20

98

20

Artificial Sequence

Antisense Oligonucleotide

98

cctcttgacc gactgcatga 20

99

20

Artificial Sequence

Antisense Oligonucleotide

99

gaattttatt tttacaaaaa 20

100

20

Artificial Sequence

Antisense Oligonucleotide

100

acactgagga ggcactcaga 20