Antisense modulation of hematopoietic cell protein tyrosine kinase expression

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

Cells in higher animals normally divide only when they are stimulated by growth factors produced by other cells and act by binding to receptor tyrosine kinases on dividing cells. Cancer cells proliferate excessively because, as a result of accumulated mutations, they are able to divide without stimulation from other cells and therefore are no longer subject to normal controls on cell proliferation. The mutated genes which lead to excessive proliferation were originally called oncogenes before their origin as normal genes was understood. The normal genes, from which they arise, are thus often referred to as proto-oncogenes.

The cellular src gene was the first molecularly-defined proto-oncogene and its product, Src, is now known to be the source of the first detected tyrosine phosphorylation event. Mutations in the src gene, captured in the genome of Rous sarcoma virus, gave rise to the prototype oncogene v-src. Since the first descriptions of the protein tyrosine kinase activity of v-Src, many other proto-oncogenes have been described (Brown and Cooper, Biochim. Biophys. Acta., 1996, 1287, 121-149).

Intracellular tyrosine kinases can now be divided into at least 8 subfamilies based on catalytic domain sequence similarity and the presence or absence of other functional domains. The members of the Src family in vertebrates include Src, Fyn, Yes, Fgr, Hck, Lck, Blk and Yrk (Brown and Cooper, Biochim. Biophys. Acta., 1996, 1287, 121-149).

Hematopoietic cell protein tyrosine kinase (also known as Hck and JTK9) was first cloned in 1987 (Quintrell et al., Mol. Cell Biol., 1987, 7, 2267-2275; Ziegler et al., Mol. Cell Biol., 1987, 7, 2276-2285) and later mapped to chromosome 20q11-q12. The hematopoietic cell protein tyrosine kinase gene has been shown to encode two isoforms, p59 Hck and p61 Hck which are derived from a single mRNA by alternative initiation of translation (Robbins et al., Mol. Cell Biol., 1995, 15, 3507-3515).

Hematopoietic cell protein tyrosine kinase expression is highest in differentiated monocytic and granulocytic cells, suggesting that the protein might function in myeloid differentiation or activation (Lichtenberg et al., Oncogene, 1992, 7, 849-858). Taguchi et al. have demonstrated that hematopoietic cell protein tyrosine kinase and Lyn are the major Src-family protein tyrosine kinases expressed in precursor lymphoblastic leukemia cells, the cell type from which childhood leukemia arises most frequently (Taguchi et al., Exp. Hematol . (N.Y.), 2000, 28, 55-64).

Cartledge et al. have recently generated monoclonal antibodies to murine hematopoietic cell protein tyrosine kinase for the purpose of pursuing investigations of the roles of hematopoietic cell protein tyrosine kinase in signal transduction (Cartledge et al., Hybridoma, 2000, 19, 323-330)

Hematopoietic cell protein tyrosine kinase has been demonstrated to interact with Bcr-Abl, a constitutively active protein tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Kinase-defective hematopoietic cell protein tyrosine kinase was found to suppress Bcr-Abl-induced outgrowth of the cytokine-dependent myeloid leukemia cell line (Lionberger et al., J. Biol. Chem., 2000, 275, 18581-18585).

Investigations of polymorphonuclear neutrophils isolated from mice deficient in hematopoietic cell protein tyrosine kinase and Fgr have identified a role for these Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and have identified Fgr and hematopoietic cell protein tyrosine kinase as potential targets for pharmacological control of the inflammatory process (Mocsai et al., J. Immunol., 1999, 162, 1120-1126).

Investigations of encephalomyocarditis virus-infected mice treated with the Src kinase inhibitor PP2 have indicated that hematopoietic cell protein tyrosine kinase plays an important role in the activation of macrophages and the subsequent production of tumor necrosis factor-alpha and nitric oxide which lead to the destruction of pancreatic beta cells, leading to diabetes. The inhibition of hematopoietic cell protein tyrosine kinase was found to prevent the onset of diabetes in this study (Choi et al., J. Virol., 2001, 75, 1949-1957).

Hematopoietic cell protein tyrosine kinase and Fgr double knock-out mice have been found to be resistant to endotoxic shock and exhibit reduced neutrophil migration, indicating that hematopoietic cell protein tyrosine kinase may be an appropriate target for therapeutic intervention in inflammatory diseases (Lowell and Berton, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 7580-7584).

Tokunaga et al. have reported inhibition of human immunodeficiency virus type 1 (HIV-1) infectivity by the expression of a dominant-negative hematopoietic cell protein tyrosine kinase protein in T293 cells, indicating a role of Src kinases in regulation of entry of HIV-1 into target cells (Tokunaga et al., J. Virol., 1998, 72, 6257-6259).

Small molecule inhibitors of Src family tyrosine kinases are well known in the art. Examples include natural products such as radiciol and geldanamycin, ring-fused pyrimidines, benzopyrans and thiol-reactive agents (Showalter and Kraker, Pharmacol. Ther., 1997, 76, 55-71).

Schindler et al. have reported the crystal structure of hematopoietic cell protein tyrosine kinase in complex with a pyrazolo pyrimidine-type Src family kinase inhibitor known as PP1 (Schindler et al., Mol. Cell, 1999, 3, 639-648).

A 21-mer antisense phosphorothioate oligonucleotide targeting the seven codons immediately downstream from the translation initiation site of the murine hematopoietic cell protein tyrosine kinase gene has been used to inhibit hematopoietic cell protein tyrosine kinase expression in investigations of tumor necrosis factor production by murine macrophages (English et al., J. Exp. Med., 1993, 178, 1017-1022) and lipopolysaccharide and interferon-gamma-mediated phosphorylation of the proto-oncogene vav (English et al., J. Leukocyte Biol., 1997, 62, 859-864).

A 20-mer phosphorothioate oligonucleotide targeting the start codon of human hematopoietic cell protein tyrosine kinase was used to inhibit hematopoietic cell protein tyrosine kinase in investigations of neutrophil apoptosis stimulated by granulocyte-macrophage colony-stimulating factor (Wei et al., J. Immunol., 1996, 157, 5155-5162).

Currently, there are no known therapeutic agents that effectively inhibit the synthesis of hematopoietic cell protein tyrosine kinase. To date, investigative strategies aimed at modulating hematopoietic cell protein tyrosine kinase expression have involved the use of antibodies, small molecule inhibitors and antisense oligonucleotides. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting hematopoietic cell protein tyrosine kinase 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 expression of hematopoietic cell protein tyrosine kinase.

The present invention provides compositions and methods for modulating expression of hematopoietic cell protein tyrosine kinase.

The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding hematopoietic cell protein tyrosine kinase, and which modulate the expression of hematopoietic cell protein tyrosine kinase. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of hematopoietic cell protein tyrosine kinase 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 hematopoietic cell protein tyrosine kinase 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 hematopoietic cell protein tyrosine kinase, ultimately modulating the amount of hematopoietic cell protein tyrosine kinase produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding hematopoietic cell protein tyrosine kinase. As used herein, the terms “target nucleic acid” and “nucleic acid encoding hematopoietic cell protein tyrosine kinase” encompass DNA encoding hematopoietic cell protein tyrosine kinase, 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 hematopoietic cell protein tyrosine kinase. 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 hematopoietic cell protein tyrosine kinase. 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 hematopoietic cell protein tyrosine kinase, 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.

It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.

Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

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.

For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in ( To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

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, aminoalkylphosphotri-esters, 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′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (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 mare 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, poly-alkylamino, 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′-dimethylamino-ethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples hereinbelow.

A further prefered 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 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 hematopoietic cell protein tyrosine kinase 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 hematopoietic cell protein tyrosine kinase, 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 hematopoietic cell protein tyrosine kinase 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 hematopoietic cell protein tyrosine kinase 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. Prefered 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. Prefered 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 prefered are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly prefered 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, polythiodiethylamino-methylethylene 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. applications Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 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 thermodynamic 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 possess 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 (PO310), 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 tri-glycerides, 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™ (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. Nos. 5,540,935 (Miyazaki et al.) and 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

cggaggcacg gaagatgagg aagatgatca ggaggatgat gaaggtgaag agggagatga 60

agacgatgac gacgatggct ctgaggggac ctcaggggct gccgagctgg gggggcgctc 120

aagctgcgag gatccgggct gcccgcgaga cgaggagcgg gcgccagg atg ggg tcg 177

Met Gly Ser

1

atg aag tcc aag ttc ctc cag gtc gga ggc aat aca ttc tca aaa act 225

Met Lys Ser Lys Phe Leu Gln Val Gly Gly Asn Thr Phe Ser Lys Thr

5 10 15

gaa acc agc gcc agc cca cac tgt cct gtg tac gtg ccg gat ccc aca 273

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

20 25 30 35

tcc acc atc aag ccg ggg cct aat agc cac aac agc aac aca cca gga 321

Ser Thr Ile Lys Pro Gly Pro Asn Ser His Asn Ser Asn Thr Pro Gly

40 45 50

atc agg gag gca ggc tct gag gac atc atc gtg gtt gcc ctg tat gat 369

Ile Arg Glu Ala Gly Ser Glu Asp Ile Ile Val Val Ala Leu Tyr Asp

55 60 65

tac gag gcc att cac cac gaa gac ctc agc ttc cag aag ggg gac cag 417

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

70 75 80

atg gtg gtc cta gag gaa tcc ggg gag tgg tgg aag gct cga tcc ctg 465

Met Val Val Leu Glu Glu Ser Gly Glu Trp Trp Lys Ala Arg Ser Leu

85 90 95

gcc acc cgg aag gag ggc tac atc cca agc aac tat gtc gcc cgc gtt 513

Ala Thr Arg Lys Glu Gly Tyr Ile Pro Ser Asn Tyr Val Ala Arg Val

100 105 110 115

gac tct ctg gag aca gag gag tgg ttt ttc aag ggc atc agc cgg aag 561

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

120 125 130

gac gca gag cgc caa ctg ctg gct ccc ggc aac atg ctg ggc tcc ttc 609

Asp Ala Glu Arg Gln Leu Leu Ala Pro Gly Asn Met Leu Gly Ser Phe

135 140 145

atg atc cgg gat agc gag acc act aaa gga agc tac tct ttg tcc gtg 657

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

150 155 160

cga gac tac gac cct cgg cag gga gat acc gtg aaa cat tac aag atc 705

Arg Asp Tyr Asp Pro Arg Gln Gly Asp Thr Val Lys His Tyr Lys Ile

165 170 175

cgg acc ctg gac aac ggg ggc ttc tac ata tcc ccc cga agc acc ttc 753

Arg Thr Leu Asp Asn Gly Gly Phe Tyr Ile Ser Pro Arg Ser Thr Phe

180 185 190 195

agc act ctg cag gag ctg gtg gac cac tac aag aag ggg aac gac ggg 801

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

200 205 210

ctc tgc cag aaa ctg tcg gtg ccc tgc atg tct tcc aag ccc cag aag 849

Leu Cys Gln Lys Leu Ser Val Pro Cys Met Ser Ser Lys Pro Gln Lys

215 220 225

cct tgg gag aaa gat gcc tgg gag atc cct cgg gaa tcc ctc aag ctg 897

Pro Trp Glu Lys Asp Ala Trp Glu Ile Pro Arg Glu Ser Leu Lys Leu

230 235 240

gag aag aaa ctt gga gct ggg cag ttt ggg gaa gtc tgg atg gcc acc 945

Glu Lys Lys Leu Gly Ala Gly Gln Phe Gly Glu Val Trp Met Ala Thr

245 250 255

tac aac aag cac acc aag gtg gca gtg aag acg atg aag cca ggg agc 993

Tyr Asn Lys His Thr Lys Val Ala Val Lys Thr Met Lys Pro Gly Ser

260 265 270 275

atg tcg gtg gag gcc ttc ctg gca gag gcc aac gtg atg aaa act ctg 1041

Met Ser Val Glu Ala Phe Leu Ala Glu Ala Asn Val Met Lys Thr Leu

280 285 290

cag cat gac aag ctg gtc aaa ctt cat gcg gtg gtc acc aag gag ccc 1089

Gln His Asp Lys Leu Val Lys Leu His Ala Val Val Thr Lys Glu Pro

295 300 305

atc tac atc atc acg gag ttc atg gcc aaa gga agc ttg ctg gac ttt 1137

Ile Tyr Ile Ile Thr Glu Phe Met Ala Lys Gly Ser Leu Leu Asp Phe

310 315 320

ctg aaa agt gat gag ggc agc aag cag cca ttg cca aaa ctc att gac 1185

Leu Lys Ser Asp Glu Gly Ser Lys Gln Pro Leu Pro Lys Leu Ile Asp

325 330 335

ttc tca gcc cag att gca gaa ggc atg gcc ttc atc gag cag agg aac 1233

Phe Ser Ala Gln Ile Ala Glu Gly Met Ala Phe Ile Glu Gln Arg Asn

340 345 350 355

tac atc cac cga gac ctc cga gct gcc aac atc ttg gtc tct gca tcc 1281

Tyr Ile His Arg Asp Leu Arg Ala Ala Asn Ile Leu Val Ser Ala Ser

360 365 370

ctg gtg tgt aag att gct gac ttt ggc ctg gcc cgg gtc att gag gac 1329

Leu Val Cys Lys Ile Ala Asp Phe Gly Leu Ala Arg Val Ile Glu Asp

375 380 385

aac gag tac acg gct cgg gaa ggg gcc aag ttc ccc atc aag tgg aca 1377

Asn Glu Tyr Thr Ala Arg Glu Gly Ala Lys Phe Pro Ile Lys Trp Thr

390 395 400

gct cct gaa gcc atc aac ttt ggc tcc ttc acc atc aag tca gac gtc 1425

Ala Pro Glu Ala Ile Asn Phe Gly Ser Phe Thr Ile Lys Ser Asp Val

405 410 415

tgg tcc ttt ggt atc ctg ctg atg gag atc gtc acc tac ggc cgg atc 1473

Trp Ser Phe Gly Ile Leu Leu Met Glu Ile Val Thr Tyr Gly Arg Ile

420 425 430 435

cct tac cca ggg atg tca aac cct gaa gtg atc cga gct ctg gag cgt 1521

Pro Tyr Pro Gly Met Ser Asn Pro Glu Val Ile Arg Ala Leu Glu Arg

440 445 450

gga tac cgg atg cct cgc cca gag aac tgc cca gag gag ctc tac aac 1569

Gly Tyr Arg Met Pro Arg Pro Glu Asn Cys Pro Glu Glu Leu Tyr Asn

455 460 465

atc atg atg cgc tgc tgg aaa aac cgt ccg gag gag cgg ccg acc ttc 1617

Ile Met Met Arg Cys Trp Lys Asn Arg Pro Glu Glu Arg Pro Thr Phe

470 475 480

gaa tac atc cag agt gtg ctg gat gac ttc tac acg gcc aca gag agc 1665

Glu Tyr Ile Gln Ser Val Leu Asp Asp Phe Tyr Thr Ala Thr Glu Ser

485 490 495

cag tac caa cag cag cca tga tagggaggac cagggcaggg cagggggtgc 1716

Gln Tyr Gln Gln Gln Pro

500 505

ccaggtggtg gctcgaaggt ggctccagca ccatccgcca gggcccacac ccccttccta 1776

ctcccagaca cccaccctcg cttcagccac agtttcctca tctgtccagt gggtaggttg 1836

gactggaaaa tctctttttg actcttgcaa tccacaatct gacattctca ggaagccccc 1896

aagttgatat ttctatttcc tggaatggtt ggattttagt tacagctgtg atttggaagg 1956

gaaactttca aaatagtgaa atgaatattt aaataaaaga tataaatgca agtcttacg 2015

4

20

Artificial Sequence

PCR Primer

4

tttgtccgtg cgagactacg 20

5

19

Artificial Sequence

PCR Primer

5

ttgtccaggg tccggatct 19

6

29

Artificial Sequence

PCR Probe

6

ctcggcaggg agataccgtg aaacattac 29

7

19

Artificial Sequence

PCR Primer

7

gaaggtgaag gtcggagtc 19

8

20

Artificial Sequence

PCR Primer

8

gaagatggtg atgggatttc 20

9

20

Artificial Sequence

PCR Probe

9

caagcttccc gttctcagcc 20

10

gatcttctcg cctcagcctc ccaaaatgct gggattatag gcctgaacca ctgcacctgg 60

cccatccctc atttgattca ttcgacaagc acatgctcat gactcttcaa gccctgtgcc 120

agcctaaggg gctgtggaaa caaataagat atatccctac cctcaaggag ctctccataa 180

cccatcgttc ttggctgtgg gttcttcttg tgtctagctt gactctaaaa atatacttca 240

gtcatccatc catccatcca tccattcatc catccatcca tccatccatc catccatcca 300

tcatccaaca gatacctgtt gattccctat tccagggtta gaagatgact tagatggggc 360

tccctgctgg agatgttctc tgtatggtgg atgaggttga tgagtaaaaa gatgactaca 420

ttgtggggtg gcaatacatc ccattaaaaa ccagccaatc aaaagttgac aaatgacagt 480

ccccattgtt ttgtctcttc tgcctcctct ttctagctag atggtattat gggtttggga 540

tgcttgcctg ttaaaaatga aaacgcttag gcttttccca ttataaagcc acctccttct 600

ggctgggcat ggtggctcac acctgtaatc ccagcacttc gggaggccaa ggcacgcaga 660

tcacctgagg tcaggagttc gagaccagcc tgaccaacaa ggtgtaaccc catctctact 720

aaaaatacaa aaaaaattag ctgggtgtgg tggtgcgtgc ctgtagtccc agctactcgg 780

gaggctgagg caggagaatc acttgactcc agggggcaga ggttgcagtg agctgaaata 840

tcatgccact gcactccagc ctgggggaca agagcgatac tctgtcaaaa aaaaacaatt 900

atgtcacccc ttcttagtga tcatggaact gggggctctt agctgtttct ctctccttct 960

tccaggcttt gccctgggtc atcccatgct ctgtttctcc actccagggt ctgaacactt 1020

acagaatccc tgggtcacct catgtagtcc ttgcagcaac catggaggag gcgtggctgg 1080

cattatttct caaaggcaac ataattgcta agtggaaaac gcagggtgta gacaacatga 1140

atggggggtt atcatttgca gacaaaggca aaataaccta gatggggggt tctcagacat 1200

gagcagcaac agcatcacct ggattgccgg ctaacaagtg ctttctgggc tccactgtca 1260

gattccgaat gtgcatttct aacaggttct tgggtgaggc agctgctgct gctgctgctc 1320

cagggaccca gcttgggaac cactgatcta aatacgcata tctggccagg tgtggtggct 1380

cacacctgta atcccagcac tttgggaagc cgagggaggt ggaacacttg aggtcaggag 1440

ttcaagacca gcctggccta catggtgata ccctgcctct actaaaaata caaaaattag 1500

ccaggtgtga tggcgggctc ctgtaatccc agctactcag aggctgaggc acgagatttg 1560

cttgatctgg gaggcagagg ttgcagtgag ccgagatcac gccactgcac tccagcctgg 1620

acaacagagc gagactctat ctcaaaaaat aataataaaa ataacaaatg aatacacata 1680

tgtgtccttt cagggcactc aggggtcctt gtgtctgaat caagaaactg ctaacagagg 1740

ttgcctctgg ggagcacaac tgggaccttg ggaatcaggg cgggatggaa tcttggtttc 1800

tctcattctc ccttgaaata ggcaatactt tcaaatgttt caaaatgcaa atgtggggaa 1860

gcatgcagaa gagtgaaaag tcttcttccc accctgtccc caaccagcca cttcccctcc 1920

ccagaagcaa ccagtttctc gtgagtcctc ccagatgctt cctccttata taagaaaaga 1980

catctcttcc cgcttatctc acacaagtgg tagataccac acacactgtt ccatgcctct 2040

cgttcagacc ttccatgcag caccttgttc ctgttttgtt ttgttttgtt ttgttttgtt 2100

ttttaggtgg agtttctctc ttgttgccca ggctggagtg caatggtgtg atctcggctc 2160

actgcaacct ccgcctcccg ggttcaagcg attctcctgc ctcagcctcc caagtagctg 2220

ggattacagg catgcgccac catacctagc taattttgta tttttaatag agacagggtt 2280

tctccatgtt ggtcaggctg gtctcgaact cctgaactcg tgatcctcct gccttggcct 2340

cccaaagtgc tgggattaca ggcgtgagcc actgctcccg gacacctcct tcctttttat 2400

accacagggc gtctgtcctg tagatgtcct accgtccatt taacctctgc cctatacagt 2460

ggtcacttaa gttgtttcca gtcatgtact gaagcgaatc tcctggtgta tatgtcattt 2520

ctcccttgtg caaggtctat gggataactt cctaggacca gaattgtcag ggagacagtt 2580

ggtttttttt gtttgtttgt ttgtttgttt ttgagacaga gtctcgctct gttgcccagg 2640

ctggagtgca gtggcgtgat ctcggctcac tgcaagctcc acctcccagg ttcacaccat 2700

tctcctgcct cagcctcccg agtagctggg actacaggct cctgccacca tgctcggcta 2760

attttttttt ttttttttgt atttttagtc gagacggggt ttcaccatgt tagccaggat 2820

ggtctcgaac tcctgacctc gtgatccacc cgcctcggcc tcccaaagtg ctgggattac 2880

aggtgtgagc caccgcgccc agccggggga gacagttctt agtcttacat agttgaatat 2940

tttaccatgg cacaagtcac tttacaacat gaaagcaaag aaggtagatg agcaacatag 3000

catggtgatt cagagcttgg gttctggagt tagactgcct agcttcaaat cctgcctcta 3060

caacttccca gctgtgtgac tctaggcaag tcacttgccc tctctgttcc tcaatttcat 3120

catccataaa atggggccag gagcagtggc tcatacctgt aatcccatca ctttgggagg 3180

ccaaggtgga tggatcacct gagttcagga gttcgagacc agcctggcca acatggtaaa 3240

accccgtctc tactaaaaat acaaaaatta gctaggtgta gtggtgggtg cctgtaatcc 3300

cagctgcttg ggaggctgag gcaaaataat tgcttgaacc cgagaagcag aggtttcagt 3360

gagcccagat cacaccactg cactccagcc tgggtgacag agcaagactc agtctcaaaa 3420

aaaaaaaaaa aaaaaaaaag ggggggaatg ataaaggtga caactccata gagctgttgg 3480

gtggatagca agatagatgt taacttcctc cccagttgat aacagaggaa ccaaaggccc 3540

aaagaggaag gcaacttgct caaagtcaca ctgcaggttt gtggcagagc caaggcaggc 3600

tgaggtcagg tctggggctt ttttccaact gctcctcacc ccagtgaagg tgggaatgga 3660

ttccactgct gctgcttgaa cttttccacc caaatatctc aatattcagg aagaatgaag 3720

aaaatgtccc aggatataat cctaagtata aaattctttc caatctcaca ttgtagctta 3780

gaagttcagg ccaactttat attcaactcc atatatgcgg aggaggattg gttgtaaagc 3840

taacaaaact caggcccttc acaggcttcc aaggtcccaa gaaggatctt ccccgtggtc 3900

ttgtatgctt gtgaaacctg caaaagtaag actttgacca cagctggtta agactgctgt 3960

ctgtttccac tctgtcttcc tgtccatctc cctgtccctc acagcgagca gcactgaaat 4020

gacctatcag ctttttgcac ttgttaatgc tgtattattt ttcttaaagg aagttccccc 4080

actccaaatt gcataggctt cagtctcagc aaacaggatt cacttcgggt aaaatgtcta 4140

ttttgatatc aaaagaatgg ctcctttacc ttctctcccc caaaaaatct ttgagtaaaa 4200

ctgatgctgc aggaagccag accctctgta tcctgacatt cccctgggac ctgcatggcc 4260

acaggcatcc tgtgtaggcc ggacaggact gcatgacccc aggttcacat ttgtcccctc 4320

ccttttccat caggtggttt ttcaagggca tcagccggaa ggacgcagag cgccaactgc 4380

tggctcccgg caacatgctg ggctccttca tgatccggga tagcgagacc actaaaggtg 4440

acaccagccc tccccacctt gtcctccctg ccgaggtgcc ccagctgggg ctggccacca 4500

ccctttcctt ggaaaatgcc ctgggaaagg ctgaaaaacc caaccaggtg ctgtggctgc 4560

caggtttctc ctgctcttgg ccacctgagc tggggagggt tgaggctctg tgcctggctc 4620

tgcccttctt atccaccata gctgccagct taggtcagtg gaactagtgc caggtggctt 4680

aggccctgaa aacaaagaaa catccagctg aagcttgatc ttcactgact tgttttttca 4740

tttgttttgg ttcttgtttt gttttgttct gttttttgag atggagtctc actctgtcgc 4800

caggctggag tgcagtggtg cgatctcggc tcactgcaac ctccacctcc caggtttgag 4860

cgattctcct gcctcagcct tccaagtagc tgggattaca agtgctcgcc accatgccca 4920

gctaattttt gtatttttag tagagacagg gtttcaccat gttggccagt atggtctcga 4980

tctcctgacc ttgcgatcca cccctcttgc cctcccaaag tgctgagatt acaggcgtga 5040

gccactgcgc ctggcctgtt ttgttttgtt tgagacaggg tctcactctg ttgcccaggc 5100

tggtatgcag tggcacaatc tcagctcact gcaacttctg cctcctgggc tcaaatgatt 5160

ctcccatctc agtctcccga gtagctggga ctacaggtac gtgccaccat gctgactaat 5220

cttttctttc tgtttttctt tctttctttt tagtagagat ggggttttgc catgttgcct 5280

aggctggcct cgaacttctg gactcaagca atctgcctac ctcagccttc caaagtgtca 5340

ggactacaaa gtgtcaggat tgtttttgtt ttggtttttt taaatacccg taacagacca 5400

agtaggggag gccaacacag gaggatcact taagcccaaa agttcgagac cagcctgggc 5460

aacatagtga gaccccatct gtatgaaaaa ttttaaaaat tagccaagca tggtggtgca 5520

catctgtact ccctgatact cagaagggtg aggccagatg atctcttgag tccaggagtt 5580

caaggctgca gcgagccata atggagccac tagactccag cctgggcaac agagcaagac 5640

cccatctcta aaaaaataaa acaaaatacc cattacaata gatcttatcc acaagcctca 5700

ttcctccttt cctccctcca taaggaaatg aggtaggatg aggtcgccag aagagactca 5760

agccagcctt ctctgggact ctgctgaaaa tgctccttgg aagcttttgg aagttttaat 5820

gtcaggtgac cagcttcctg gaatttctgt gctgcaaggg tggattcatg ctgattgggt 5880

gcatggatgg caagaacagg cctggaaaac atcgcgtagc tcaaactggc atcgattgag 5940

gctcattgcc tcaaaggaat gaacatcagt aggattctta tgatttcaga tttcatgtgc 6000

taagtgcacc tttactaata catttaggtt cacaactgct tgactcaaat aagccaagta 6060

tattgtataa tatgtgacct cacggtaccc tgaagcaaac agatttgggg acattgatta 6120

aaggcatgtt cctcatgtgg ccttctactt gaccctcagc tatctgaatt ggcgaagctt 6180

tcatgtggct aggataatgc tacccacaat cagtgtggca gctcagtgaa tgcctgggct 6240

ttgtctcttc ccacccaacg agccccattc aagcccccag ggggaagaaa aggagataaa 6300

aagacaaagc tgtgcacagc ccagcgcttc tcctctctag ctgtgcccag gacagctttt 6360

ggcttgggct ggtccatcct gcagacaagg gcagagaaat caaagaaacc atcacagatc 6420

tgtggggcag gaaaatgagc ctggtccagc tttcacagct ctctgagatg gggcatgttg 6480

ggaattttag ccgatttaat aaaagttgca gcatgagacc tgtgaatccc accctgctgc 6540

ttcctggatc ctgccacacc ccatccagca gcaaccaagc cagtctcgcc cctgactggg 6600

acagagtggc tgagaggggc tctggagcca gctgcctgga tttgaatccc agctgtgcca 6660

cttaccagct gtgtgactgt aggtgagtta tttcacctct ctgggcttca gtttcctcat 6720

ccgtaaatga ggatgatgat attataaaac ccttacccca tgaggttatt cattcattaa 6780

ataaataata ttataattta tatagttata attcattata atgaatatta ttcttattca 6840

ttcattgttc atctacgcag agtgctttga acttgcctgg catgtacggt aagctattat 6900

tcattcaaca gtataaactg gccaggtgag gtgactcaag cctgtaatcc cagcagtttg 6960

ggaggctgag gcatgtggat cacctgagct caggagttgg agaccagcct ggccagcatg 7020

gtgaaactcc atctctacta aaaatacaaa aattagccgg gcgtagtggc gggcgtctgt 7080

aatcccagct actcaggagg ctgaggtagg agaattgctt gaacctggga ggcagagatt 7140

gcagtgagcc aagattgcgc cactgcactc cagcctggat gacacagcga gactctgaca 7200

cacacacaca aaaaataaag tttaaacttc ccaaatccta tgacattgta ctattatcat 7260

ggtaaagaac actgagacag agggactaat aacttgccca agggcacaca gctcacaaat 7320

agcaaaacgg tggtcatgag ctgaaagact tatgtgtttc tgctctatgt gataatatag 7380

ggtggtgcaa aagtaatttc agttttttcc actacttttg aaggcaaaaa ctgcaattac 7440

ttttgcacca gcctaatatg tatgttaggt tcccattgct gctgtaacaa attaatacaa 7500

acttagtggc ttaaaacaac acccgtctat tctctaacag ttctggggat cagtagtctg 7560

acctcactgg gctcaagatg tcagcagggc tggttccttc tggaggcttc aggggagaat 7620

tcattttctt gcctttttta gtttccagag gctacagcat tccttggctc atagtccctt 7680

cctgacatct gtccaagctc ttgtttccat cttcacctct tccactttga ccttcctacc 7740

tccttctaac aagaactcaa gggattacac tggggctacc tggatcatct ggaataatct 7800

ccccgtctcc agatccttaa cttaatcaca tctctaaagt accttttacc atgtaaggta 7860

acatattccc aggttccagg gtttagggca tgactatctt gagggaccac tattcagcct 7920

accacagcat gttcacattt caataactgg actggatttc tgggtttttt tccagaaaag 7980

tcatagcctg agtttttatg aaaaagtaaa aagtatacta tggtctcttg gctttttgta 8040

gggtagatga tagaaagatg gaacaataga tcagaaataa tgaagatgat gatgatgata 8100

gccaacattt gcagagtgtt cagcttccaa gtagtttaca ggcattactt cttttagccg 8160

tcaggcaata ggatcaggta gggaggcgtg agaactattc ccattttaca gatgagaaaa 8220

ctgaggcata gagaggtgac atagtcacat ggccaaggtc tcacgggtgg atgtggtaga 8280

gcctggatga gagcccagga tgcctggctc ccacagccca ctcagaccct cgggcacttc 8340

tgcccaggtg tcaggacggt gccagcggta gccttacagg gtgtcagagt gctaatgcaa 8400

ggtggcaggc ctccaagatg ccattctgag gggtttctct ttggtcaatt caggaagcta 8460

ctctttgtcc gtgcgagact acgaccctcg gcagggagat accgtgaaac attacaagat 8520

ccggaccctg gacaacgggg gcttctacat atccccccga agcaccttca gcactctgca 8580

ggagctggtg gaccactaca agagtgagtc ccaccccagg ggtgacatcc ccaccacgat 8640

gggcccacag actcctagtc acggatgcac tgtggcccct gagacctgct gtgtccttct 8700

tggccatccc ctagacagat agttgctttg gatgcttctg aaggcttagg actgttgagc 8760

aggagggtga ggtggccaag atgtagaggt ggcaccccgt cacactctgc ttgcttggga 8820

atgtctctga tggtggcaac caggtggaac actggagaga tccagtggac caggtagggc 8880

ggcctccaag gagcaacctc tggctggctc ggtgcttgtt gctctcaatt gaccagggac 8940

tctgttccag aggggaacga cgggctctgc cagaaactgt cggtgccctg catgtcttcc 9000

aagccccaga agccttggga gaaagatgcc tgggagatcc ctcgggaatc cctcaagctg 9060

gagaagaaac ttggagctgg gcagtttggg gaagtctgga tgggtaagga cccagggcca 9120

cagcccacag ggccagaggg tggaggggag agggaggcca cttgcttcca ggaacacctt 9180

atggcaaagc gggaatgcta cccaaggcag agggggagat ttaaataata gccataaaga 9240

agcagttccc tgatagcaaa ccaaattgtc ttgctgtgca aacactgatt tgcctttgag 9300

accagtcctg tagtggtcaa aaacttcctt ctgggaagca ggtgtcatgg atgaggaatc 9360

cctccctcat acggagtggg gactgcctaa cagtttggga ttgagataac cagagacctt 9420

ttcagatgat gaaaattaag aatggggaaa tgcagcttgg agatttccag gcaactgatg 9480

aacaggttat tctgagcacc tgctgtgtgc ccagcactgt tagggtcttg tgggaaaatc 9540

agaaaggcac atctcacacc tgctgatttt ccccaagaac ttaagaacaa cactgcctgc 9600

tttgcagggc tgtggggtag atgcagtgcc agcccagtgt ggggcagcct gctgtgttaa 9660

tacgttccaa atgggagcgt tccaaatggg agcgaagaca acttacaggg atgcaaacaa 9720

ctcagccaga taaaataagt ttaaaatcca tgtatttcga ctgtatttat tgcgattctg 9780

ctgcatgcaa ggccgggcac agtggctcac gcctgtaatc ccagcacttt gggaggccga 9840

ggcaagtgga tcaatcaagg tcagcagttc gagaaaaacc tggatagtat agtgaaatcc 9900

cgtctctacg aaaaaataca acaattagcc aggcgtggtg gtgggagcct gtaatcccac 9960

ctactcggga ggctaaggca ggagaatcgt ttgaacccag gaggtggaag ttgcagggaa 10020

ccgagatcgc accactgcac tccagcctgg gagacagagt gagactctgt ctaaaaaata 10080

ataataaaat taaaaagaat ttgacaaaaa agaaaatatg gagaggaagg taagacagat 10140

ctgagtggtg agatttgaaa gccaaaaata tcatataaga cgctagactc ctactacagg 10200

aagcccacgg ttctggattg gagcttcctg gaagccaagg caaaatggaa acccaagcaa 10260

ttataaaatt gacaagaccc accaaatgaa gcagggtggg gtgaggggag ggaacagcat 10320

gtgccaaatc cctgtggtgg gaggtgagtt gggtgcttga gggactgagt gacctgcagg 10380

gtgactggca cataaaaggc aaggggagtt tagtggctga tgaggctaga ggggtggacg 10440

aggctgggtc atgtagggcc tgggggtcat agtaagtctg gcatttattt aagagcagtg 10500

ttaagattaa agtgggcggt gtatgtgtgt gacataacct gagtagtgtt ttaaagctat 10560

ttttatttgt ttgggttttt tgtttgtttg tttgtttgag acagagtctc gctctgtcac 10620

ccaggctgga gtgcagtggc tcaatcttgg ctcactgcaa gctccgtctc ctgggttcac 10680

accattctcc tgcctcagcc tccccagtag ctgggactac aggcgcccac aactgcgcct 10740

ggctaatttt ttgtattttt agtagagacg gggtttcacc gtgttagcca ggatggtctc 10800

gatctcctga ccttgtgatc cgcccacctc ggccccccaa agtgctgaga ttacaggcgt 10860

gagccactta gagctatttt taaatccttg caacagcact tcaaaggcaa agcttaccat 10920

ttccacttta cagatgagga agctgaggaa gccacggctc agagacgtta agccacttgc 10980

cctagtgtgc acagctggga agtggtgaac ccgcctttca acccacagtt gtctgaggcc 11040

taagcccatg atttcgcttc cctctctgag agttgagatg agcaggaaag actttctgga 11100

ggaggcgggt ctgcagctgg gccttctagg gtggtacggg agaccagtgg gctctgacca 11160

ccttccctgc tctctatccc cctccagcca cctacaacaa gcacaccaag gtggcagtga 11220

agacgatgaa gccagggagc atgtcggtgg aggccttcct ggcagaggcc aacgtgatga 11280

aaactctgca gcatgacaag ctggtcaaac ttcatgcggt ggtcaccaag gagcccatct 11340

acatcatcac ggagttcatg gccaaaggtg ctgcgtgctg gggctggggg tgcaggctgt 11400

ggcctatact ggtcaattgc gggcccaagg gtggcttgga catggttctt gcccttgaga 11460

tggccccaat ctggccagga gctcttccaa caagtactca ttgagaatct aatgtggaac 11520

aatccagccc tcctggttag tggggtgaaa tcatgttaag gggtcaccaa ggggaaggga 11580

gaacaagggg ctaccaggag aagggaatgg ggccagaata ctcctgggca atgggagaag 11640

gggcttatgg ttcacagtga gccttagagg ccagggtgtg gcatgggtgg tgggggtgcc 11700

actctcccgc atcagccacc ttccctgtac ttgccaaagc cctgggagag ggagctggca 11760

aagcagcagt tctgccccca cccccgccct gccacaactc aggatttctt ggggaatttt 11820

tttaggccac tgtttcacaa ccattttcaa cccctgccat tttttgtttg tttgtttttt 11880

agggacagga acttgctctg ttactcaggc tggagtgcag aggtgcaatc ataactcctg 11940

ggctcaagtg atcctcccac cccagccccc cgagtggctg ggaccacagg cccatgccac 12000

cacactcagc taattttatt tatttattta catatttatt tttagagaca gggttgccct 12060

ctgcctaccc aggctggagt gcagtggcgt gatcatagtt cactgtagcc tcaaactctc 12120

aggctccagg atcctcccga cttagtctcc ccagtagctg gcactacagg catgcactac 12180

cacacccagc taatttctaa aaaaattttt tttttttttg agacagagtc tcactctgtt 12240

tcccaggctg gagtgcagtg gcacaatctc ggctcaatgc aagctccacc taccaggttc 12300

acgccattct cctgcctcag cctcccaagt agctgggact acaggtgcct gccaccatgc 12360

ctggccaatt tttttgtatt tttagtagag acggtgtttc accgtgttag ccaggatggt 12420

ctcaatctcc tgaccttgtg atccgcccgc ctcagcctcc caccatgctg gaattacagg 12480

cgtgagcaac tgcgcccagc ttctaaaatt tttttctaga gacaaggtct cgccatcttg 12540

ccccagctgg tctcaaactc ctgggttcaa ctgatcctcc caaagctttg ggattatagg 12600

catgaaccac tgagcctggc ctaacatata tatttgtaaa tttcttctat agatggggtc 12660

ttgcgatgtt gcccaggctg gtctcaaact cttggcctca ggtgatcctc tcacctcggt 12720

ctcctaaagt gttggaatta caggagtgag cctcgtgcct agccactctg cccatttttt 12780

gataaacaga atcatctcat gcaatcccat ttagtgcgat ttgaaacctc aagccttttt 12840

cattttaact atgcaattat gccagtcctg tgaaggcatg atcccagcca tccaggctca 12900

ggggctgtct cagtcacaga atccatctag caatcaaaaa gtcacttcac taaaactcaa 12960

ttctctttct caaacactag gaattgcaaa caaatattcg tatttaaatc tactactatg 13020

aaaaatcttc accacacact aaagtactag attggtgcaa aagtaattgt ggtttttgcc 13080

attaaatagt aaaagtagta aatgttcctc ccctctcccc catataggaa gcttgctgga 13140

ctttctgaaa agtgatgagg gcagcaagca gccattgcca aaactcattg acttctcagc 13200

ccaggtgaga gcctaacgag gaaacgggga agggaaacag gaattcgatt tttttacttg 13260

ccaaatattt actgaccaca tactatgatg atagcagtaa taataatggg taaaatgtat 13320

tgagagctca gtataaaact atgttttttg catatatttt attatacatg tactcattca 13380

tttaatcatc ctataagctt tataggctgg gatttttgtt atctattttg gtataacaaa 13440

ccatccaagt cctagtagct tgaaacaaca gcaattgtta tttctcatga tcctgtgggt 13500

tgcctgggtt cagctgggtg gttcttctgc tcgtcatggt aaagtgaggt gcagatgttg 13560

gcacttgccc aggctagaat gtccaagaca tggctcagcc tccagggtct ctccacatag 13620

cccctcacca cccaatcacc cagcctgagc ttcttcacgg cgcagcagct gggttccaag 13680

acagagctgg accaggggcc aagccttcat gagcaggctc tcattaagcc tctgcctgca 13740

tcaggcttgc taatggccca ttggccaagt cacatagcca tgcccagaat cagtgtggga 13800

ggggactgac caaggacaca aatcccaggg accccctcaa tgtaacagtc tggccacaga 13860

gtaggtaatt atcatcccat tttacagaat aggaaattga agcatagagc agaagtaaag 13920

tacagatgtg gaaactgagc cccagtgtgg ttaagtggct tgcacaagtt actgtgctaa 13980

ttatggggga tatagcagtg aacaaaatga aaatgttcct gtcctaatag agttcacaat 14040

ccaggtggga agatgagtat taaataacca aatctcttag agaccatgac ccaagtggtc 14100

ctggctgagt caaaagagca cagtatctaa caaagtaaat aatgttcaca aatcgcaaaa 14160

acactgctga aaacctaatt ttctttctca tgttccccac gccccttgca gctcctctca 14220

cccccagcct aagcagtttt ttcacttctg tcctctctca tgctctctct gatgtctggg 14280

aaaacagagt gtaaggacgg ggtggtatta gagtgcatag ctagagcatt tagcctggtc 14340

caggagcaat cagagaatgc ttcctggagg aagtggcctc taggccgagc ctgaaggatg 14400

aatagaagtt cagtagatga agagaaaagg taaaggtctt ttagacaaaa gaaacagcct 14460

gtgcaaaagt aggtgaagga gttcagccta ctcagcagga gcccagggat agtgggagtt 14520

gagtgagacc ctgcctgggc ctcagctggg cctgctggag atgccaaggg cagggctcat 14580

ttgagtatgc aaattcaggg gcccagagga caccacaaag tgtaacaaaa gactctactt 14640

tcagagcctg gaatgccaga ggcatatgga aatgtgtttt cactcctatg ggccaggtcc 14700

tctgggagac aatagactgg tctgttgcta aattatcatc ttcaaactag cagaagccct 14760

gctgggcagc agtcaccgac cagggcaaga gccactctgc ccgtttcaca gaggggaaaa 14820

caaaccaagg gaggaaaaga ggttctgggc cttcatctgt ccagaagcag aggtagattc 14880

agagcccagg gacagaaagg agcccacctg gggctttggt cttcacctcc agactcagag 14940

aggcagcttg gagcagcagg tggagaactg gaccacacat gtggggtttc tgtagtcaca 15000

gctttgtggg tgacatgaaa tgttattcag tagctgcata tatacaaaca ggtcaaaaca 15060

gagctgctct gactgaagca gggtgggggg cttatctgtg gcctttcttt tgtctccccc 15120

acccccatat aaccttgagg catccatagg attcagttgg aagagctcta caggtccttc 15180

cagctctgat gctgtgactc cctgatactc tttggaagca aacccaaggg tgcccaacct 15240

catggcaccc tatctggggg tcatgaaatc tcaccgcctt tatcaacccg cagtgcttac 15300

acctgggccc aataaaccta ctcgttattc attcagtcag cagaccctgg agaagcaaca 15360

ctattagcat ctgggcttag gagtcacaca gagttttgtt ttgatctcag ctctgcctgt 15420

gtagcctcaa gcaagttgct taacctctct gagtgccact ttcccccact gggagcactg 15480

ggagccactc tttaaataaa tacttaacta tgtgccaaac actctactaa gcccttgatg 15540

taggcttagg ggattcatct tcacatgacc ccttggaacc aggtgctatt acagagaaga 15600

aaagtgaggt acagagagat gacacaactt gcttgacatc acagagctgc agcgctcaag 15660

ctgagttggt ccagctccag agccatttag ctattaccct ctgctgtctc ctaagtgggg 15720

tgtcatccct gccccatcga gctcctgtga aaatcagtag catatgagca tctgccagtg 15780

agcatcaaac agtggtaact accagcatta gtccttgctc cagaagaaaa gcaaggcaaa 15840

aaagctgggc caagtaacag gtaagtgcgt ggaccccaag cagcgctttt ggagctccag 15900

ccctggctcc gcaacttgca agctgtgtaa gctggggcca cttacctatt tctctgggct 15960

tcagttttct tatctgtgga aatggggctg ataacaaaag tgcctcctcc aaagcacatg 16020

gtaaatgtca cataagtgtt taagtatcat cttataaata agctatgatg ccttccatta 16080

gcatcataac ttttgttaac tgttcatcta aggactcata aatttcccac actcccaggg 16140

ccaagatgct gtctcactcc accctggcaa cctcatcccc atttcgtccc caccccccac 16200

tattctaaac aaaatcagag tctcttccaa gtttttactg aaaaatttgg gtctctgggt 16260

ctcccttggg catgacagct caggcctgta atcccagcac tttgggaagc tgaagcagga 16320

ggatccttta agcccagcct gggccacata gtgggacctc ttctctatta aaaaaaaaaa 16380

aaattctttt taaatttaat ctggcatagt ggcatgcacc tatagtacca gctgcttggg 16440

aagctgaggt gggaagactg cttgagccca ggaggcacag gttgcagtaa gctgtgatcc 16500

caccactgta ctccgtgaca gagcaaggcc ctgtctcaaa agaaaaaaaa aaagaagaaa 16560

gaaaagaaaa aagcaaagca tgggtctcca ttctaactgc ctcgggtgcc ccctgctgtc 16620

caccccttgc aagtgcgcca cccacttgcc tcagaccttg agaagccggc cggtggtgct 16680

ttgattgttc tgtctagtca gacgccctca cttccagaat aatttcatca gactgcaccc 16740

agctgcagtc tgccggagca ctggagtgct gggttattta gaggcagcag gatgcactgg 16800

attgggagtc ctcagactgg tttgagcctg acattcagaa ttttcctggt ggtgtgatct 16860

ggaacaagtg gttccatgtc tgtaaaacag agattatgat cattcctgcc tcaaagagct 16920

caagcccagt ttgtaaacca aaagtgctgc tttctgggca tctgagggtt catcttcaac 16980

cgcttttgtc caaacccttg taagtaaaaa tgtctgcaag atataatccc tgtgttttca 17040

tcagaggctt ctagggctcc tggtgtgaat aagaatcctc cacgcagtct gttagtcact 17100

cagcagccat ggtgatcttt aaatgtacat cagaacatgt cacgctcctg ctcgaaggcc 17160

tctaatatct ccccaacacg ctcagaataa aatccaccct cctctcctga cctacaaggc 17220

agttcatgat tggcggcctc cctctcaact tcaactctcc ccactctctc tgtggcatct 17280

tctgctggcc acactgacct ctgtgctgtt ccatgaacac ccaagctctt acctacctcc 17340

acgcctttgc atctcatgtt ctctctgcct agaatattct tatgcgacac acttattatt 17400

atcatcattc agatctttgc taaaatctca ccccctcaaa gcaggtttcc ctgaacgccc 17460

aatccaaaaa agctctctac aggtcatatt ttgttacttt gttttatctt ttttttcttt 17520

ttgagacaga gtcttgctct gtcgcccagg ctggaggaca gtggcacgat ctcggctcac 17580

tgcagcctct gcctcctggg tccaagcaat tctcgtgcct caacctcctg agcagatggg 17640

attacaggcg tgcaccacca tgctcatctt ctttttgtat ttttagtaga gacggggttt 17700

cgccatgttg gccaggctgg tctcgaactc ctggcctcaa gtgatccgcc cacctcggcc 17760

tcccaaagtg ctgggattac aagtgtaagc caccatgccc ggcctgtttt atcttttctt 17820

tatggtactt accacctgca ggggctttgc cgggctcatc acctgttcaa gcaacatgtc 17880

tggcacccac aagacactca gtggatttct gttgagtaaa taagtgaatt ttccctaccc 17940

aggctggggc atgaggaaga tatgagaggt atagcagaat atgacatgaa cctgggttgt 18000

ccccagaaag agagttgaag gtgaaagctc ttcatagtat cctagggttc gtgtgtgtgt 18060

gtgtgtgtgt gtgtgtgtgt gtgcatgtgc acgtgtgtgt gtatgtgtgt tcctggaaga 18120

ataggtcctg gagaaatagt cccattgctt tttccctgag aacatttcaa agtgctctct 18180

ggaagtcttg gaattctaga gaatccccca aactggccca ggcctcctta gggatccccc 18240

taacctgaat gggttgatgg aggaatgcca ccctgagccc tggggccctc ccgacacaaa 18300

agggagggct ggtgcagaca tttcgcattt tcttcacttg aacacctctc tgctgctttt 18360

gggtggggcc atcttggcgt aggccaggtc tgaggacaaa ggtgtctctg tttggggtgc 18420

agattgcaga aggcatggcc ttcatcgagc agaggaacta catccaccga gacctccgag 18480

ctgccaacat cttggtctct gcatccctgg tgtgtaagat tgctgacttt ggcctggccc 18540

gggtcattga ggacaacgag tacacggctc gggaaggtag ggaacgctgc caagcagccc 18600

cacgttgccc atttggatgc ttgtgagtgt tgagagttga tacttgtgag agcgattggt 18660

aaaatgcaag ggactgcccc agtactagct gtgcattctt gagcttggtg gatccttctg 18720

gataatgtcc tgaacttcag agtctcactc agagattttg aggagatttt agatcagatt 18780

gagaagtact gatagatttt agagggagtg ggcaaatata tgtcgaaatt aagggtaaga 18840

gcagctttgt gcataacaga caaaaatggg gagggtatcc aaatgtccat agcaggagga 18900

tggattgtgg tatattcaca caatataata ccacgcagtg atgaaaaagc acaaactggc 18960

caggctcagt ggctcacgcc tgtaatccca gcactttagg aggccaagac tggaggactg 19020

cttgagccca gaagctcaag accagcctgg gcaacacagg aagacctagt ctctacaaaa 19080

atttaagaaa ttagccaggc atggtggcac atgcctgtat gcctgtagtt gtggctactc 19140

aggaggttag ggtgagagga tcacttgagt ctaggaagct gaagctgcag taagccatga 19200

tcatgccact gcacccagcc tggtggacag agcaagatct tgtcaaaaga aaagaaaaga 19260

aaagaaaaga aagaaagaaa gaaagaaaga aagaaagaaa gaaagaaaga aagaaagaaa 19320

gaaggaaaga aagaaagaga gagaaagaga aagaaagaaa gaaagaaaga aagaaagaaa 19380

gaaagaaaga aagaaagaaa gaaagaaaga aaagaaagaa agaggcacaa gccaatgcta 19440

aatgcaccaa aatgggtaac tctcacaggt ataatatcaa aatgaaagaa attagaccaa 19500

aaaaaaagag tagatgccac ttggtttcgt ttttgtgaaa ttctaaaaga gacaaaacat 19560

gtaattggac attcatggca gcattcttcc taatagccaa aaaatggaaa caacactctt 19620

taatgtccat caattgatga acagacgaac aaaatgtgat atatccatac aatggaatat 19680

tatttggcca taaaatggaa tgaagtacta cgccaggcta gaacacagat gacccttaaa 19740

aacattttgt taaaggaaag ccaacatgaa gaccacatgt tgtataattc cccttctatg 19800

aaatgtccag aagaggcaat cttttttttt tctttgagat ggagtcttgc tgtcacccag 19860

gctagagtgc agtggctcaa tctcagctca ctgcaacctc tgcctcccag gttcaagcga 19920

ttcttgtgcc tcagccttct gagtagctgg gattacaggc gcacaccacc atacccagct 19980

aatttttgta tttttagtag ataacagggt ttcaccatgt tggccaggct gaactcaaac 20040

tcctgacctc aagtgatccg cccgccttgg cctcccaaac tgctgggatt acaggtgtga 20100

gccaccatgc ccggcccaga acagacaaat ctaaagagac agaaagtaga ttattggtgc 20160

ctacagctgg tggtggggag ttgggagaaa tgaggagtga cttctgtggg ttttttgcag 20220

ggaaatgtga tgaaagtatt ctaaaatcca caactttgta aatattctga aagccacgga 20280

attatacact ttaaatgggt gaaatgtatg gtctgtgaat tctatctcga taaagctgtt 20340

aaagaaagaa acaggcaaaa tttatttata gcactagagg tcagaattgc agtcaccttt 20400

agtttctgtt agcctaatat ttatttattt atttatttat ttatttattt atttatttat 20460

ttattttgag acggagtctc gctctgctgc ccaggctgga gtgcattggc gccatctcag 20520

ctcactgcaa actccacctc ctgggtttaa gtgattgtcc tgcctcagcc tcccaagcag 20580

ctgggattac aagcatgtgc catcatgccc agctagtttt tgtattttta gtagagacag 20640

ggtttcacca tgttggccag gctggtctcg aactgatgac ctcaagtgat tcacccacct 20700

gggcctcccg aagtgctggg attacagatg cgagccactg tgccagtcct atttattttt 20760

ttaaagagac gggtgcggcc gggcacagtg gctcacacct gtaataccag cactttggga 20820

ggccaaggca ggcggatcac aaggtcagga gtttgagacc agcctggcca atatggtgaa 20880

accctgtctc tactaaagat acaaaaatta gctgggcgtg gtggcaggtg cctgtagtcc 20940

cagctactcg ggaggctgag gcaggagaat catttgaacc cgggaggcag aggttgcagc 21000

aatgagccga gattgtgcca ttgcactcca gcctgggtga tagagcgaga ctccgtctca 21060

aaaaaaaaaa aaaaaaaaaa agataggtgc ttgctctgtt gcccaggctg tagtggaggg 21120

gtgagatcat agctcactgt agcctccaac tcttgggctc aagcaatctt ccagcctcag 21180

cctcctgagt acctgaaact acagatacgt gctaccatgc ccagctaatt ttttaattat 21240

ttgtagagat agtgtctcac tgtgttgccc aggctggtct caaactcctg gacttacgtg 21300

atcctcctgc ctcaggctct gaaagtgcta ggattacagg catgagccac tatgcacagg 21360

ctgcagtcac cttaagtaag ggggtattaa ctggaaggac gcagaaagga gcgttctgaa 21420

atgctgaaaa tgttctacag cccaatctga tggtgattac aagggtatat atatatatat 21480

aaagctgcag caaaatggat attaaagatt tgtgcatatt atttcatgta cgttataact 21540

cgattaaaaa aaatttagac cgggcacagt ggctcatgac tataatccta gcactttggg 21600

aggccaaaat gggcagattg cttgagccca ggagttcaag accagcctgg gcaatatagc 21660

aagaccccat ctctacaaaa aatttaaaaa taagtcaggc gtggtggtgt gagcctgtgg 21720

tccctgctac tcaggaggct gaggcgggag aatcgcttga gcccaggagg ttgaggctgc 21780

agtgagccaa gatcatgtca ctgcactcca gcctgtgtaa cagagcaaga ctttgtctca 21840

aaaaagtaaa taaatagggc cgggcacgtt ggctcatgcc tgtaattcca gcactttggg 21900

aggctgaggt gggaggatca cgaggtcagg agatcgagac catcctggct aacacggtga 21960

aaccccatct ctactaaaaa tacaaaaaat tagccaggcg tggtggcggg cacctgtagt 22020

cccagctact tgggagactg aggcaggaga atggcgtgaa agccgggagg cggaacttgc 22080

agtgagctga gatgacacca ctgcactcca gcctgggcaa acagagcgag accctgtctc 22140

aaaacataaa taaataaata aataaataaa tgtaatgact aaaggaataa aagtagaatg 22200

tataatttct aaaccagaag aagataagca ggaaatcaag aaaagtctgt agatccaaag 22260

gaggaaggga gaaaacatat acacacacac acactctcac acacacaaac acacacatag 22320

aacattttca aaacttgacc atgtactcag tcataaagca ggtctcaaca aattccaaag 22380

aacatatatt ctatagacca tgttcaccaa ccatgatata attaaactaa acacttttta 22440

aaaaaggata actatgccag gtgcggtggc tcacacctat aatcccagtg ctttaggagg 22500

ccaagatggg aggatcactt gagcccagga gttttgagaa caggctgggt aacataataa 22560

aacactgtct ctacaaattt ttctattttt ttatatttta ttttatttta ttttatttta 22620

tttttgagac ggagtctgac tctatcatcc aggctggagt gcagtggtgt gatctcggtt 22680

cactgcaacc tccatcccct gggttcaagt gattctcctg tctcagcctg tagctggaac 22740

tacaggcacc tgccaccatg cccagctaat tttgtatttt tagtagagat ggagttggcc 22800

aggctggtct cgaattcctg acctcaggtg atcctcctcc ttggtctccc aaagtgctgg 22860

gattacaggc acaagccacc atacctggcc aaaaaaattt ttttaattag cttggcataa 22920

tggggcacac ctgtaacccc aggggcttga gaggctgagg caggaggata gcttgaggtc 22980

aggagttcga ggcttcagtg aactatgatt gtgccactgc gctccagcct gggcactctg 23040

tctctaaaag aaaaaaaaaa tgaaaaggct aactaaaccc atgtcagact aggtgggagg 23100

aagggcgggt aaagggagag acacatagtg actggtggga acctcatggg ctgagagcca 23160

gaatcctccc ttttcccatc gcccaggatg agaggactga ggcatcagct gcaacctgga 23220

ctcaaggccc cctgaagtcc ttctgtcccc aaagacctgt gacctctggg attccactct 23280

tcggagttgc agttaactgg ggtatcagct gaatcaacga ggaggattct agagtgaact 23340

tccacaccat accccaggcc cctaagccca ctcctccttg tttagccatc tgtcctcagg 23400

atggatgtcc cttgcttcca cagggaggcc acgtatcagg gaaattgcag gtctgcaggg 23460

gcagatgttg gcagctcttg cccttgcctg ttcccccacc ttagcagagc caaccctcac 23520

tactccccag ccttccccga ctctgctctg ttcaaccctg caggggccaa gttccccatc 23580

aagtggacag ctcctgaagc catcaacttt ggctccttca ccatcaagtc agacgtctgg 23640

tcctttggta tcctgctgat ggagatcgtc acctacggcc ggatccctta cccaggtagg 23700

gaaggggcat cagctcaggg ctgctaccag ggcccagtct ggcaatgggc tcatctcaac 23760

aacatgtcca ttcaaactga gttcttgatc ctcaccccca accttccctc acctttcctg 23820

tcttagttaa aggcacctcc atccatctaa tgtcttaagc ctgaaatctg ggggcttcct 23880

tggcccttcc ttctctctca ctcctatgac tttttttttt tttttttttt tttttgagac 23940

ggagtctccc tctgtcaccc aggctggagt gcagtgacgt gatctcggct cactgcaacc 24000

tccgcctccc aggttcaagc gattctcctg cctcagcctc ctgagtaact gggattacaa 24060

gcatccacca ccatgcctgg ctaatttttg tatttttagt agagacgggg tttcaccatg 24120

ttggccaggc tggtctcgaa ctgctgacct taggtgatgc gcctgcctcg gcctcccaaa 24180

gtgctgggat tacaggcgtg agccactgtg cctggcctca cccctatgac atctgagcag 24240

tcacagggtt ttggccactc gactccaaaa catatcccaa gtctcaccac tttgaacccc 24300

acagtctcca ctgcatccag gccagcgtca tctcccatag atggtgcagc ggcatcctca 24360

tcagtcttac tgctttctcc ctcctcgtcc tacagcctat tctccacttg cagccagaag 24420

gataattcta aactttaaat cagatcttgt cctttctcca cctttttttt tttttttaag 24480

acaaggtctc actctgtcac tgaggctgta gtgcagtggt gtaatcatag ctcatggcag 24540

cctcgacttc ctaggctcaa atgatcctcc cgcttcagcc tcccgagtat ctgggaccat 24600

aggcacatgc caccatgtct ggctattttt tattttttta atttttttgt agagacttgg 24660

tctcactacg ttgtgcagcc tggtctctaa cttccaggct caaacgatcc tccagccttg 24720

gcctcccaaa atgctgggat tacagacaca caccaccatg cccagctaat tctttaaatt 24780

ttttgagaga caaggtattg ctatgttgcc gggctggtga tttccatttt taagcgatgc 24840

tttcagcatg gagagtgaat gggagggaaa gggaacaggg tggcagccag tgaggaggcc 24900

acagcagtgg cccaggcaga ggtgatgatg gtctggacag ggtggtggca ttcgtgactg 24960

gcatacgttg atgaactggt gctacgtttc aaaaggagaa aagatctgac ttgctgactt 25020

aggaggtgag agagagagaa taaaggatga ttaccctggc tttgcaccac agggtgggta 25080

atggtctagg gatagaaatt ggagagctct gttttagctc tgctaaattt gagttgactt 25140

aaatatagag acatccaagt aaaggaagca cttgagttca tgagtttgga gtttgagact 25200

aataatataa aattggggat cacagacatt gaatggctat tcttatgaat agctaataga 25260

aacttgaact gaacaaaaaa atggtcccac ccataggtta acattgaatc tggccacctg 25320

aatgttttgt cttgagggtg cactagtcaa aaccaggcaa cacacaatag acgctgaatc 25380

cattaggatg tgttccacag caagcaacaa aataacagac aaagagtaac tgaaatagaa 25440

gcaatctatt actcccaatc ttccattggt gactggttag ttcagcaact cagcagctgt 25500

caggactctg ggttcctgtc tctatgatgg tcttctccat gttgtcagct ccaagtatca 25560

taagtatctc acagtgttca aaggaagaga tgaagggtgc gttctccaaa gactctcctc 25620

taagcgggag gaaaaacctt tccatcactt tcccagcaga cctccccatc agctcccatt 25680

ggcccaaact gggtcccagg cccatgtcct agctgcaagt gaagctgagg gtggaagtct 25740

gtctttcaca gtggaagggg ctcagcttgc aaggtgaggt ggggagtgat gcctgctggg 25800

gaggccacag ggcctgccac ccctgggctc tcatttccca actgcttccg tttctaattc 25860

cacggctcct tttcagggat gtcaaaccct gaagtgatcc gagctctgga gcgtggatac 25920

cggatgcctc gcccagagaa ctgcccagag gagctctaca acatcatgat gcgctgctgg 25980

aaaaaccgtc cggaggagcg gccgaccttc gaatacatcc agagtgtgct ggatgacttc 26040

tacacggcca cagagagcca gtaccaacag cagccatgat agggaggacc agggcagggc 26100

cagggggtgc ccaggtggtg gctgcaaggt ggctccagca ccatccgcca gggcccacac 26160

ccccttccta ctcccagaca cccaccctcg cttcagccac agtttcctca tctgtccagt 26220

gggtaggttg gactggaaaa tctctttttg actcttgcaa tccacaatct gacattctca 26280

ggaagccccc aagttgatat ttctatttcc tggaatggtt ggattttagt tacagctgtg 26340

atttggaagg gaaactttca aaatagtgaa atgaatattt aaataaaaga tataaatgcc 26400

aaagtcttta ccaaaacgtt ggttttcctg tccttccaat tcctgaatat ctatttgcct 26460

tcttgctgta tgacaagtca gaaatggtga aggactctcc cggggaccag aaatacaaag 26520

acaagtgggg ttcagactgg gtgctgtggc tcccacctat aataccagtg ctttgggaga 26580

ccgaggagcg ggggattgct tgagcccagg aggtcgaggc cacagtgagc tatgatcgtg 26640

ccactgcact ctagcctgag cgacacagtg agaccctgtt taaaaaaaaa aaaaaaagat 26700

gaagaagaag aaagaaagga aaggaaagaa aattggggtt caatgcttga ggttttctaa 26760

ctgccctaaa atagttactg gctatattag tttcctcttg ctcctgtaac aaattaccac 26820

atatctggta gcttaagaca acaaaactgc ataacattac ttggccgggc ataatggctc 26880

atgcctatag ttccagcatt ttgggagact gaggtgggtg atcacttgag atcaggagtt 26940

cgagaccagc ctggccaaca tggtgaaacc ccgtctctac taaaaataca aaaattagcc 27000

aggcatggtg gtgggcgcct gtaatcctag ctactggaga ggatgaggca ggagaattgc 27060

ttgaacccgg gaggtggagg ttgcagtgag ccgagattgt accactgcac tccagcctgg 27120

gcaagacagc gagaccctgt ctcaatacat acatacatac atacatacat gtataacctt 27180

actgttccag aaaagcaaga ccctgtctca atcaatcaat caatcaatca gtcaatacat 27240

gtataacctt actgttccag aagtcagagg gactgaaatg ggttggcagg gctgcattct 27300

ttttggaggc tctaggggat aatttgtttc cttgcctttc ccagtttcta gaggctgctc 27360

ctattccttc actcatcagc ccctttcgtt cattactcca aactttgctt tcatcttcat 27420

atctcctctg agtcttcttc ccctctttta tttgtaagaa ctccttggac ccacctggat 27480

aattcagaat aatcttctca tctcaggatt cttaatcacc tttgcaaagt cccttttgcc 27540

atgtgaggta acattttcac acgctctgag ggttaggaca tgaacatctt tcgacggggg 27600

gcacttttct gccttctcca ctggtatcca gatgctggat gggttttgcc tccaaggaga 27660

tgggtcttcc aggatgacag gaatggtttc tcatgatctg ggaccctgac tttcctgtta 27720

ggccacattg ctataagagc tggtcagttt acagctcccc agagctgggg gctgcctgca 27780

gacctgacgt ttcccatggg tcaagcagcc atcatacttg tcgaagtggt atttgccatt 27840

gcaggaatcc tggtctggag atatggctgc tgagaggtgt ctagtaccct acaggggaag 27900

ctctcagtgg aggggtggga gacctgcgta ggaagcagag aagacgaagc agtgcgggct 27960

tcctgaatgt tgcctcagct gaaagaccct ggcagctagt ttttatcata gagagaaaga 28020

caaaagaggt ggacaatggg tgaaaacgaa acaccacctt tctatattat tgcatcccct 28080

gaaggttgta catcacatca ccccccaggg tcccatggaa tcctatacta acttgattct 28140

gtacttctga gtagggtggg gttctaacct taggaactga atataagaac agtggaaaat 28200

aaagctagca gtgtttcaat ttacctagtt ggtaatttct ttcttgcttt ctagtttggg 28260

gtatggcttg ggtacatgaa agcagagccc agggtcttct gatggaaatg ttcccttatg 28320

atttgtgata ataatccaat gggaggagta catttccctg ctccatcagt gttgagcttg 28380

accgtgagac ttgctttagc caatgggatg ttagcagatg tgatgcaagc agaggcttga 28440

aacgtgcttg tggggctggg cttgccttcc agtcttttgc cctgaaaaga acatggtccg 28500

aggaagatga gtgacagatg gagcagacct agaccaacct gcaggcctga gcccagccta 28560

gatcaactga gctccatctg acctgtggac atgtgagaga gaaatagatg cttttgtgtt 28620

ctggggttgt tttgttacat aaaagtatga cagccacagc aactgaggca cgatctcagg 28680

agttggagcc tattgcttat gaacaaggtt aagagatcaa tttctgcttc ttactctttc 28740

aatttatacc gcccaaacca acactgattt gtctctctgg tcaccgaaaa gacaggtaaa 28800

aacatgggga tagatacaac accaaaagca tgatccacaa aagaaaaaaa agataaactg 28860

tacctcttta aaattaaaaa ctctgctctg ccaaagacac tattaagaaa atagaaacat 28920

ggccaagcgt ggtggctcac gcctgcaatc ccagaacttt gggaggtcga ggtgggtgaa 28980

tcaccagagg tcaggaattc gagaccagcc tggccaacat ggtaaaaccc tgtctcaact 29040

aaaaatacaa aaattagcca ggcatggtgg cgggcgcctg taatcccagc tactcgggag 29100

gctgaggcag gagaattgct tgaacccagg aggcggaggt tgcaatgagc caagatcaca 29160

ccattgcact ccagtctggg tgacaagagc aaacctctgt cttaaaaaaa aaaaaaaaga 29220

aaaagaaaaa agaaaagaaa agaaaagaaa aatgtgattc ccaagcttgt agaaaatatt 29280

tgcaaaagat atctctgatg aaggactggt atctgcaata tataaagaac tcttaaaacc 29340

caacaattaa aaaaaaccgt atctgctaaa atagctgaga ttcaaaacac tgatgacacc 29400

agatgctgag gaggatgtgg agcaacagga actctcattc attgctggtg ggaatgcaca 29460

acggtacagc cactttggaa ggcatctggc agtttattac caaactaagc ataacctact 29520

acacaatcca gcaattgtgc tcccatatga gttgaaaact tacgtctaca cacactcaca 29580

aaaatggcac atggatattt acagcagctt tattcataaa tgccaaaact tggaaacaat 29640

taagatgtcc ttcaataggt gaatggataa acaacttgtg gtatattcac ataatgggat 29700

attattcagc actaagaaaa aagagttgac caggtgcagg tgcagtggct cacacctata 29760

atcccagcac tttgggaggc taaggtggga ggattgtttg agcccagaag tttgagacca 29820

gcttgggcaa caagtgggac cccccccccc accatatcta caaagagtta aacatttagc 29880

tggttgtggt ggcatgcaac tgtggtccca gctacacagg aggctgaggc aggagaaaca 29940

cttgagccca ggagattgag gttgcagtga gccgtgattg taccactgca ctccagcctg 30000

11

20

Artificial Sequence

Antisense Oligonucleotide

11

tcgtcatcgt cttcatctcc 20

12

20

Artificial Sequence

Antisense Oligonucleotide

12

cctcagagcc atcgtcgtca 20

13

20

Artificial Sequence

Antisense Oligonucleotide

13

cctgaggtcc cctcagagcc 20

14

20

Artificial Sequence

Antisense Oligonucleotide

14

cagcccggat cctcgcagct 20

15

20

Artificial Sequence

Antisense Oligonucleotide

15

gggcagcccg gatcctcgca 20

16

20

Artificial Sequence

Antisense Oligonucleotide

16

tggatgtggg atccggcacg 20

17

20

Artificial Sequence

Antisense Oligonucleotide

17

tggctattag gccccggctt 20

18

20

Artificial Sequence

Antisense Oligonucleotide

18

atgtcctcag agcctgcctc 20

19

20

Artificial Sequence

Antisense Oligonucleotide

19

ttctggaagc tgaggtcttc 20

20

20

Artificial Sequence

Antisense Oligonucleotide

20

cccttctgga agctgaggtc 20

21

20

Artificial Sequence

Antisense Oligonucleotide

21

tagccctcct tccgggtggc 20

22

20

Artificial Sequence

Antisense Oligonucleotide

22

ctgatgccct tgaaaaacca 20

23

20

Artificial Sequence

Antisense Oligonucleotide

23

gcccagcatg ttgccgggag 20

24

20

Artificial Sequence

Antisense Oligonucleotide

24

aaggagccca gcatgttgcc 20

25

20

Artificial Sequence

Antisense Oligonucleotide

25

atgaaggagc ccagcatgtt 20

26

20

Artificial Sequence

Antisense Oligonucleotide

26

gatcatgaag gagcccagca 20

27

20

Artificial Sequence

Antisense Oligonucleotide

27

ccggatcatg aaggagccca 20

28

20

Artificial Sequence

Antisense Oligonucleotide

28

cggacaaaga gtagcttcct 20

29

20

Artificial Sequence

Antisense Oligonucleotide

29

cagggtccgg atcttgtaat 20

30

20

Artificial Sequence

Antisense Oligonucleotide

30

tcgttcccct tcttgtagtg 20

31

20

Artificial Sequence

Antisense Oligonucleotide

31

gacagtttct ggcagagccc 20

32

20

Artificial Sequence

Antisense Oligonucleotide

32

gacatgcagg gcaccgacag 20

33

20

Artificial Sequence

Antisense Oligonucleotide

33

ggcttggaag acatgcaggg 20

34

20

Artificial Sequence

Antisense Oligonucleotide

34

agctccaagt ttcttctcca 20

35

20

Artificial Sequence

Antisense Oligonucleotide

35

cccagctcca agtttcttct 20

36

20

Artificial Sequence

Antisense Oligonucleotide

36

ctgcccagct ccaagtttct 20

37

20

Artificial Sequence

Antisense Oligonucleotide

37

ggtggccatc cagacttccc 20

38

20

Artificial Sequence

Antisense Oligonucleotide

38

ttgttgtagg tggccatcca 20

39

20

Artificial Sequence

Antisense Oligonucleotide

39

tgtgcttgtt gtaggtggcc 20

40

20

Artificial Sequence

Antisense Oligonucleotide

40

tggtgtgctt gttgtaggtg 20

41

20

Artificial Sequence

Antisense Oligonucleotide

41

gacatgctcc ctggcttcat 20

42

20

Artificial Sequence

Antisense Oligonucleotide

42

ggaaggcctc caccgacatg 20

43

20

Artificial Sequence

Antisense Oligonucleotide

43

gctgcagagt tttcatcacg 20

44

20

Artificial Sequence

Antisense Oligonucleotide

44

aagtttgacc agcttgtcat 20

45

20

Artificial Sequence

Antisense Oligonucleotide

45

ctccgtgatg atgtagatgg 20

46

20

Artificial Sequence

Antisense Oligonucleotide

46

catcactttt cagaaagtcc 20

47

20

Artificial Sequence

Antisense Oligonucleotide

47

ctgcttgctg ccctcatcac 20

48

20

Artificial Sequence

Antisense Oligonucleotide

48

ggctgagaag tcaatgagtt 20

49

20

Artificial Sequence

Antisense Oligonucleotide

49

cttctgcaat ctgggctgag 20

50

20

Artificial Sequence

Antisense Oligonucleotide

50

tgccttctgc aatctgggct 20

51

20

Artificial Sequence

Antisense Oligonucleotide

51

aggccatgcc ttctgcaatc 20

52

20

Artificial Sequence

Antisense Oligonucleotide

52

tgctcgatga aggccatgcc 20

53

20

Artificial Sequence

Antisense Oligonucleotide

53

tagttcctct gctcgatgaa 20

54

20

Artificial Sequence

Antisense Oligonucleotide

54

tcggtggatg tagttcctct 20

55

20

Artificial Sequence

Antisense Oligonucleotide

55

tcagcaatct tacacaccag 20

56

20

Artificial Sequence

Antisense Oligonucleotide

56

gacccgggcc aggccaaagt 20

57

20

Artificial Sequence

Antisense Oligonucleotide

57

tcgttgtcct caatgacccg 20

58

20

Artificial Sequence

Antisense Oligonucleotide

58

aacttggccc cttcccgagc 20

59

20

Artificial Sequence

Antisense Oligonucleotide

59

gcttcaggag ctgtccactt 20

60

20

Artificial Sequence

Antisense Oligonucleotide

60

tctgacttga tggtgaagga 20

61

20

Artificial Sequence

Antisense Oligonucleotide

61

gggatccggc cgtaggtgac 20

62

20

Artificial Sequence

Antisense Oligonucleotide

62

ggtttgacat ccctgggtaa 20

63

20

Artificial Sequence

Antisense Oligonucleotide

63

catccggtat ccacgctcca 20

64

20

Artificial Sequence

Antisense Oligonucleotide

64

gcgcatcatg atgttgtaga 20

65

20

Artificial Sequence

Antisense Oligonucleotide

65

agcacactct ggatgtattc 20

66

20

Artificial Sequence

Antisense Oligonucleotide

66

tccagcacac tctggatgta 20

67

20

Artificial Sequence

Antisense Oligonucleotide

67

tcatccagca cactctggat 20

68

20

Artificial Sequence

Antisense Oligonucleotide

68

atggctgctg ttggtactgg 20

69

20

Artificial Sequence

Antisense Oligonucleotide

69

cctccctatc atggctgctg 20

70

20

Artificial Sequence

Antisense Oligonucleotide

70

ccttcgagcc accacctggg 20

71

20

Artificial Sequence

Antisense Oligonucleotide

71

cagtccaacc tacccactgg 20

72

20

Artificial Sequence

Antisense Oligonucleotide

72

ggattgcaag agtcaaaaag 20

73

20

Artificial Sequence

Antisense Oligonucleotide

73

gtcagattgt ggattgcaag 20

74

20

Artificial Sequence

Antisense Oligonucleotide

74

cctgagaatg tcagattgtg 20

75

20

Artificial Sequence

Antisense Oligonucleotide

75

gctgtaacta aaatccaacc 20

76

20

Artificial Sequence

Antisense Oligonucleotide

76

acagctgtaa ctaaaatcca 20

77

20

Artificial Sequence

Antisense Oligonucleotide

77

actattttga aagtttccct 20

78

20

Artificial Sequence

Antisense Oligonucleotide

78

tcatttcact attttgaaag 20

79

20

Artificial Sequence

Antisense Oligonucleotide

79

taagacttgc atttatatct 20

80

20

Artificial Sequence

Antisense Oligonucleotide

80

agatgtgcac caccatgctt 20

81

20

Artificial Sequence

Antisense Oligonucleotide

81

tgcaccaccc tatattatca 20

82

20

Artificial Sequence

Antisense Oligonucleotide

82

aacatacata ttaggctggt 20

83

20

Artificial Sequence

Antisense Oligonucleotide

83

gagtagcttc ctgaattgac 20

84

20

Artificial Sequence

Antisense Oligonucleotide

84

tcgttcccct ctggaacaga 20

85

20

Artificial Sequence

Antisense Oligonucleotide

85

gggtccttac ccatccagac 20

86

20

Artificial Sequence

Antisense Oligonucleotide

86

ggccagcaga agatgccaca 20

87

20

Artificial Sequence

Antisense Oligonucleotide

87

tttgacatcc ctgaaaagga 20