Patent Publication Number: US-2003232440-A1

Title: Antisense modulation of STAT1 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of STAT1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding STAT1. Such compounds have been shown to modulate the expression of STAT1.  
       BACKGROUND OF THE INVENTION  
       [0002] Many important cellular processes are regulated by cytokines, hormones and growth factors which interact with cell-surface receptors. Receptors such as type I and II interferon (IFN) receptors are associated with members of the Janus kinase (JAK) superfamily of cytoplasmic tyrosine kinases. Upon cytokine activation, the receptor-associated JAKs then phosphorylate the family of dual function proteins known as signal transducers and activators of transcription (STATs). Phosphorylated and activated STATs then dimerize and translocate to the nucleus, and bind to DNA or act with other DNA binding proteins in multiprotein complexes to regulate gene transcription in a cascade of intracellular signaling events that ultimately affects cell growth and differentiation, the immune response, antiviral activity, or homeostasis (Akira,  Stem Cells,  1999, 17, 138-146; Ramana et al.,  Oncogene,  2000, 19, 2619-2627).  
       [0003] To date, seven STAT family members have been described: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STAT6. The STATs were originally discovered as critical players in interferon signaling mediated by cytokine receptors lacking intrinsic tyrosine kinase domains and employing the JAK kinases. The STATs were found to be activated upon stimulation of cells with interferons alpha, beta and gamma (IFNα, IFNβ and IFNγ) . More recently, it was discovered that STATs are also activated by receptor tyrosine kinases such as the epidermal growth factor receptor (EGF-R) and platelet derived growth factor receptor (PDGF-R), which are capable of directly phosphorylating STATs in the absence of JAK activation. G-protein-coupled receptors such as the angiotensin II and serotonin 5-HTA receptors, as well as the T-cell receptor complex and the CD40 receptor also activate STATs (Akira,  Stem Cells,  1999, 17, 138-146; Ramana et al.,  Oncogene,  2000, 19, 2619-2627) .  
       [0004] Two distinct pathways of STAT transcription factor activity have been described. In response to IFNγ stimulation of cells, the STAT1 protein homodimerizes and translocates to the nucleus to bind to the IFNγ-activated sequence (GAS) in the promoters of IFNγ responsive genes. Alternatively, STAT1 is also a component of the multiprotein complex known as ISGF-3 which binds to the interferon stimulated response element (ISRE) in the promoters of interferon-inducible genes. In response to IFNα or IFNβ stimulation of cells, the ISGF-3 DNA-binding complex is formed, translocates to the nucleus, and specifically binds ISRE. The ISGF-3 complex consists of 84, 91 and 113 kDa proteins, termed collectively the ISGF-3□ proteins, which translocate from the cytoplasm to the nucleus in IFN-α-activated cells and join a 48 kDa protein, the ISGF-3γ (p48) subunit, which is capable of weak, site-specific DNA binding on its own; the ISGF-3 complex forms a tight DNA-binding transcription factor. This four component ISGF-3 complex binds with high affinity to the ISRE site in the nucleus and acts as an interferon-dependent transcriptional modulator (Akira,  Stem Cells,  1999, 17, 138-146; Ramana et al.,  Oncogene,  2000, 19, 2619-2627).  
       [0005] The ISGF-3 multisubunit transcription factor was purified, its four protein components separated, and peptide sequences were obtained. Degenerate oligonucleotide probes were subsequently designed, PCR products amplified from a HeLa cell cDNA library, and cDNAs encoding the components of ISGF-3 were thus cloned. From these human cDNA clones, it was determined that the 91 kDa and 84 kDa components of ISGF-3 represent two isoforms of a previously unknown gene, later named STAT1 (also known as signal transducer and activator of transcription 1, STAT-1, STAT91, and ISGF-3) (Fu et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1992, 89, 7840-7843.; Schindler et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1992, 89, 7836-7839).  
       [0006] The STAT1 gene was mapped with the STAT4 gene to human chromosomal bands 2q32.2-q32.3 by fluorescence in situ hybridization. This region is associated with lung carcinoma and ependymoma, suggesting that these STAT family members may play a role in the pathogenesis of human cancer. The expression patterns of these two genes differ; human STAT1 is expressed ubiquitously, whereas STAT4 is expressed in certain tissues, including spleen, heart, brain, peripheral blood cells, and testis (Yamamoto et al.,  Cytogenet. Cell Genet.,  1997, 77, 207-210). Genomic structure analysis resulted in the determination that the 2 isoforms, STAT1α (p91) and STAT1β (p84), expressed from the STAT1 gene are generated by alternative splicing after exon 22 (Yan et al.,  Nucleic Acids Res.,  1995, 23, 459-463).  
       [0007] The ability of STAT1 to activate transcription is regulated by post-translational modifications. Dimerization, nuclear import and DNA-binding ability of these STAT transcription factor complexes are dependent upon tyrosine phosphorylation of STAT1 by JAK kinases (Mowen and David,  Mol. Cell. Biol.,  2000, 20, 7273-7281). Furthermore, activation of IFNα/β-induced transcription requires arginine methylation of STAT1, and alteration of arginine methylation may be responsible for the lack of IFN-mediated gene induction and the impaired antiproliferative response observed in many malignancies (Mowen et al.,  Cell,  2001, 104, 731-741).  
       [0008] A family of protein inhibitors of activated STAT (PIAS) proteins has been isolated; PIAS1 associates with only phosphorylated STAT1 and blocks its DNA-binding ability, inhibiting STAT1-mediated gene activation. Because the PIAS3 protein similarly binds and inhibits STAT3 function, it has been suggested that specific PIAS inhibitors may modulate each STAT signaling pathway (Akira,  Stem Cells,  1999, 17, 138-146).  
       [0009] The role of the STAT1 gene has been investigated in knockout mice generated by two groups (Durbin et al.,  Cell,  1996, 84, 443-450; Meraz et al.,  Cell,  1996, 84, 431-442). Both groups found that cells and tissues from STAT1-deficient mice were generally insensitive to interferons, and were unable to resolve infections by microbial pathogens and viruses. Although Stat1-/- knockout mice were born at normal frequencies and displayed no overt developmental defects, they were extremely susceptible to opportunistic infections by viral pathogens (Durbin et al.,  Cell,  1996, 84, 443-450). Furthermore, a small proportion of these mice died with enlarged spleens and livers that contained small white foci of undetermined origin (Meraz et al.,  Cell,  1996, 84, 431-442). Thus, STAT1 is primarily important for IFN-dependent signaling pathways.  
       [0010] Interferons are not only involved in immunity, but also inhibit cell growth. Transcriptionally active STAT1 is required for this antiproliferative activity. IFNγ suppresses c-myc expression, and deregulated expression of c-myc is likely to contribute to the abnormal proliferation of Stat1-null mouse embryonic fibroblasts in response to IFNs (Ramana et al.,  EMBO J.,  2000, 19, 263-272). Abnormal proliferation and genomic instability are two hallmarks of tumor development and even a transient excess of c-myc can promote genomic instability (Ramana et al.,  Oncogene,  2000, 19, 2619-2627). It has also been observed that, while STAT1-deficient mice do not display heightened spontaneous malignancy (Levy and Gilliland,  Oncogene,  2000, 19, 2505-2510), mice lacking the IFNγ receptor or STAT1 develop tumors more rapidly and with greater frequency following challenge with chemical carcinogens (Kaplan et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1998, 95, 7556-7561).  
       [0011] STAT1 is involved in a variety of growth regulatory processes, and, paradoxically, STAT1 is constitutively activated in several malignancies. Loss of STAT1 may contribute to oncogenesis both by leading to deregulation of the cell cycle and by impairing the ability of tumors to be recognized and rejected by the immune system. STAT1 appears to play a role in the rejection of transplanted tumors through a variety of mechanisms, including maintenance of natural killer (NK) cell cytolytic activity and tumor recognition, as well as production and response to IFN (Levy and Gilliland,  Oncogene,  2000, 19, 2505-2510). NK cells are cytotoxic lymphocytes that exhibit cytolytic activity toward tumor cells, and STAT1-deficient mice show a decreased basal NK cell activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells, normal levels of molecules involved in activation and lytic function, and despite a normal response to cytokines. Thus, a STAT1-dependent and partially IFN-independent pathway for NK-mediated antitumor activity was defined (Lee et al.,  J. Immunol.,  2000, 165, 3571-3577).  
       [0012] STAT1 also appears to mediate apoptosis. STAT1 is a modulator of the growth inhibitory effect of fibroblast growth factor (FGF) in vitro and of its negative regulatory effect on bone growth in vivo. Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. When transgenic mice overexpressing FGF were crossed with STAT1-null mice, the loss of STAT1 function led to a significant reversal and correction of the chondrodysplasic phenotype (Sahni et al.,  Development,  2001, 128, 2119-2129).  
       [0013] Lymphocytes derived from mice deficient in STAT1 show reduced apoptosis and enhanced proliferation in vitro. Levels of caspases 1 and 11, two enzymes involved in both cytokine processing and induction of apoptosis, were reduced in lymphocytes from STAT1-null mice, and STAT1-null animals were more susceptible to carcinogen-induced thymic tumors, possibly a result of altered T-cell growth and/or survival (Lee et al.,  J. Immunol.,  2000, 164, 1286-1292). STAT1 also participates in TNF-α-induced apoptosis in a STAT1-deficient human cell line. STAT1 was required for efficient expression of the caspases Ice, Cpp32, and Ich-1, and as a consequence, STAT1-null cells were resistant to apoptosis in response to tumor necrosis factor (TNF) or IFNγ due to low constitutive expression of these caspases, conferring a selective advantage to tumor cells that lack STAT1. Furthermore, STAT1 point mutations that inactivate domains required for dimer formation were able to restore protease expression and apoptosis, indicating that the apoptotic functions of STAT1 do not require dimerization and thus differ from functions inducing gene expression (Kumar et al.,  Science,  1997, 278, 1630-1632).  
       [0014] A natural heterozygous germline STAT1 mutation was identified, and is associated with susceptibility to mycobacterial but not viral disease. The mutation causes a loss of GAF and ISGF-3 activation, but is dominant for the former and recessive for the latter, thus impairing the nuclear accumulation of GAF but not of ISGF-3 in heterozygous human cells deficient in STAT1 stimulated by IFNs. This suggests that the antimycobacterial, but not the antiviral, effects of human IFNs are principally mediated by GAF (Dupuis et al.,  Science,  2001, 293, 300-303).  
       [0015] To date, investigative strategies aimed at modulating STAT1 function have involved the use of electroporated antibodies as well as antisense expression vectors and oligonucleotides.  
       [0016] In macrophages, hepatocytes, astroglial cells, and vascular smooth muscle cells (VSMC), (NO) synthesis is induced by bacterial bacterial endotoxins or cytokines. The role of the JAK/STAT pathway on inducible nitric oxide synthase (iNOS) nitric oxide induction was examined by electroporating a neutralizing anti-STAT1 antibody in rat aortic VSMC, and it was demonstrated that STAT1 is involved in suppression rather than activation of IFNγ and lipopolysaccharide-stimulated induction of iNOS (Marrero et al.,  Biochem. Biophys. Res. Commun.,  1998, 252, 508-512).  
       [0017] Exposure of cardiac cells to ischemia or INFγ treatment results in apoptosis and is accompanied by phosphorylation and increased expression and transcriptional activity of STAT1. A vector construct expressing STAT1 in the antisense orientation reduced both ichemia-induced and overexpressed-STAT1-induced cell death in cardiac cells (Stephanou et al.,  J. Biol. Chem.,  2000, 275, 10002-10008).  
       [0018] A phosphorothioate antisense oligonucleotide, 20 nucleotides in length, complementary to the initiation codon in the STAT1α mRNA, was used to inhibit both constitutive and IFNγ-enhanced STAT1α expression specifically, and to demonstrate that STAT1α is involved in IFNγ induction of class II transactivator and class II MHC gene expression (Lee and Benveniste,  J. Immunol.,  1996, 157, 1559-1568). The same antisense oligonucleotide was also used to show that STAT1 is involved in the regulation of eosinophils by IFNγ. The STAT1 antisense oligonucleotide significantly inhibited IFNγ induced CD69 expression on IL-3- and IL-5-induced eosinophils, suggesting that STAT1 plays a role in eosinophil regulation (Ochiai et al.,  Int. Arch. Allergy Immunol.,  2001, 124, 237-241).  
       [0019] Activation of STAT1 and STAT3 is constitutive in transformed squamous epithelial cells, which produce elevated levels of TGFα. A phosphorothioate antisense oligonucleotide, 21 nucleotides in length, directed against the translation start site of the STAT1 gene was used to show that STAT1, in contrast to STAT3, had no effect on cell growth, and thus, TGFα-mediated autocrine growth of transformed epithelial cells is dependent on STAT3 but not STAT1 (Grandis et al.,  J. Clin. Invest.,  1998, 102, 1385-1392)  
       [0020] Lung fibrosis is a fatal condition of excess extracellular matrix deposition associated with increased transforming growth factor-beta (TGFβ) activity. A phosphorothioate antisense oligonucleotide, 18 nucleotides in length, corresponding to the 5′-end of the STAT1 mRNA, was used to decipher the molecular mechanism of this TGFβ-mediated fibrosis and show that in IFNγ exerts an antagonistic, antifibrotic activity via STAT1 (Eickelberg et al.,  FASEB J.,  2001, 15, 797-806)  
       [0021] Disclosed and claimed in U.S. Pat. No. 5,731,155 are compositions and methods for inhibiting cytokine-induced intracellular activation of the STAT family of transcription factors, including STAT1, comprising an isolated peptide or derivative thereof, wherein the peptide contains an amino acid sequence derived from a receptor for a cytokine, wherein the peptide contains a phosphorylated tyrosine, and wherein the protein specifically binds to a member of the STAT family to inhibit activation of the transcription factor by the cytokine (Schreiber et al., 1998).  
       [0022] Disclosed and claimed in U.S. Pat. No. 5,976,835 is an isolated nucleic acid encoding a receptor recognition factor (RRF) selected from the group consisting of STAT1α and STAT1β, a method of expressing a recombinant RRF in a cell containing an expression vector comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the RRF by the cell, and wherein said RRF is selected from the group consisting of STAT1α and STAT1β. Also disclosed is a recombinant DNA molecule, wherein the recombinant DNA molecule expresses antisense RNA or ribozymes which would attack the mRNAs of any or all of said STAT DNA sequences (Darnell et al., 1999).  
       [0023] Disclosed and claimed in U.S. Pat. No. 6,030,780 is a method for identifying a drug that modulates the ability of adjacent STAT protein dimers to interact and bind to adjacent DNA binding sites (Vinkemeier and Darnell, 2000).  
       [0024] Disclosed and claimed in U.S. Pat. No. 6,159,694 is an antisense compound 8 to 30 nucleobases in length targeted to the coding region of human STAT3, a region which is also found in STAT1, as well as a method of inhibiting the expression of human or mouse STAT3 in human or mouse cells or tissues comprising contacting said cells or tissues in vitro with the antisense compound so that expression of human or mouse STAT3 is inhibited (Karras, 2000).  
       [0025] Disclosed and claimed in U.S. Pat. No. 6,160,092 is a crystal of the core portion of the STAT1 protein in dimeric form with an 18-mer duplex DNA that contains a binding site for the STAT1-dimer. Corresponding structural information obtained by X-ray crystallography is also provided. Further disclosed are methods of using the crystal and related structural information in drug screening assays (Chen et al., 2000).  
       [0026] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of STAT1.  
       [0027] Consequently, there remains a long felt need for additional agents capable of effectively inhibiting STAT1 function.  
       [0028] 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 STAT1 expression.  
       [0029] The present invention provides compositions and methods for modulating STAT1 expression, including modulation of both alternatively spliced forms, STAT1α and STAT1β.  
       SUMMARY OF THE INVENTION  
       [0030] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding STAT1, and which modulate the expression of STAT1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of STAT1 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 STAT1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0031] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding STAT1, ultimately modulating the amount of STAT1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding STAT1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding STAT1” encompass DNA encoding STAT1, 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, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, 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 STAT1. 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.  
       [0032] 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 STAT1. 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 STAT1, regardless of the sequence(s) of such codons.  
       [0033] 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.  
       [0034] 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.  
       [0035] 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. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0036] 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.  
       [0037] 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.  
       [0038] 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.  
       [0039] 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.  
       [0040] 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.  
       [0041] 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 activity, 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. It is preferred that the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al.,  J. Mol. Biol.,  1990, 215, 403-410; Zhang and Madden,  Genome Res.,  1997, 7, 649-656).  
       [0042] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as “preferred target regions” and are therefore preferred sites for targeting. As used herein the term “preferred target region” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target regions represent regions of the target nucleic acid which are accessible for hybridization.  
       [0043] While the specific sequences of particular preferred target regions are set forth below, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target regions may be identified by one having ordinary skill.  
       [0044] Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well.  
       [0045] Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.  
       [0046] 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.  
       [0047] 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.  
       [0048] 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.  
       [0049] 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).  
       [0050] 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.  
       [0051] 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.  
       [0052] 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 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, 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.  
       [0053] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.  
       [0054] Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.  
       [0055] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.  
       [0056] 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. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. 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.  
       [0057] 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.  
       [0058] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-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.  
       [0059] 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.  
       [0060] 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.  
       [0061] 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.  
       [0062] 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.  
       [0063] 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.  
       [0064] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta,  1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 , also described in examples hereinbelow.  
       [0065] 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.  
       [0066] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH 2 —) n  group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.  
       [0067] 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 &amp; 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.  
       [0068] 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.  
       [0069] 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 conjugate 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. U.S.A.,  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 &amp; 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.  
       [0070] 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.  
       [0071] 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, increased stability 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. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. 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.  
       [0072] 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.  
       [0073] 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.  
       [0074] 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.  
       [0075] 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.  
       [0076] 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.  
       [0077] 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.  
       [0078] 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.  
       [0079] 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.  
       [0080] 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 STAT1 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.  
       [0081] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding STAT1, 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 STAT1 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 STAT1 in a sample may also be prepared.  
       [0082] 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.  
       [0083] 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.  
       [0084] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, 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. application 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 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0085] 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.  
       [0086] 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.  
       [0087] 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.  
       [0088] 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.  
       [0089] 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.  
       [0090] Emulsions  
       [0091] 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&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or 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 phase provides an o/w/o emulsion.  
       [0092] 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).  
       [0093] 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).  
       [0094] 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.  
       [0095] 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).  
       [0096] 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.  
       [0097] 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.  
       [0098] 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 ease of formulation, as well as 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.  
       [0099] 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&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 271).  
       [0100] 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.  
       [0101] 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 (DA0750), 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.  
       [0102] 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.  
       [0103] 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.  
       [0104] Liposomes  
       [0105] 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.  
       [0106] 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.  
       [0107] 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.  
       [0108] 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.  
       [0109] 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 and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.  
       [0110] 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.  
       [0111] 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.  
       [0112] 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).  
       [0113] 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).  
       [0114] 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.  
       [0115] 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).  
       [0116] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.  S.T.P. Pharma. Sci.,  1994, 4, 6, 466).  
       [0117] 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).  
       [0118] 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.).  
       [0119] 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.  
       [0120] 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.  
       [0121] 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.  
       [0122] 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).  
       [0123] 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.  
       [0124] 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.  
       [0125] 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.  
       [0126] 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.  
       [0127] 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).  
       [0128] Penetration Enhancers  
       [0129] 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.  
       [0130] 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.  
       [0131] Surfactants:  
       [0132] 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).  
       [0133] Fatty Acids:  
       [0134] 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).  
       [0135] Bile Salts:  
       [0136] The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman &amp; Gilman&#39;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&#39;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).  
       [0137] Chelating Agents:  
       [0138] 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).  
       [0139] Non-Chelating Non-Surfactants:  
       [0140] 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).  
       [0141] 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.  
       [0142] 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.  
       [0143] Carriers  
       [0144] 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 &amp; Nucl. Acid Drug Dev.,  1996, 6, 177-183).  
       [0145] Excipients  
       [0146] 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.).  
       [0147] 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.  
       [0148] 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.  
       [0149] 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.  
       [0150] Other Components  
       [0151] 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.  
       [0152] 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.  
       [0153] 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.  
       [0154] 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.  
       [0155] 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.  
       [0156] 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.  
     
    
    
     EXAMPLES  
     Example 1  
     Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0157] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.  
     [0158] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH 2 Cl 2 ), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).  
     [0159] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al.,  Nucleic Acids Research,  1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:  
     [0160] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0161] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (R f  in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH 2 Cl 2  were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH 2 Cl 2  (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH 2 Cl 2  (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH 2 Cl 2  (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.  
     [0162] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0163] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R f  0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R f  0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).  
     [0164] TLC indicated a complete reaction (product R f  0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.  
     [0165] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.  
     [0166] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0167] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH 2 Cl 2 -EtOAc; CH 2 Cl 2 -EtOAc 4:1; R f  0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.  
     [0168] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g) ,dissolved in CH 2 Cl 2  (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.  
     [0169] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0170] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na 2 SO 4 ), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).  
     [0171] 2′-Fluoro Amidites  
     [0172] 2′-Fluorodeoxyadenosine Amidites  
     [0173] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al.,  J. Med. Chem.,  1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a S N 2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.  
     [0174] 2′-Fluorodeoxyguanosine  
     [0175] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyryl-arabinofuranosylguanosine. Alternatively, isobutyryl-arabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides &amp; Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.  
     [0176] 2′-Fluorouridine  
     [0177] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0178] 2′-Fluorodeoxycytidine  
     [0179] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0180] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0181] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).  
     [0182] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0183] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.  
     [0184] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).  
     [0185] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.  
     [0186] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0187] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.  
     [0188] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the frations were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.  
     [0189] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0190] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).  
     [0191] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0192] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R f  0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R f  0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight  
     [0193] TLC indicated a complete reaction (CH 2 Cl 2 -acetone-MeOH, 20:5:3, R f  0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH 2 Cl 2  (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH 2 Cl 2  (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.  
     [0194] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0195] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of &gt;99.7%.  
     [0196] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)  
     [0197] 5′-O- (4,4′-Dimethoxytriphenylmethyl) -2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).  
     [0198] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)  
     [0199] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L) . The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).  
     [0200] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G Amidite)  
     [0201] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).  
     [0202] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0203] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0204] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.  
     [0205] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0206] O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (R f  0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH 2 Cl 2  (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.  
     [0207] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0208] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure&lt;100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R f  0.67 for desired product and R f  0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.  
     [0209] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0210] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.  
     [0211] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0212] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH 2 Cl 2  (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH 2 Cl 2 , and the combined organic phase was washed with water and brine and dried (anhydrous Na 2 SO 4 ). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation.  
     [0213] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0214] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH 2 Cl 2 ) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.  
     [0215] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0216] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH 2 Cl 2 ). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH 2 Cl 2 ) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.  
     [0217] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0218] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 5  under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH 2 Cl 2  containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.  
     [0219] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0220] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P 2 O 5  under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N 1 ,N 1 -tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). The EtOAc layer was dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.  
     [0221] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0222] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.  
     [0223] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0224] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].  
     [0225] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0226] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.  
     [0227] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0228] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O 2 -,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH 2 Cl 2 /TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.  
     [0229] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0230] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers were washed with saturated NaHCO 3  solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH 2 Cl 2 /TEA) to afford the product.  
     [0231] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl Uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0232] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH 2 Cl 2  (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.  
     Example 2  
     Oligonucleotide Synthesis  
     [0233] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.  
     [0234] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with &gt;3 volumes of ethanol from a 1 M NH 4 OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.  
     [0235] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0236] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0237] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0238] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.  
     [0239] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0240] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0241] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.  
     Example 3  
     Oligonucleoside Synthesis  
     [0242] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.  
     [0243] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.  
     [0244] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     PNA Synthesis  
     [0245] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential  
     [0246] Applications,  Bioorganic &amp; Medicinal Chemistry,  1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.  
     Example 5  
     Synthesis of Chimeric Oligonucleotides  
     [0247] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.  
     [0248] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0249] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligo-nucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH 4 OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0250] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides  
     [0251] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.  
     [0252] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0253] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.  
     [0254] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.  
     Example 6  
     Oligonucleotide Isolation  
     [0255] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH 4 OAc with &gt;3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al.,  J. Biol. Chem.  1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.  
     Example 7  
     Oligonucleotide Synthesis—96 Well Plate Format  
     [0256] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.  
     [0257] Oligonucleotides were cleaved from support and deprotected with concentrated NH 4 OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.  
     Example 8  
     Oligonucleotide Analysis—96-Well Plate Format  
     [0258] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.  
     Example 9  
     Cell Culture and Oligonucleotide Treatment  
     [0259] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.  
     [0260] T-24 Cells:  
     [0261] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy&#39;s 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.  
     [0262] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.  
     [0263] A549 Cells:  
     [0264] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.  
     [0265] NHDF Cells:  
     [0266] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.  
     [0267] HEK Cells:  
     [0268] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.  
     [0269] Treatment with Antisense Compounds:  
     [0270] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.  
     [0271] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.  
     Example 10  
     Analysis of Oligonucleotide Inhibition of STAT1 Expression  
     [0272] Antisense modulation of STAT1 expression can be assayed in a variety of ways known in the art. For example, STAT1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology , Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology , Volume 1, pp. 4.2.1-4.2.9, John Wiley &amp; Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer&#39;s instructions.  
     [0273] Protein levels of STAT1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to STAT1 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.12.1-11.12.9, John Wiley &amp; Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.4.1-11.11.5, John Wiley &amp; Sons, Inc., 1997).  
     [0274] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 10.16.1-10.16.11, John Wiley &amp; Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 10.8.1-10.8.21, John Wiley &amp; Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.2.1-11.2.22, John Wiley &amp; Sons, Inc., 1991).  
     Example 11  
     Poly(A)+ mRNA Isolation  
     [0275] Poly(A)+ mRNA was isolated according to Miura et al., ( Clin. Chem.,  1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 1, pp. 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.  
     [0276] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     Total RNA Isolation  
     [0277] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer&#39;s recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.  
     [0278] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.  
     Example 13  
     Real-Time Quantitative PCR Analysis of STAT1 mRNA Levels  
     [0279] Quantitation of STAT1 mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;s instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.  
     [0280] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.  
     [0281] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5× PCR buffer (-MgCl2), 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).  
     [0282] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0283] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0284] Probes and primers to human STAT1 were designed to hybridize to a human STAT1 sequence, using published sequence information (GenBank accession number M97935.1, incorporated herein as SEQ ID NO:4). For human STAT1 the PCR primers were:  
     [0285] forward primer: TGGGCTACTTTGTCCTTTTTG (SEQ ID NO: 5)  
     [0286] reverse primer: CAGCGAAACATATGCAGTTCTC (SEQ ID NO: 6) and the PCR probe was:  
     [0287] FAM-CTGACAACTTGAATAATACACCAGAGATAATATGAGAATCAGATCATTTC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:  
     [0288] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)  
     [0289] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     Northern Blot Analysis of STAT1 mRNA Levels  
     [0290] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer&#39;s recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer&#39;s recommendations for stringent conditions.  
     [0291] To detect human STAT1, a human STAT1 specific probe was prepared by PCR using the forward primer TGGGCTACTTTGTCCTTTTTG (SEQ ID NO: 5) and the reverse primer CAGCGAAACATATGCAGTTCTC (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0292] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.  
     Example 15  
     Antisense Inhibition of Human STAT1 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap  
     [0293] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human STAT1 RNA, using published sequences (GenBank accession number M97935.1, incorporated herein as SEQ ID NO: 4, GenBank accession number BC002704.1, incorporated herein as SEQ ID NO: 11, and a genomic sequence representing nucleotides 298372-346390 of GenBank accession number NT — 005413.5, incorporated herein as SEQ ID NO: 12). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human STAT1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.  
               TABLE 1                          Inhibition of human STAT1 mRNA levels by chimeric       phosphorothioate oligonucleotides having 2′-MOE wings and a       deoxy gap                                                     TARGET                                   SEQ                   CONTROL               ID   TARGET       %   SEQ ID   SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   NO   NO                                                     153699   Coding   4   742   ttgccacaccattggtctcg   76   13   2               153704   Coding   4   2206   aggcatggtctttgtcaata   76   14   2               204461   Start   4   188   cactgagacatcctgccacc   55   15   2           Codon               204462   Coding   4   222   caggaattttgagtcaagct   82   16   2               204463   Coding   4   416   tgtagcaagaagttattctc   58   17   2               204464   Coding   4   453   atcctgaagattacgcttgc   82   18   2               204465   Coding   4   559   ccgactgagcctgattaaat   57   19   2               204466   Coding   4   1408   ctttcaattgcaggtgccga   74   20   2               204467   Coding   4   1716   acctcttttggtgacagaag   63   21   2               204468   Coding   4   1904   ccatcattccagagagggag   80   22   2               204469   Coding   4   2028   cacccatgtgaatgtgatgg   72   23   2               204470   Coding   4   2060   aagtcaggttcgcctccgtt   49   24   2               204471   Coding   4   2322   agaagggtgaacttcagaca   32   25   2               204472   Coding   4   2398   cagagcccactatccgagac   67   26   2               204473   Stop    4   2439   attcatgctctatactgtgt   73   27   2           Codon               204474   3′UTR   4   2869   taagaattctcccacagaaa   70   28   2               204475   3′UTR   4   2952   attgtatataaacttggctt   79   29   2               204476   3′UTR   4   3982   gaaacaatattgtttttaat   17   30   2               204477   5′UTR   11   2   gcaggaaagcgacctcgtgc   21   31   2               204478   5′UTR   11   18   cctccgcagactctgcgcag   77   32   2               204479   5′UTR   11   32   ggtgcagccgagcccctccg   77   33   2               204480   5′UTR   11   151   atgaaacttttctgcgcgca   57   34   2               204481   Stop   11   2443   gtgttcacttacacttcaga   74   35   2           Codon               204482   3′UTR   11   2490   agccaggagcaaggctggct   55   36   2               204483   3′UTR   11   2569   gggatctcaacaagttcagc   68   37   2               204484   3′UTR   11   2592   aaatgctgataggcagtaac   64   38   2               204485   intron   12   4612   ttttcaagtagggcatggaa   63   39   2               204486   exon   12   6697   caggtcatacctgaagatta   42   40   2               204487   intron   12   24158   tcccaaaacctaatagggac   41   41   2               204488   intron:   12   24339   cacaaacgagctgcaaatac   15   42   2           exon           junction               204489   intron:   12   25985   caccaacagtctggaaagaa   43   43   2           exon           junction               204490   intron   12   32119   tttgtcacttctcccttaac   26   44   2               204491   intron   12   37717   caagaagagtattcctgaaa   31   45   2               204492   exon:   12   39848   gttcacttacacttcagaca   52   46   2           intron           junction               204493   intron:   12   40728   tagaagggtgactaaaatgg   20   47   2           exon           junction               204494   exon:   12   40830   tggtactcaccatactgtcg   22   48   2           intron           junction               204495   intron:   12   44942   ctgtgttcatctgtaaaaag   44   49   2           exon           junction                  
 
     [0294] As shown in Table 1, SEQ ID NOs 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 26, 27, 28, 29, 32, 33, 34, 35, 36, 37, 38, 39 and 46 demonstrated at least 50% inhibition of human STAT1 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found.  
               TABLE 2                          Sequence and position of preferred target regions identified       in STAT1.                                             TARGET           REV               SITE   SEQ ID   TARGET       COMP OF       SEQ ID       ID   NO   SITE   SEQUENCE   SEQ ID   ACTIVE IN   NO                                                 69240   4   742   cgagaccaatggtgtggcaa   13     H. sapiens     50               69245   4   2206   tattgacaaagaccatgcct   14     H. sapiens     51               122175   4   188   ggtggcaggatgtctcagtg   15     H. sapiens     52               122176   4   222   agcttgactcaaaattcctg   16     H. sapiens     53               122177   4   416   gagaataacttcttgctaca   17     H. sapiens     54               122178   4   453   gcaagcgtaatcttcaggat   18     H. sapiens     55               122179   4   559   atttaatcaggctcagtcgg   19     H. sapiens     56               122180   4   1408   tcggcacctgcaattgaaag   20     H. sapiens     57               122181   4   1716   cttctgtcaccaaaagaggt   21     H. sapiens     58               122182   4   1904   ctccctctctggaatgatgg   22     H. sapiens     59               122183   4   2028   ccatcacattcacatgggtg   23     H. sapiens     60               122186   4   2398   gtctcggatagtgggctctg   26     H. sapiens     61               122187   4   2439   acacagtatagagcatgaat   27     H. sapiens     62               122188   4   2869   tttctgtgggagaattctta   28     H. sapiens     63               122189   4   2952   aagccaagtttatatacaat   29     H. sapiens     64               122192   11   18   ctgcgcagagtctgcggagg   32     H. sapiens     65               122193   11   32   cggaggggctcggctgcacc   33     H. sapiens     66               122194   11   151   tgcgcgcagaaaagtttcat   34     H. sapiens     67               122195   11   2443   tctgaagtgtaagtgaacac   35     H. sapiens     68               122196   11   2490   agccagccttgctcctggct   36     H. sapiens     69               122197   11   2569   gctgaacttgttgagatccc   37     H. sapiens     70               122198   11   2592   gttactgcctatcagcattt   38     H. sapiens     71               122199   12   4612   ttccatgccctacctgaaaa   39     H. sapiens     72               122206   12   39848   tgtctgaagtgtaagtgaac   46     H. sapiens     73                  
 
     [0295] As these “preferred target regions” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these sites and consequently inhibit the expression of STAT1.  
     [0296] In one embodiment, the “preferred target region” may be employed in screening candidate antisense compounds. “Candidate antisense compounds” are those that inhibit the expression of a nucleic acid molecule encoding STAT1 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target region. The method comprises the steps of contacting a preferred target region of a nucleic acid molecule encoding STAT1 with one or more candidate antisense compounds, and selecting for one or more candidate antisense compounds which inhibit the expression of a nucleic acid molecule encoding STAT1. Once it is shown that the candidate antisense compound or compounds are capable of inhibiting the expression of a nucleic acid molecule encoding STAT1, the candidate antisense compound may be employed as an antisense compound in accordance with the present invention.  
     [0297] According to the present invention, 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.  
     Example 16  
     Western Blot Analysis of STAT1 Protein Levels  
     [0298] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to STAT1 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).  
    
     
       
         1 
         
           
             73  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

gtgcgcgcga gcccgaaatc                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            3 

atgcattctg cccccaagga                                                 20 

 
           
             4  
             4003  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (197)...(2449)  
             
           
            4 

attaaacctc tcgccgagcc cctccgcaga ctctgcgccg gaaagtttca tttgctgtat     60 

gccatcctcg agagctgtct aggttaacgt tcgcactctg tgtatataac ctcgacagtc    120 

ttggcaccta acgtgctgtg cgtagctgct cctttggttg aatccccagg cccttgttgg    180 

ggcacaaggt ggcagg atg tct cag tgg tac gaa ctt cag cag ctt gac tca    232 
                  Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser 
                    1               5                  10 

aaa ttc ctg gag cag gtt cac cag ctt tat gat gac agt ttt ccc atg      280 
Lys Phe Leu Glu Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met 
         15                  20                  25 

gaa atc aga cag tac ctg gca cag tgg tta gaa aag caa gac tgg gag      328 
Glu Ile Arg Gln Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu 
     30                  35                  40 

cac gct gcc aat gat gtt tca ttt gcc acc atc cgt ttt cat gac ctc      376 
His Ala Ala Asn Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu 
 45                  50                  55                  60 

ctg tca cag ctg gat gat caa tat agt cgc ttt tct ttg gag aat aac      424 
Leu Ser Gln Leu Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn 
                 65                  70                  75 

ttc ttg cta cag cat aac ata agg aaa agc aag cgt aat ctt cag gat      472 
Phe Leu Leu Gln His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp 
             80                  85                  90 

aat ttt cag gaa gac cca atc cag atg tct atg atc att tac agc tgt      520 
Asn Phe Gln Glu Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys 
         95                 100                 105 

ctg aag gaa gaa agg aaa att ctg gaa aac gcc cag aga ttt aat cag      568 
Leu Lys Glu Glu Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln 
    110                 115                 120 

gct cag tcg ggg aat att cag agc aca gtg atg tta gac aaa cag aaa      616 
Ala Gln Ser Gly Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys 
125                 130                 135                 140 

gag ctt gac agt aaa gtc aga aat gtg aag gac aag gtt atg tgt ata      664 
Glu Leu Asp Ser Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile 
                145                 150                 155 

gag cat gaa atc aag agc ctg gaa gat tta caa gat gaa tat gac ttc      712 
Glu His Glu Ile Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe 
            160                 165                 170 

aaa tgc aaa acc ttg cag aac aga gaa cac gag acc aat ggt gtg gca      760 
Lys Cys Lys Thr Leu Gln Asn Arg Glu His Glu Thr Asn Gly Val Ala 
        175                 180                 185 

aag agt gat cag aaa caa gaa cag ctg tta ctc aag aag atg tat tta      808 
Lys Ser Asp Gln Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu 
    190                 195                 200 

atg ctt gac aat aag aga aag gaa gta gtt cac aaa ata ata gag ttg      856 
Met Leu Asp Asn Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu 
205                 210                 215                 220 

ctg aat gtc act gaa ctt acc cag aat gcc ctg att aat gat gaa cta      904 
Leu Asn Val Thr Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu 
                225                 230                 235 

gtg gag tgg aag cgg aga cag cag agc gcc tgt att ggg ggg ccg ccc      952 
Val Glu Trp Lys Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro 
            240                 245                 250 

aat gct tgc ttg gat cag ctg cag aac tgg ttc act ata gtt gcg gag     1000 
Asn Ala Cys Leu Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu 
        255                 260                 265 
agt ctg cag caa gtt cgg cag cag ctt aaa aag ttg gag gaa ttg gaa     1048 
Ser Leu Gln Gln Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu 
    270                 275                 280 

 cag aaa tac acc tac gaa cat gac cct atc aca aaa aac aaa caa gtg    1096 
Gln Lys Tyr Thr Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val 
285                 290                 295                 300 

 tta tgg gac cgc acc ttc agt ctt ttc cag cag ctc att cag agc tcg    1144 
Leu Trp Asp Arg Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser 
                305                 310                 315 

 ttt gtg gtg gaa aga cag ccc tgc atg cca acg cac cct cag agg ccg    1192 
Phe Val Val Glu Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro 
            320                 325                 330 

 ctg gtc ttg aag aca ggg gtc cag ttc act gtg aag ttg aga ctg ttg    1240 
Leu Val Leu Lys Thr Gly Val Gln Phe Thr Val Lys Leu Arg Leu Leu 
        335                 340                 345 

 gtg aaa ttg caa gag ctg aat tat aat ttg aaa gtc aaa gtc tta ttt    1288 
Val Lys Leu Gln Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe 
    350                 355                 360 

 gat aaa gat gtg aat gag aga aat aca gta aaa gga ttt agg aag ttc    1336 
Asp Lys Asp Val Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Lys Phe 
365                 370                 375                 380 

 aac att ttg ggc acg cac aca aaa gtg atg aac atg gag gag tcc acc    1384 
Asn Ile Leu Gly Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr 
                385                 390                 395 

 aat ggc agt ctg gcg gct gaa ttt cgg cac ctg caa ttg aaa gaa cag    1432 
Asn Gly Ser Leu Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln 
            400                 405                 410 

 aaa aat gct ggc acc aga acg aat gag ggt cct ctc atc gtt act gaa    1480 
Lys Asn Ala Gly Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu 
        415                 420                 425 

 gag ctt cac tcc ctt agt ttt gaa acc caa ttg tgc cag cct ggt ttg    1528 
Glu Leu His Ser Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu 
    430                 435                 440 

 gta att gac ctc gag acg acc tct ctg ccc gtt gtg gtg atc tcc aac    1576 
Val Ile Asp Leu Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn 
445                 450                 455                 460 

 gtc agc cag ctc ccg agc ggt tgg gcc tcc atc ctt tgg tac aac atg    1624 
Val Ser Gln Leu Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr Asn Met 
                465                 470                 475 

 ctg gtg gcg gaa ccc agg aat ctg tcc ttc ttc ctg act cca cca tgt    1672 
Leu Val Ala Glu Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys 
            480                 485                 490 

 gca cga tgg gct cag ctt tca gaa gtg ctg agt tgg cag ttt tct tct    1720 
Ala Arg Trp Ala Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser 
        495                 500                 505 

 gtc acc aaa aga ggt ctc aat gtg gac cag ctg aac atg ttg gga gag    1768 
Val Thr Lys Arg Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu 
    510                 515                 520 

 aag ctt ctt ggt cct aac gcc agc ccc gat ggt ctc att ccg tgg acg    1816 
Lys Leu Leu Gly Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr 
525                 530                 535                 540 

 agg ttt tgt aag gaa aat ata aat gat aaa aat ttt ccc ttc tgg ctt    1864 
Arg Phe Cys Lys Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu 
                545                 550                 555 

 tgg att gaa agc atc cta gaa ctc att aaa aaa cac ctg ctc cct ctc    1912 
Trp Ile Glu Ser Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pro Leu 
            560                 565                 570 

 tgg aat gat ggg tgc atc atg ggc ttc atc agc aag gag cga gag cgt    1960 
Trp Asn Asp Gly Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg 
        575                 580                 585 

 gcc ctg ttg aag gac cag cag ccg ggg acc ttc ctg ctg cgg ttc agt    2008 
Ala Leu Leu Lys Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser 
    590                 595                 600 

 gag agc tcc cgg gaa ggg gcc atc aca ttc aca tgg gtg gag cgg tcc    2056 
Glu Ser Ser Arg Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser 
605                 610                 615                 620 

 cag aac gga ggc gaa cct gac ttc cat gcg gtt gaa ccc tac acg aag    2104 
Gln Asn Gly Gly Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys 
                625                 630                 635 

 aaa gaa ctt tct gct gtt act ttc cct gac atc att cgc aat tac aaa    2152 
Lys Glu Leu Ser Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys 
            640                 645                 650 

 gtc atg gct gct gag aat att cct gag aat ccc ctg aag tat ctg tat    2200 
Val Met Ala Ala Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr 
        655                 660                 665 

 cca aat att gac aaa gac cat gcc ttt gga aag tat tac tcc agg cca    2248 
Pro Asn Ile Asp Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro 
    670                 675                 680 

 aag gaa gca cca gag cca atg gaa ctt gat ggc cct aaa gga act gga    2296 
Lys Glu Ala Pro Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Thr Gly 
685                 690                 695                 700 

 tat atc aag act gag ttg att tct gtg tct gaa gtt cac cct tct aga    2344 
Tyr Ile Lys Thr Glu Leu Ile Ser Val Ser Glu Val His Pro Ser Arg 
                705                 710                 715 

 ctt cag acc aca gac aac ctg ctc ccc atg tct cct gag gag ttt gac    2392 
Leu Gln Thr Thr Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Phe Asp 
            720                 725                 730 

 gag gtg tct cgg ata gtg ggc tct gta gaa ttc gac agt atg atg aac    2440 
Glu Val Ser Arg Ile Val Gly Ser Val Glu Phe Asp Ser Met Met Asn 
        735                 740                 745 

aca gta tag agcatgaatt tttttcatct tctctggcga cagttttcct tctcatctgt  2499 
Thr Val 
    750 

gattccctcc tgctactctg ttccttcaca tcctgtgttt ctagggaaat gaaagaaagg   2559 

ccagcaaatt cgctgcaacc tgttgatagc aagtgaattt ttctctaact cagaaacatc   2619 

agttactctg aagggcatca tgcatcttac tgaaggtaaa attgaaaggc attctctgaa   2679 

gagtgggttt cacaagtgaa aaacatccag atacacccaa agtatcagga cgagaatgag   2739 

ggtcctttgg gaaaggagaa gttaagcaac atctagcaaa tgttatgcat aaagtcagtg   2799 

cccaactgtt ataggttgtt ggataaatca gtggttattt agggaactgc ttgacgtagg   2859 

aacggtaaat ttctgtggga gaattcttac atgttttctt tgctttaagt gtaactggca   2919 

gttttccatt ggtttacctg tgaaatagtt caaagccaag tttatataca attatatcag   2979 

tcctctttca aaggtagcca tcatggatct ggtaggggga aaatgtgtat tttattacat   3039 

ctttcacatt ggctatttaa agacaaagac aaattctgtt tcttgagaag agaatattag   3099 

ctttactgtt tgttatggct taatgacact agctaatatc aatagaagga tgtacatttc   3159 

caaattcaca agttgtgttt gatatccaaa gctgaataca ttctgctttc atcttggtca   3219 

catacaatta tttttacagt tctcccaagg gagttaggct attcacaacc actcattcaa   3279 

aagttgaaat taaccataga tgtagataaa ctcagaaatt taattcatgt ttcttaaatg   3339 

ggctactttg tcctttttgt tattagggtg gtatttagtc tattagccac aaaattggga   3399 

aaggagtaga aaaagcagta actgacaact tgaataatac accagagata atatgagaat   3459 

cagatcattt caaaactcat ttcctatgta actgcattga gaactgcata tgtttcgctg   3519 

atatatgtgt ttttcacatt tgcgaatggt tccattctct ctcctgtact ttttccagac   3579 

acttttttga gtggatgatg tttcgtgaag tatactgtat ttttaccttt ttccttcctt   3639 

atcactgaca caaaaagtag attaagagat gggtttgaca aggttcttcc cttttacata   3699 

ctgctgtcta tgtggctgta tcttgttttt ccactactgc taccacaact atattatcat   3759 

gcaaatgctg tattcttctt tggtggagat aaagatttct tgagttttgt tttaaaatta   3819 

aagctaaagt atctgtattg cattaaatat aatatcgaca cagtgctttc cgtggcactg   3879 

catacaatct gaggcctcct ctctcagttt ttatatagat ggcgagaacc taagtttcag   3939 

ttgattttac aattgaaatg actaaaaaac aaagaagaca acattaaaaa caatattgtt   3999 

tcta                                                                4003 

 
           
             5  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

tgggctactt tgtccttttt g                                               21 

 
           
             6  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

cagcgaaaca tatgcagttc tc                                              22 

 
           
             7  
             50  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

ctgacaactt gaataataca ccagagataa tatgagaatc agatcatttc                50 

 
           
             8  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            8 

gaaggtgaag gtcggagtc                                                  19 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            9 

gaagatggtg atgggatttc                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            10 

caagcttccc gttctcagcc                                                 20 

 
           
             11  
             2638  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (316)...(2454)  
             
           
            11 

ggcacgaggt cgctttcctg cgcagagtct gcggaggggc tcggctgcac cggggggatc     60 

gcgcctggca gaccccagac cgagcagagg cgacccagcg cgctcgggag aggctgcacc    120 

gccgcgcccc cgcctagccc ttccggatcc tgcgcgcaga aaagtttcat ttgctgtatg    180 

ccatcctcga gagctgtcta ggttaacgtt cgcactctgt gtatataacc tcgacagtct    240 

tggcacctaa cgtgctgtgc gtagctgctc ctttggttga atccccaggc ccttgttggg    300 

gcacaaggtg gcagg atg tct cag tgg tac gaa ctt cag cag ctt gac tca     351 
                 Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser 
                   1               5                  10 

aaa ttc ctg gag cag gtt cac cag ctt tat gat gac agt ttt ccc atg      399 
Lys Phe Leu Glu Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met 
         15                  20                  25 

gaa atc aga cag tac ctg gca cag tgg tta gaa aag caa gac tgg gag      447 
Glu Ile Arg Gln Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu 
     30                  35                  40 

cac gct gcc aat gat gtt tca ttt gcc acc atc cgt ttt cat gac ctc      495 
His Ala Ala Asn Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu 
 45                  50                  55                  60 

ctg tca cag ctg gat gat caa tat agt cgc ttt tct ttg gag aat aac      543 
Leu Ser Gln Leu Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn 
                 65                  70                  75 

ttc ttg cta cag cat aac ata agg aaa agc aag cgt aat ctt cag gat      591 
Phe Leu Leu Gln His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp 
             80                  85                  90 

aat ttt cag gaa gac cca atc cag atg tct atg atc att tac agc tgt      639 
Asn Phe Gln Glu Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys 
         95                 100                 105 

ctg aag gaa gaa agg aaa att ctg gaa aac gcc cag aga ttt aat cag      687 
Leu Lys Glu Glu Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln 
    110                 115                 120 

gct cag tcg ggg aat att cag agc aca gtg atg tta gac aaa cag aaa      735 
Ala Gln Ser Gly Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys 
125                 130                 135                 140 

gag ctt gac agt aaa gtc aga aat gtg aag gac aag gtt atg tgt ata      783 
Glu Leu Asp Ser Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile 
                145                 150                 155 

gag cat gaa atc aag agc ctg gaa gat tta caa gat gaa tat gac ttc      831 
Glu His Glu Ile Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe 
            160                 165                 170 

aaa tgc aaa acc ttg cag aac aga gaa cac gag acc aat ggt gtg gca      879 
Lys Cys Lys Thr Leu Gln Asn Arg Glu His Glu Thr Asn Gly Val Ala 
        175                 180                 185 

aag agt gat cag aaa caa gaa cag ctg tta ctc aag aag atg tat tta      927 
Lys Ser Asp Gln Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu 
    190                 195                 200 

atg ctt gac aat aag aga aag gaa gta gtt cac aaa ata ata gag ttg      975 
Met Leu Asp Asn Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu 
205                 210                 215                 220 

ctg aat gtc act gaa ctt acc cag aat gcc ctg att aat gat gaa cta     1023 
Leu Asn Val Thr Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu 
                225                 230                 235 

gtg gag tgg aag cgg aga cag cag agc gcc tgt att ggg ggg ccg ccc     1071 
Val Glu Trp Lys Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro 
            240                 245                 250 

aat gct tgc ttg gat cag ctg cag aac tgg ttc act ata gtt gcg gag     1119 
Asn Ala Cys Leu Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu 
        255                 260                 265 

agt ctg cag caa gtt cgg cag cag ctt aaa aag ttg gag gaa ttg gaa     1167 
Ser Leu Gln Gln Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu 
    270                 275                 280 

cag aaa tac acc tac gaa cat gac cct atc aca aaa aac aaa caa gtg     1215 
Gln Lys Tyr Thr Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val 
285                 290                 295                 300 

tta tgg gac cgc acc ttc agt ctt ttc cag cag ctc att cag agc tcg     1263 
Leu Trp Asp Arg Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser 
                305                 310                 315 

ttt gtg gtg gaa aga cag ccc tgc atg cca acg cac cct cag agg ccg     1311 
Phe Val Val Glu Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro 
            320                 325                 330 

ctg gtc ttg aag aca ggg gtc cag ttc act gtg aag ttg aga ctg ttg     1359 
Leu Val Leu Lys Thr Gly Val Gln Phe Thr Val Lys Leu Arg Leu Leu 
        335                 340                 345 

gtg aaa ttg caa gag ctg aat tat aat ttg aaa gtc aaa gtc tta ttt     1407 
Val Lys Leu Gln Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe 
    350                 355                 360 

gat aaa gat gtg aat gag aga aat aca gta aaa gga ttt agg aag ttc     1455 
Asp Lys Asp Val Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Lys Phe 
365                 370                 375                 380 

aac att ttg ggc acg cac aca aaa gtg atg aac atg gag gag tcc acc     1503 
Asn Ile Leu Gly Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr 
                385                 390                 395 

aat ggc agt ctg gcg gct gaa ttt cgg cac ctg caa ttg aaa gaa cag     1551 
Asn Gly Ser Leu Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln 
            400                 405                 410 

aaa aat gct ggc acc aga acg aat gag ggt cct ctc atc gtt act gaa     1599 
Lys Asn Ala Gly Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu 
        415                 420                 425 

gag ctt cac tcc ctt agt ttt gaa acc caa ttg tgc cag cct ggt ttg     1647 
Glu Leu His Ser Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu 
    430                 435                 440 

gta att gac ctc gag acg acc tct ctg ccc gtt gtg gtg atc tcc aac     1695 
Val Ile Asp Leu Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn 
445                 450                 455                 460 

gtc agc cag ctc ccg agc ggt tgg gcc tcc atc ctt tgg tac aac atg     1743 
Val Ser Gln Leu Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr Asn Met 
                465                 470                 475 

ctg gtg gcg gaa ccc agg aat ctg tcc ttc ttc ctg act cca cca tgt     1791 
Leu Val Ala Glu Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys 
            480                 485                 490 

gca cga tgg gct cag ctt tca gaa gtg ctg agt tgg cag ttt tct tct     1839 
Ala Arg Trp Ala Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser 
        495                 500                 505 

gtc acc aaa aga ggt ctc aat gtg gac cag ctg aac atg ttg gga gag     1887 
Val Thr Lys Arg Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu 
    510                 515                 520 

aag ctt ctt ggt cct aac gcc agc ccc gat ggt ctc att ccg tgg acg     1935 
Lys Leu Leu Gly Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr 
525                 530                 535                 540 

agg ttt tgt aag gaa aat ata aat gat aaa aat ttt ccc ttc tgg ctt     1983 
Arg Phe Cys Lys Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu 
                545                 550                 555 

tgg att gaa agc atc cta gaa ctc att aaa aaa cac ctg ctc cct ctc     2031 
Trp Ile Glu Ser Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pro Leu 
            560                 565                 570 

tgg aat gat ggg tgc atc atg ggc ttc atc agc aag gag cga gag cgt     2079 
Trp Asn Asp Gly Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg 
        575                 580                 585 

gcc ctg ttg aag gac cag cag ccg ggg acc ttc ctg ctg cgg ttc agt     2127 
Ala Leu Leu Lys Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser 
    590                 595                 600 

gag agc tcc cgg gaa ggg gcc atc aca ttc aca tgg gtg gag cgg tcc     2175 
Glu Ser Ser Arg Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser 
605                 610                 615                 620 

cag aac gga ggc gaa cct gac ttc cat gcg gtt gaa ccc tac acg aag     2223 
Gln Asn Gly Gly Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys 
                625                 630                 635 

aaa gaa ctt tct gct gtt act ttc cct gac atc att cgc aat tac aaa     2271 
Lys Glu Leu Ser Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys 
            640                 645                 650 

gtc atg gct gct gag aat att cct gag aat ccc ctg aag tat ctg tat     2319 
Val Met Ala Ala Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr 
        655                 660                 665 

cca aat att gac aaa gac cat gcc ttt gga aag tat tac tcc agg cca     2367 
Pro Asn Ile Asp Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro 
    670                 675                 680 

aag gaa gca cca gag cca atg gaa ctt gat ggc cct aaa gga act gga     2415 
Lys Glu Ala Pro Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Thr Gly 
685                 690                 695                 700 

tat atc aag act gag ttg att tct gtg tct gaa gtg taa gtgaacacag      2464 
Tyr Ile Lys Thr Glu Leu Ile Ser Val Ser Glu Val 
                705                 710 

aagagtgaca tgtttacaaa cctcaagcca gccttgctcc tggctggggc ctgttgaaga   2524 

tgcttgtatt ttacttttcc attgtaattg ctatcgccat cacagctgaa cttgttgaga   2584 

tccccgtgtt actgcctatc agcattttac tactttaaaa aaaaaaaaaa aaaa         2638 

 
           
             12  
             48019  
             DNA  
             Homo sapiens  
             
 
           
            12 

cttgactcaa atcccgtcta cttgaaggaa ctaattgatt ttatgagcat atagtccaaa     60 

aagatttagt ttgtctggct tcagcaatgc ctacctggag aagtgacatt taagctgaga    120 

tttttaaact aagatctaaa agatgagtta gccagcgaag agttgggtga agcagatgtt    180 

ggctcaaggg gttttccaaa ctcagggtga gaagtccctg agtttggaga gtgtggttta    240 

tttgaagaac taaatgctga agtaagcaaa gcagagtggc acattatggg gttggacaaa    300 

gagatcgggc aaccagggct ttatagtatg tattaagaaa ctgaatttta taagagcaaa    360 

gaaaagccat ggaaggattt tcaggttggc aaatgacatg atctgctttt caattaaaaa    420 

aaaaaacact caaactcttg tatggagaat ggattaaaag gtagataagg gctgaagtga    480 

ggcagccatt cgggaatcta ctgcagaagg aggcagcaga agagatggag cagagtaaat    540 

gagacttggg aaatatttag gaggaagaat caattaagat ttgaggatca ggtgttgcga    600 

ggtgagggag aaggcaatgt caactatgaa tcctaggtat ctgtcatgtg ccatttctag    660 

aaaaagagca ggtttgagga gaaagatgtt tggttctgaa catagtttgt agagctcact    720 

gcacatacaa gtggagaggc aagtgggagt tgtaggtgtg aagcccagag gagaggtgtg    780 

gacgggataa gcatttaaga ctcctccatc tagaaggaaa ctgaagctgt gggtgaggtc    840 

atcacagcac agcgtttagg agaagcccag gtaaagaagc tgacgaatgt ctggaccctg    900 

acaaccttaa catataatgg tttgatagtg gaggtggagg caatgtagaa agaatgccag    960 

aggcaggaaa aagcaagaag gatgtgttat catcatgacc aaggaagaaa cgtgtttcaa   1020 

gaacaaaggc gtcaactctg ccccatgctt ccgagctgtc aagtaaagtg agaaaaacag   1080 

aaaagcgttc cctgggttta gcaacacgga ggtcagttgc taaagggagc ttctagaatg   1140 

acgacgtcgc caaatctgtc ctctgcctgg attctcggcg atgaaactac tacagagacc   1200 

tccaagtttg ggcttctgca aacacagcac gtccttctga tcgttctcta agatatgtaa   1260 

acagaacgcc agttcccagc gtggcaacac gggactgggc tgcagctcac ccagccggcg   1320 

gcccccgccg gaagccggcg gaaatacccc agcgcgtggg cggagcagcg gcccgcagag   1380 

ggaggcggtg gcgcccacgg aacagccgcg tctaattggc tgagcgcgga gccgcccggt   1440 

gattggtggg ggcggaaggg ggccgggcgc cagcgctgcc ttttctcctg ccgggtagtt   1500 

tcgctttcct gcgcagagtc tgcggagggg ctcggctgca ccggggggat cgcgcctggc   1560 

agaccccaga ccgagcagag gcgacccagc gcgctcggga gaggctgcac cgccgcgccc   1620 

ccgcctagcc cttccggatc ctgcgcgcag agtgagtggc cgtgaggttc cgggtgccgg   1680 

gggtgggacg cgcagggaca gagtcctcgg gccgtgcagt tggacgccgg gcgaggacgg   1740 

gccgtctgtg ctgacccgcg aatagtgatc ccagaggaag aacgcgtcct gagtattcca   1800 

agtgcgagca gtgccacctg ttctcggaga tggcactgct cagcacgatt gtccttgcca   1860 

gtcgtgctct ggcagtgaag aggacttgtg aaatataatt ttcctctaga aggcactgca   1920 

ccttcccatt taccgtcact ttctcccgtt tccacccctt tccatcagtc acgttttctt   1980 

cttttcgcag aaagtttcat ttgctgtatg ccatcctcga gagctgtcta ggttaacgtt   2040 

cgcactctgt gtatataacc tcgacagtct tggcacctaa cgtgctgtgc gtagctgctc   2100 

ctttggttga atccccaggc ccttgttggg gcacaaggtg gcaggtcagt aaatgttcat   2160 

ggaatcagat gaatgagtac atgacttcgg aatcttaatt atgtttctat gttcaaagat   2220 

ggaagggctt aatgtcttcc tagagtttaa aagtgatgtt tagtttttgc agacccccca   2280 

ccccaaaata atatagtctt aaaaaaattt ctggccggta ctgaagagtt ctgtggtagg   2340 

tatgcagagg tgtggttgat tgtgctttga atgttgtttg tatagataag gagttatttt   2400 

catagaacag aaacaggact tgagcctcca gcacagtcca ttgtgcaacc attgcttaat   2460 

gaggatgctg gactcacact tgtgagatca taatatggtt tgctttttac tttctaattc   2520 

cgtctgaaat atacataatt tcaattcaac aaactgtttc gagcacatag tatatgcagg   2580 

gcactgtcca aggtccataa gatgttaaaa agcaaaatac cccactactc taccccctcc   2640 

ccacccacct tcccccgccg gggactctgg aggcatcaca gagctttaca aggaaaactg   2700 

gaaccgatgc tctaatagag gtacagaatt cagtgagacg caagtcaaag aaagcttaat   2760 

tgtgagtaag tctcagacaa ctttatgaaa gaaggggctt ttgtacatag ctttgaatgc   2820 

tataggattt agagacgcaa aaatttgtgg agaggctgtt tccaagggcg agtagctgga   2880 

gcaaagcaga aatgttgggc acagttgaaa aacagtaaat gatttggtga ctgggttgct   2940 

ttgttgatgg atgaagatgg ggtggggtgg aaatctgcct aggaatggac agatcatagg   3000 

atttttgcac aggagattga tgtggtccaa tctgtgggca gcagaggctg gactgggaag   3060 

gtagaatgtt ggagacagga aggagcaggt cttgggatgg agtggaagga atcaacccta   3120 

aatctgagat ttgagctcag tgactgggag atggccatgc cactccctga gttggtaaca   3180 

ggttttgtga gtagatgaga ctgactccag atttgtgaac atgagcttat ttattcattg   3240 

tttttttttt ttagagacag agtctctctc tgtctcccag gctggagtac agtggcgcga   3300 

tctcagctca ctgcaacctc cgcctcctgg gttcaagcaa ttcccctgcc tcagcctcac   3360 

gagtagttgg gactacaggt gcacaccacc atgccctgct aatttttttt tgtattttag   3420 

tagagacaag gtttcaccat gttgcccagg ctggtcttga actccggagc tcaggcaatc   3480 

cgcccacctt ggcctccgaa agtgctggga ttacaggcgt gagccacctg gccagcccaa   3540 

acatgagctt attaattcac ctcttctgtg ccaggtgcta catatgtact agctcaacca   3600 

gccttaagaa ccaactgtgg cagggtgcgg aagctcacgc ctgtaatccc agcactttgg   3660 

gaggctgagg tggaaggatt gcctgaggtc aggtgtttga gaccagcctc agaaacataa   3720 

cgagaggctg tctctacaga aaatttaaaa attagccagg cttcttggct cgtgtttatt   3780 

gtattagtct gttctcatgc tgctataaag aactgcccga gactaggtaa tttataaagt   3840 

gaagaggttt aattgactca cagttctgca tggcagaagg catctcttca cggggcagca   3900 

ggagagagaa tagatgcaag caggggaaat gccagtggct tataaaacca tcagatgtca   3960 

tgaggctcac tcattatcac gagaacagca tgggggagac tgcccccatg attcagttac   4020 

ctccactggt cccgccctta acacatggga attatattac aattcgaggt aagatttggg   4080 

tgggggcata gagccaaacc atatcacttg cagtctgagc tccttgggag gctgaggtgg   4140 

gaggattgct ctagtccagg ggttggaggc tgcagtgaac tgcgctccac tgccactgcg   4200 

ctccaacctg gatgacagag caagttcctg tctactaaac acacacacac acaccttgtt   4260 

gataggggag taagttacct gggttcgctt ggctaataag aggcgttgca gctcccattg   4320 

cctgacggtc cagcatatct gctttaggta gatccttatt agttaccaag cagggaggga   4380 

gacagccaca gatttttcca ctcaggtaaa ataaaactga actgaaaaat caaattgctt   4440 

cagcaggagt gccaaggggc tgggcttggg tccgtttcta ctggtatata tgaggataac   4500 

tagctttata ggccagacat ttcctcacac tcctttggaa ggacatttgt ggagtaggcg   4560 

tgacctcatg cctgcataca tgtgctcggc tacaagtatg catacacact cttccatgcc   4620 

ctacttgaaa atccagactc cttccccaga ggtctggcag gacaagattc acttgtgtct   4680 

gctctgcata ttgccacctg tgtgctgata taaatcctaa gttacatgat ggtcatggga   4740 

gactctccgt tttcctaatg aattttagga cttgcttata tcaagttttt gttgtgattt   4800 

tgaatcttta ggatcactat gtaaaaatgg acttaacagt tatccaaaaa gctaagtgtt   4860 

actttatcct tttaaattac atttaatgat gttttaatct ttaaaaatca ttacttcttt   4920 

atgccagatt tattgtttgt agtaggcaaa gaagaaaata tatcttttta aatgtgtata   4980 

agaaaaataa tgaattttac catatgaatc ataaatataa aacagaagtt tgaaaatatt   5040 

tgagttaaat tacaaatgta ctgtgatttg taggagagca attttgggag agtcttgtga   5100 

tttgtcttaa agcaagtcta attaaaataa tgtttttcat gtatttattt gataaaatca   5160 

caaactcatt ttaaaggcat atttattgga tacctactat gtggttgaca ttgttctagg   5220 

gatacaccag tgcacagaat cctccaccaa catccatccc ttcaagtggt gaaaacagac   5280 

aataatgaaa ataaaatatg tggtaggtca gatggtggtg taagtactat gaagaaaaat   5340 

aaaccaggga aaagagagag catgtttcca gggtgggagg tggttgcagt ttaaataaaa   5400 

gaggccagag aaggctgccc tgatatgttg acaataagca aagacatgaa gatgtgtttc   5460 

gtggaggaga aaaaaagttc atccccaaac tcaattaaaa acaggcttta gactataaat   5520 

gccatttaaa taaaatattc ctgggacaaa ctatgttttg gattaagcaa gatatttaag   5580 

agtacaacct gatgctagga aggctttctt tggagctatg gtttccatat acatagaaag   5640 

aatgtatatt ctttttccct ataggatgtc tcagtggtac gaacttcagc agcttgactc   5700 

aaaattcctg gagcaggttc accagcttta tgatgacagt tttcccatgg aaatcagaca   5760 

gtacctggca cagtggttag aaaagcaaga ctggtaagga aaattcacta gacagaagaa   5820 

gaagtttaca ccttaaaagt actggttgat taagtgactt ggggccatgt ttactggaag   5880 

atgaattcca tcaatggcca ctggaattat ttgttgtaga aaagtttgtt gatttaaaaa   5940 

atgactcaac aaagttgagc caaaaagtgt ctcaacaaag ttaagtatat tagtcattaa   6000 

gcacttttat acagttttgc caaatttaat ttatgataga tattcctaaa tgctttggac   6060 

aagttacgga atactgacac cgattatttt ccaacttagg atccagacta tagaaagaag   6120 

taatttaaaa gaaataaagc caaattaatg gttaaacatt gattagaaat atgaagaagg   6180 

tagtaaaatt aaggggattt tttttaactg ataggtgttc tactttaaaa tttattattt   6240 

gctaatttaa aaacgtcctg aaagtatttg acatagaaag tgaagggtcc cctgttgact   6300 

tttccccctt cagagatagt caccatgaac actgtcatgc acaatctctt agagtttaga   6360 

attttttgtg ctaattgcac ataagagaaa taccacataa actgctgcta aaagcaatgt   6420 

cagtttcaaa agaagaaaat aaacctaaga aaatacttaa attattttta ataatttcaa   6480 

taaggaaacc aacatttgtt ttgtctgttt tacatagaca tttagttcta gaatgaaatg   6540 

tgtaaatgtt aaatctccta gggagcacgc tgccaatgat gtttcatttg ccaccatccg   6600 

ttttcatgac ctcctgtcac agctggatga tcaatatagt cgcttttctt tggagaataa   6660 

cttcttgcta cagcataaca taaggaaaag caagcgtaat cttcaggtat gacctggttt   6720 

ttgtatttta gttgaaatgt tctcatgttt atggaaggca gttttcttca ttcagcaaat   6780 

acaattgagc acacacttat tggtatttag agaagtaata gagactaact ccatttatgt   6840 

tttctacagc atcagtaaat atagcataag taaaataaga taccaattta ttttataaac   6900 

acagaaggaa tttttagggt taaatgtatc acctcttagg tatcagtaat cctgagaaaa   6960 

atgtgatgct ataattttgt tttactaaaa attgtcaata tttaaaccaa agaaaagggg   7020 

ctacatttta agagcaatga tctatagtaa tacattaaag ctccagggaa cattagtggt   7080 

taggttcagg tccttgctat gcaaatgagg aataaaacag gaaaggtttg actttcacct   7140 

aaggaagtca ggtgacttca ggaactagac tccaccttgc cttgcttctg gtggagttgg   7200 

cctctcctgg aagagaaagt caaagaaaga tccagacatg agagaacatg ctatagagtt   7260 

ttaggggttc ttcagatgta ctcccagcat gcccttgggg tggctatcat atttatgttg   7320 

tgtaataaca gaacttttct gttttaatgg aggtgaaagc tatcattttc ttctttcata   7380 

caatacaact gaacttttca gcttaggtaa ttggaagttg ttttttttcc tttgtgaatg   7440 

tatttatgat tttatttacg agtggggaaa tcaaattaac tttacagaaa tttgttctca   7500 

tacagacctt gcatgaatgt acctgtataa agagccatta ttgctattgg tagtgttttg   7560 

caaactagct ttgacaatgg caacagaaaa ccctagatga atatctttgg tctagttcaa   7620 

agcttagacc aacagatgca ttcattgagg aatgtcatgt atagttttta tatcatttgc   7680 

attgtctcgg gatcctaacc aaagataatt ggccttaaga aatatggaaa gatattaaag   7740 

taaaaatatt atgtgaacca tatgtgaaaa tacttgtttt taaatcaaaa agtacgttag   7800 

gtacctgcac ttagaaagtt aaagcaattt ttgtcataat tcgtgaaaga aatattttga   7860 

ttttaacaaa catagcttat atagaattta atagatgaga gatgaggttt cccggagaat   7920 

ataaacaaat tcatatcaac ttacaataca caataaagta aacattctgc attttaattt   7980 

atttttgttc taaccactca aatctaggat aattttcagg aagacccaat ccagatgtct   8040 

atgatcattt acagctgtct gaaggaagaa aggaaaattc tggaaaacgc ccagagattt   8100 

aatcaggtac ttttttctca ttgaacacat aaactgtaat aaaataaaaa tgtattcatt   8160 

catcaaatac tattagggcc catgtcaaag caatgatgaa tatctctcta attttatagc   8220 

cccaagaagt gggagaatca gatttagaga ttttatagaa ccaggcttta agaaagatcc   8280 

taatctaatg gaattaagtt ctaacgagga gagagaacat ataaacaaga tacttctggg   8340 

tagcataaag tgctgagaag ataacatgct gaattcatgg agtggctggg agtggggttg   8400 

ttcctttaga tcagggtcag gaaaggcctc tgagttgctg gcacaggaat ggaagcaggc   8460 

agatagaggg ttcttgcagc tgtccaaggt agaggcagtg gaagcttgcg ctcagtagaa   8520 

tcagtggagg taggaagagg tggacagatt caggttatgc tttggatgtg ataccaatat   8580 

agaaggatcc ttatgttgaa ggattagatg caggagtgag agcagaaagt caaggatggc   8640 

tctacagcag ctactagaac cccttgaaaa atggtaatgc tgtacacgga gctgggaagg   8700 

caaggagggg gcaggggcag gagttttgta ggactcaata ggtttgtgga gagaaaagga   8760 

ggctgatggg aaaatcaagg gctttgtttt gagcaacatc aagataccta ttaaatattc   8820 

aaggcatcaa acagtaaatg agtcagagtt gtactagtat caagattttt gatcattgtt   8880 

ttattaatcc taatacaaac cttcctagtt gtcagggttc agcagagttt tgaatttgct   8940 

atggcattta gtattccttt cttgagcact ggggtggtga caacaagcca acaggcaggc   9000 

agtgtgaagc caccatgtta tggattgttc tggaggagcc cattgctata tggatgccag   9060 

aggacacagt atgcatcctg tgtgcatttc accaacaaat aactaccgaa gtgaactgtg   9120 

tgacagtgga gagggaggga gtttaggtgt ggtggaggat gccattctcc acacactgcc   9180 

tgagcatttg tcccagtcct tgtaatcctg tcacttccct aggcgtatag ttcccatctt   9240 

cttgtcacac tgatagcatc ttgctgaact gagcatgtaa agatacatat tgcttattct   9300 

aaactcaagc tggctagctc atctggtacc cagcctaggg aacaaaggtg tatatcaacg   9360 

ctggaggaga agctggccag gttgtctcct ctgaaagagg tgtgcctgtc tctaacctgg   9420 

catatacctg ggagattttc agccggcagt tttgattata tggggcttcc atcctaaaag   9480 

tagtatgcgt gggcctcctc tgtatttcta atagacttga aaggacagcc aggagcaaag   9540 

atgggcagaa ggacaacctg tttcccccag ctgcaagcat gtcatcctca catttggccc   9600 

cttggcccag agatggcatt ggcagcagag taggtgaccc ctgctctccc acatgaacct   9660 

catcccctgg ctttggatct tgaaggttta ccagcttttg agctccctcc ccattaaaaa   9720 

tgtaggtaat ctggggattg ttttgagcct ctcagaggac acctgagtcc gtgaacacct   9780 

ttgcaaagcc ttcttggggt gtgagggaag tttctgggtg tctggggatg tcagggaaag   9840 

ggaaaaggca caataggtgc ctcctttcca aagttatcca gccaactatt ggctaggtag   9900 

gtcagaaaca tttcttcacc atttcccccc ttgttgaagt aactttattt tattaataca   9960 

gacatgcatc acttaatgac acagatacat tctgagaaat gcaccactag gtgatcgcgt  10020 

ttttgtgcaa acattataga gtgcacttac acaaacccag gtggtataac ctaccataca  10080 

cctacactat atggtaaagc ctattgctcc taggctacaa acctgtacaa tatgaagttg  10140 

tactgaacgc tatgggcaat tgtaacacaa tgttatttgt gtatctaaac atagaaaagg  10200 

tacagtacag atagtgtgta aaagataaaa ggtggtaccc ccataaagct ccattctaat  10260 

cttatggggc cactgtcata tacgcggtcc atcgttgact gaaccattgt tatgtggtac  10320 

atgactgtgt atattctatt catgctaaag tatatgtgta tggaatggtg tgatgtctgg  10380 

tatttgcttc aaataatccc atttggggac tagaagagta aggagaatag ggatgaaaca  10440 

aggccatcca tgaatgctaa ttgttgaaac tgtgataggt acatggggat tcaacagatt  10500 

caacaagtta aaaacatgca aaattccgag ttagttcaga tcatttagag accacaaaat  10560 

tctatagaca tgccgagatg ggaaattatt gttcccattg ttttttaata taagagcttt  10620 

tagaaaatac atttaagaga atattatgct atacacactg tagtagacta taaatgtttc  10680 

gtgtgctgtc ttacagcaca ctaagttttt ttctttcttt ttttctttgt ggtgaaaaag  10740 

ctagttgcat gtctagacaa aaatttaggt ccatagggtc tgaagggatg gggagaattt  10800 

ctttcaggtg ggagggaaag acatgaggtg gattctaagc ttcctttgct gttccatctt  10860 

tgtgttctta aggactaatg gttaccatat gctgagtgct tatcatgttt caggccctgt  10920 

gctaagcact ttataaacat tatttcattt gagtaaatac tttcgaaact atcactgaag  10980 

ttaggtattc ttatccccat tttaaagatg agaaaactaa ggtttagagg tcaggtactt  11040 

cactcagagc cacagagcta atgagaagta ggtttggaat ttaaacccag tattgtccaa  11100 

ctctaaaact agttcactgg agaagtcact tttcagtttt ctaatctgtg ggatgggata  11160 

tgagggcttt ctggctggaa cttctcacac ccatggagtg cctattccct tacttggaca  11220 

actgctcact ccattggaaa ggaccccctt cctgagcaga agacttttct ttgctctggg  11280 

gtctggagta accaagttcc catccaaaca aggtctctag aggtacagta gtgaaacgag  11340 

aaagtaagag tcaatgacag ccgaagtccc ttccaacttc agtgtttgaa attcccgtgg  11400 

tttcagtact cctggtttct tagaaagtta aaaagttaag gactcactgt gaaagtggta  11460 

tcagtactaa agtgtacagg agcagccaag catctgctta gcagatgggg gcagggtgga  11520 

gggaagcagg gagggatgag tggaaggggt aagagagcac caagggtttg agtgacttgg  11580 

agggactact gcccaagttg gaagcaggag atttcttagg ccctgccaca gtcaccaggt  11640 

ttttccacca agcatctccc agcaagtcag tagaaagggg atgtagaagc tggggatgta  11700 

gctgctgttt tacagccatt gtttatatcc ctgttctcta aaccgagcct tggtcatttg  11760 

ggaggaggcc tgggaagtca gaattctcca gtggcctggt ccctctctct aggtgtagtg  11820 

tcccagctgt tacaaagccc acaggacacc gtgcccacat cctgctggct tagaagttgt  11880 

attagtctgt tcttgcatgg ctataaagaa atacctgaga ctgggtaatt tataagaaaa  11940 

gaggtttaat tgacttatgg ttctgcaggc tgttacagga agcatggtgc tggcatctgc  12000 

tcagtttctg gggaggcctc aggaaactta cagtcatagc agcaggtgaa ggggaagcac  12060 

acatatcaca tggccagagc aggaggaagc aggggagagg tgctccacac ttaaccagat  12120 

cttgtgagaa ctcgtgatca caaggatagt accaagagga tggtgctaaa ctattcatga  12180 

gaaactgtcc ccgtgatcca gtcaccttcc accaggcccc acctccaaca atggggatta  12240 

caattcatga gatttgggtg gggacacaga tccaaaccat gtcagaatta gacaacagca  12300 

aagatttccc tgcacaggca gaaagctgta tggaactcct caaagtgaca acttattcca  12360 

accgcccaga gaaacccctt ttgtcactga gtgatatgtg gcaccttgga cttacagagg  12420 

ataccagtaa cattcatgta tttctaatac acactttaca gctgatacaa ttcacatgct  12480 

ttgaacttct gctgttggag aatatgtaac atgaaggaaa acttcctatg aagagagaat  12540 

aactttgatg acgtatgcct gataaagata aaacaaaaaa ttatggacca attttactta  12600 

tgagtatcaa tataaaagtc ctcaataaga tattagggat cagaattaaa tactacatta  12660 

agaaaggaca tgaccaaata tgatttatta taggttacaa gggtagtcca tgactgggca  12720 

acctattaat gcaattcact aaattaatag ctacaaggag aaaaaagcat ttacttcctt  12780 

ccacagaagc tgaaaagatc taacagaatt caatactcat tcctcataaa aacattgaat  12840 

aagataggaa ttgatgggta cctctttaac acaaaaaaat atatatatct ccattctaga  12900 

gacagtatct tacctaactg ggaaaactcc agtaagtcag aaacacagca aggatgctgt  12960 

gtttccactg ctatttagca tcgtattggt ggtattggcc aatgcaatta gacaagagaa  13020 

aaccattaga agcataagaa ttaaaaacaa catcataaag atgttagctc tccctgagtt  13080 

aacttgtaat tttaatgtta ttccaataaa aataccaaca agttgttttc tggagctaca  13140 

caaatttgtt ctgaaattta tgaaaaataa acatgcaaga gtagcgaggc aacttctgaa  13200 

caagtagagc aatgaaatgg gactggccct actagacaga ctataaatcc tctatagtca  13260 

aacatccttc aagggttcag gcttatgaat aaaaaggcca atgggaaaga agagaaaaat  13320 

ctagaaggaa acccgtgtgc atatggaaat tcatgtgata aagttggcag ctcagagcac  13380 

tggggaaaat aatggacttt tcagtaaatg ctgttaagac aaatggaaag ctaactggaa  13440 

aaaaagtgaa agcaaaaagt ggacccatac ttcactctga tcatgagaat aaacaaatat  13500 

atcagcgatc taaaagttaa aaagaaaaaa aggaaacctt gcaagtgcta gggaaatgag  13560 

tgaatgatgt aatgtcaata gggaaaggct ttctaatata aggaaaattc cagacatgaa  13620 

gaaaagatga tacaattgca catataaaaa cacccccgta gaatctgtgc tgtatagggc  13680 

actgtgtaag aaaggttgac aagcacagtt tttgctgtgt gggggattta actctaaaaa  13740 

aataacatct ttctagaatt taatctctat gagaacaggg atcactggtt accactctaa  13800 

cacccgagcc tagagcattg cctgacattt acttagtacc tagtatgtgc agggtacatc  13860 

tggtgtatga agcaagtaga gaccctgtca cttctgagtg ttctgattat caaaaatagg  13920 

actgaatact cccaggcaag tctgggttta tattcattat tgactttata gtagcagatt  13980 

ttcattgatg gccttaatgg agtgatttca tatttcagta tacctctgga tcacccggaa  14040 

ttgtgttaaa atagggtttt ctgattgcct aatcatatta gcccttgaga ttttccaaca  14100 

agatcctagg tggtgagatg ctgctggtct gaggaccaca ctttgaatca gaaagcttta  14160 

agataaataa ggactgagtt aatgagagta atcttgtgta tagtatctga gaaaccgtga  14220 

actaaattcc agcgtggtac catgcaactg tatgaattaa tggaaactcc cagaaatact  14280 

gtttatttta gacatgaatt gagcctagtt tttttgtaac cttgttaaca gtttgccagc  14340 

attttccagg gccttgggaa aaatttcata ttgacacata agcaggaagc ctgtagtaca  14400 

tgtcagaaga tctggaaggc aagtggggac ttgactttgg agtgtaacca gcaagtacac  14460 

accctgaaga aaacgatggc tataatcatg cccttcccat tacaggctca gtcggggaat  14520 

attcagagca cagtgatgtt agacaaacag aaagagcttg acagtaaagt cagaaatgtg  14580 

aaggacaagg ttatggtgag tattgagtaa acacatgcat ttattctgaa actttctgta  14640 

ggggaaaaaa ggtttcaatt gcttggggtg taaacctcgt aactgagttc agtcttttaa  14700 

gtagttggga agaaaaattt tgcagaagtc tcaagctata agtaaacaaa ttggcaaaat  14760 

caatgaccag atagactttt tgaatggcct cttataactg ccaaacattg agtttagctt  14820 

tttcttccct gaaagatcat gcagtaaatg ttgactttgc ccagtaccca gtaaatgaga  14880 

tgttgctgct ctaaatgggc ctttatgtcc acacctctca aatcacagtg ccccaggata  14940 

aacatggccc atccttccca gtcatctgtt ttatcctaag acttggaaga ggaacaaaaa  15000 

tacattttat tgaaagaaat ttggatacat ttctgattaa gacatccaca gcaattttag  15060 

gatacaactt ccaagtcaat ttatggaagc agagggctgc tctggcgtcc tcgatggtgc  15120 

gcatttactc ctctccactg ttagggctgg tgtggggagg gtttgaggag ttctttttca  15180 

cagaggcctg tgtttgtgga aaaggccttc cagcttaggg gtttggtcct ctgtggatct  15240 

ctgggtaata gcagtatggc cttgacttac atgcttcaga cagcatcttt ccaaactcca  15300 

cattctttgg agcaacttca tcttgcggtc tcctagggaa acatttcatt aaagtagaaa  15360 

ccgagtgagc ccagtccaga aatagccatg gaaatccaga gaagaatatg tggaaatgtg  15420 

accaaatccc aaacaaatga ggcttatttg tagaatgaac atacaattta ttgtccagat  15480 

ggagatgcat ttgagccgcc aagagggcac tgtttataat tacagcaagt atgaaaccag  15540 

gtctatcccg gtaaagcagg gatgtatggt cacttttctt acttggaagg aatgaggaaa  15600 

ttttctgtga gctttttccc cgcctgctgt gggcttcaca gtggagttct taaatcatat  15660 

ctcagaggta gtctcaaaaa tgatgaggaa gaatcaaaat ttaggcgctg gaaccaggtt  15720 

aaggacatac aaatcagttt gagaattatt gtacgtttgc ttttttaaaa atgttatgac  15780 

atacttatta atgaaaccga aattatttca tgtatatgat tagaactgtc caagtcagat  15840 

ctctttttgg gggagatgga attgttttcg tgtttctctg ggttccatag aagcaatact  15900 

ctgttgccta aagtctttgg aagttgctga tcagtagaaa acatgtttac atctttgttt  15960 

gtagtgtata gagcatgaaa tcaagagcct ggaagattta caagatgaat atgacttcaa  16020 

atgcaaaacc ttgcagaaca gaggtaaggg ttcacaactg aagtggtgcc cgttggctgc  16080 

aattttttct gttcacacta gataacgaag atgattactc ctttctattt gccgagtatt  16140 

ttatgactct gaattcttcc ttgataaccc aggccagggc ttctcaacat ccaatattat  16200 

tgacatttcg ggctaatttg ttgttggggc tgtcctggca ttgtagcgtg tttaacagca  16260 

tccccgactt ttacccacca gatgccagta gcactaccca ggagccctca ctagtgacaa  16320 

ccaaaactgt ctccagagat tgccaaaagt cccctagggg tcagtgaccc ctacttgata  16380 

accactggtt atgtgttact caccatattt attgcgccta gcccactgac tgacacagag  16440 

cgatagatat gtggagttcc cctgccctaa tatctcaggt ttcttcaaac gttgcagggc  16500 

cacgtgtata cacagcagat aagctcagta tttttctcat gttttaaagc agttttactg  16560 

tattttctta ctgttgcgtg tttttaatga gagtagcaac agaaaagatc tcaagaatgt  16620 

acattgtgta ggacacgtag ggtactgctg ttactctatt catcatctgg ggtataccat  16680 

ggttcatagg cagagattat cagtatgatt tgggtagaaa gtataagaga cagcaacttc  16740 

aactaccact tcctactcat ctacttctct ttgaaagcta cagctatgct ttgtatacat  16800 

tttttatctg aattctgttc agataaaaca ttttattatt tacatttatt tttataataa  16860 

taataacatt attattaatg ttatttacat ttatttaggt aattacctga cagtgtctta  16920 

agtggcagat actactatgt ctgatttaca tgggtcactg aaaacaagtt ttttttgttt  16980 

tttttttgtt ttttttgttt ttttttggag acggagtctc gctctgtcgc ccaggctaga  17040 

gtgcagtggc gagatctcgg ctcactccaa gctccgcctc ccgggttcat gctgttctcc  17100 

tgcctcagcc tcccgagtag ctgggactac aggcgtctgc cactgcgccc ggctaatttt  17160 

tgaaaacaag tattttgttt cagtgtgtct agttgttata cttaggactt tttttcatgt  17220 

tcattaaaga tcgaagaaag ccaaactttg acctgtcact aggcagcatt tgtgtcatat  17280 

ttatcctaaa tttatatgaa tcttggcttt tgttggtttt gtcttcttta tatatttact  17340 

ggctgtctct caatttatag aacacgagac caatggtgtg gcaaagagtg atcagaaaca  17400 

agaacagctg ttactcaaga agatgtattt aatgcttgac aataagagaa aggtagttat  17460 

ttactttcca gaatagcatg ccacttttgt tatacactgt aaataatgga ctcaaagttt  17520 

agaagagagg aaaaattata gccacctggc ttagagcccc agttgagaat gaaatgatat  17580 

ttgcttttct tttaaaaaat attttaaaat tcagtgatta aaaatgaatt tatttcactt  17640 

tgtttcttct attgccttta ggaagtagtt cacaaaataa tagagttgct gaatgtcact  17700 

gaacttaccc agaatgccct gattaatgat gaactagtgg agtggaagcg gagacagcag  17760 

agcgcctgta ttggggggcc gcccaatgct tgcttggatc agctgcagaa ctggtaagat  17820 

tctccaaagc agaaaactgg cagctgactg gctaaccaca taaacacata aatgtacctt  17880 

tgagctgtgt tagttgaatg gaccctgtca aaacattagt atagagtttg acatagtcca  17940 

gcttctgctt accctggaaa cactcccttg gaaagcacag tgttattcat gactctcgcc  18000 

acgttcagcc acgtctgctt ggtttggagc actcctgtac catggggtgt cgtttgactg  18060 

tacaaaacat cagtgacatg gtcgttatag aacacattta atgacttgac tcttaatgtt  18120 

tccttaaaaa caagatagct tgaggcattt ttctaggctt acagaatagt tttttaggaa  18180 

acattcagtc atccatccag taatacttat taaatgccta ctgtgtgccg ggcactgtag  18240 

gacctgggca cacaaggatg agtgattctt ggcccttgcc aacaaggagt tctcagtgtg  18300 

gaggggggga tgtagacatg gtaataattc gtgtggtaaa agtatccagg tgctctagca  18360 

aaaggccatc agaattctgt ggaggacaaa ggaaagcatc tccagggaag gtttcataga  18420 

ggaagtggcc ttgtgctgac tcaaaagtca ctaggattac tccacgtaga tcaaggggaa  18480 

gtgctttcca gcacagagca cagcgtgtgc aaaggcacac atgtgggaga ctgcaggggc  18540 

tgtattgggt ggctggagag tggggtgtcc agggagatgg gaaggaaagt ggacccggtg  18600 

ggagatgctg gagcctgatg gtgcaggttc tcagcaccat gctgggaatg ggggatgtac  18660 

gcagctgtac tgctgagcca aggcaggctt ttttcttaag caggagatgt atttctctga  18720 

tctttgattt agaaagggca ctctggccac agcctggagg ataggataga gacagaaaga  18780 

ccagttggaa tactattagc atattagtgt ggtccaggtg aggggcaaca ggagctgaac  18840 

ttggaatcac agagtgaaca tgagggaagg tggcctggga caattgctaa gggtccaagg  18900 

accagagatc tcacgaggtt attttctata ttgccagttt ttaaaaaaat cacaattaat  18960 

attcagccat ttatttttgt tctgtgaatt gcctgtaggt accccagatt tcttgtgggt  19020 

tagagcagag cttctgagcc cagtgcaccc catgtgcctt gagggataca tacaacccta  19080 

aagcccttca ggtggccagg gaggccctgg ggtgtttaga gccccctagt ggtcctccta  19140 

gaccacagga gcctcttgta gggacacaca tgccctctag ttttcccact gtgctgtcat  19200 

tttctctgtg tgcaagcaga gttggggaca ctggtttagc ttctccgctg gaactcagcc  19260 

agccgttatc ctgccttctc ctttcctgac gaagggctgc tgtcccaggg ctttgaactg  19320 

gaatcgagca aaatcagaaa gtgaagtaga tacttttccc agggaagaag acacttttaa  19380 

aaggttttct tctgtcatta ctgcaagtga ataaaacagc gtttccccag cctgtgttgc  19440 

tgttagcagt tgactgcaag gaaggaagac taagagcaat cactgggttt actcattggc  19500 

tttttatgtt tagggtctag actgagcata ctgccttctc ctttcagttg agtgaaacca  19560 

gaatcatcat tccttcctct cttcctgtcc ccggccttgt ctcccatctc tgtctctcct  19620 

cctgcccctc tgccccacat ctctgactgg tcctgccgag acactgcttg ctgatgccac  19680 

tctcttgaca gaacctgcag tggctctttg ctgcctcttg catcacctgg aagcacctgc  19740 

tttgctgttt tccctgcctc tggcctcctc tttgccctgc accagtcaca gatcacctag  19800 

aagccctgcc tccacccatc tctcctcaca taggcctcag tggtgtgtag aagtttttga  19860 

tgtaggctgt aatcattcag tgcatgttag ttgtactcct caaataacaa agcttctact  19920 

gctggcacaa gcctcgttcc ctttgttttt ccctagaggg cagagcatgg catcctaggt  19980 

gacattttgt gttgcatata accacttgtt ggtttccatg ccatatggtt tggccgtgtc  20040 

cccacccaaa tctcaccttg aattgtaata atccccatgt gtcaagggca gggccaggtg  20100 

gagataattg aatcatgggg gtggttcccc caatactgtt ctcatggtag tgagtaagtc  20160 

tcacgagatc tgatggtttt ataagtggga atttccctcc acaaactctc ttgcctgcca  20220 

ccatgtaaga tgtgactttg ctcctcattt gccttctgcc atgattgtgg ggcctcctca  20280 

gccatgtgga actatgtgtc aattaaacct ctttccttta taaattctcc agtctcgagt  20340 

atgtctttat tagcagcatg agaatggact aatataccat gattcccttt aatccaggct  20400 

gcttctggac tgtttctcat agagacatct gtgtcttgtg tcttcccagg ttcactatag  20460 

ttgcggagag tctgcagcaa gttcggcagc agcttaaaaa gttggaggaa ttggaacaga  20520 

aatacaccta cgaacatgac cctatcacaa aaaacaaaca agtgttatgg gaccgcacct  20580 

tcagtctttt ccagcagctc attcagaggt aactcaaggg acatttattt gtaccttctg  20640 

taatcggtct atacagagga acattttacc gttaattcag atgatttaca agggtttaat  20700 

aggtgttttt tatatatata tatatatata tatatatata tttttttttt ttttgagata  20760 

gagtctcacc tatcacccag gctagagtgc agtggcgtga tcttggctca ctgcaacctc  20820 

cacctcccaa gttcaagcaa tcctcctgcc tcagcctccc aagtagctgg gactacaggc  20880 

gcatgccacc ccacctggct aatttttttt agtagagact gggtttcgcc atgttggcca  20940 

ggctggtctc caactcctga actcaggtga tctgccctcc tcggcctccc aaagtgctgg  21000 

gattacaggc gtgagccact gtgcctggcc tcaaattttt taatgacaat gactcttcct  21060 

gaattatttg gttataccag tggttcttca ctcttgtggc cagctaggct ggaagctata  21120 

gtaaccttcc actcctcctt cccctttggc tgagtctccg ccgtgaccct aacatgcctc  21180 

acctcctcct ctcccctcct catgtctcag gctctcaaat cctcctttct ggactcttcc  21240 

ccagcctccc caccagtcct ccttcccagt ccttcctccc tgtgaccagt gctgtcccag  21300 

agcagagttc cattctggtt cctcatcacc tgctgatcca agtgaatgcc cagctgggca  21360 

ttcacggccc cccttctgca gcctggacct agctgaacac tccacgcttc acgtccacac  21420 

acgccccttt ctaggcaaac ttgacctctt gctgattcct ataggggcca tgccgcatac  21480 

ttgcttttct gtggctctcc tgaggatact tcctcttcct cagagccttc ttttcatgta  21540 

cctgctttca aaattgtatc atcccgaaag cttcaccttg tatctcatca tttaatcttc  21600 

ccagatttcc caaacagatg ttatctccct tcgtggccta cagtcaccct tcgttccttt  21660 

cctactctgc cttttgttat atagttgtta tgtatttctt tatatttctt gaatatcata  21720 

aagttctgga aggcagagac atgcctttgt cacccttttc ccccagcagg acctagcgca  21780 

gtgccttgtg aacagtaggc tctctagaca cacttagtag atagataaat gtcactgagt  21840 

ggaaaagaag ggttcatatt gcaagttttt ttttttttaa tgcaaacttg acatgataaa  21900 

aatgtacttg attttgggtg gaagcaatag ctgagtggca gcggctgctg attgcgttcc  21960 

agggggcaga gtcggggaag acatttactc tcccagaaca gcctatctca ttcctttcct  22020 

tcagttaccc gtggtgcaga gagtcagggc agcagctgta gcagcaaggg tggccgtggc  22080 

agcggtggca gctacagtga catgtccagc atgaatccca aattgattat ttattcaaat  22140 

tacttctgat tggcgactct ggggttggaa agtcttgcct ccttcttagg tttgcagatg  22200 

ataacatata cagaaagcta catcagcaca attggtatgg atttcaaaat aagaactata  22260 

gagttagacg ggaaaacaat caagcttcaa atatggaaca cagcagaaca ggaaaggttt  22320 

cgaacagtca tctccagtta ttacagagga gccaatggca tcctagtggt gtatgatgtg  22380 

acagatcagg agtccttcaa taatattaaa cagtgactgc agaaaataga tcattatccc  22440 

agtgaaaacg tcaacaaatt gttggtaggg aacaagtgtg atctgaccac agagaaagta  22500 

gtagactaca caacagccaa ggaatttgct gattcccttg gaattctgtt tttggaaact  22560 

agtgctagga atgcaacaag gcccggcacg gtggctcatg cctgtaatcc cagcactttg  22620 

ggaggccgag gtgggtggat cacctgaggt caggagttcg agagcagcct ggccaacatg  22680 

atgaaacccc atccctacta aaaatacaaa aattagccag gcatggtggc gggcgcctgt  22740 

aatcccagct acttgggagg ctgaggcagg agaattgctc gaacccaggt ggcggaggtt  22800 

gcagtgagcc gagattgcgc cattgctctc cagcctagga gacggagcaa gactctgtct  22860 

caaaaaaaaa aaaaagaaag aatgcaatga atgtagaact gtgttccatg atgatggcag  22920 

ctgagattaa gaagctaatg ggtcctggag caacagctgg tggtgctgag aagtccagtg  22980 

ttaaaattca gagcactctg gtcaagcagt caggtggagg ttcctgctaa aatttgcctc  23040 

catccttttc tcacagtaat aaatttgcaa tctgaaccca agtgaagaaa caaaattgcc  23100 

tgaattgtac tgtatgtagc tgcactacaa cagattctta ccatatccac agaggtcaga  23160 

gattgtaaat ggtcaatact gacttttttt ttttattctc ttgactcaag acagctaact  23220 

tcattttcag aactgtctta aacctttgtg tgctggttta taaaataatg tattttcctg  23280 

atatcagact gttttctcgt ggttggttag aatatatttt gttttgatgt ttatattggt  23340 

gtgtttagat gtcaggtata gtcttctgaa gatgaagttc agccatttta tatcaaacag  23400 

cacaagcagt gtctgtcact ttccatgcgt aaagtttagt gagatgttat atgtaagatc  23460 

tgatttgcta gttcttcctt gtagagttat aaatggaaag attacactgt ctgattaata  23520 

gcttcttcat actctgcata taatttgtgg ctgcagaata ttgtaatttg ttgcatacta  23580 

tgtaacaaaa caactgaaga tatgtttaat aaatatcgta cttattggaa gtaatattta  23640 

aaaaaatgta cttggttccc tatggctaat gttcaaacaa gaagtcctgt atgttgggtt  23700 

ttttaaaatt taaaaacatt acttgtactt tgagtgcaca ttttattctt aattctgaca  23760 

tgtaaatgaa cataattaaa tacggggttt ttatttttta ttttggattt ttgagacagg  23820 

gcctcactct gtcatccaga ctggagtgca gtggtgtgat catggctcac ttcagcctca  23880 

acctcctggg ctcaagtgat cttcccactt caaccctcca gtagttggga ctaattagcc  23940 

acgcctggct aattaaaaaa aaaaaaacaa attgtaaaga tacggtctca ctatgttgcc  24000 

tgagctggtc ttgaactcct gggctcaagc aatcttcaca cctcagcctc ttaaagtgct  24060 

gggattacag acatgggcca ctgtgcctag ccttcattgt atgtttgttt caatgtaata  24120 

gtttcacaga tcattacagt tattctgttt ctgttaagtc cctattaggt tttgggattt  24180 

ctttactaat tagaaaatat cactttgttc atccaacttg tcagaatctt tcaaatgatt  24240 

ttttagatcg tactctctaa cccccttttt tctttcagga aaatcattgt gattgcctca  24300 

accttaatgg aaatgctaac ttatctatcc tttgctgggt atttgcagct cgtttgtggt  24360 

ggaaagacag ccctgcatgc caacgcaccc tcagaggccg ctggtcttga agacaggggt  24420 

ccagttcact gtgaagttga ggtaacaagg gaaagatggc atcccataca tcacctgtca  24480 

cgtagtttgt acccctagtt tccttttata aaaactgtta tatagggtgc ttttgaggaa  24540 

ataatttttc agggggcact ttaatatgaa ctcttactga gaatatctga gagtctttta  24600 

ttaatatatt catagtagaa atgtaactga tatacatata tgtacacact cctggttttt  24660 

ttccaatcat aaatgtaatg cgtgttcaca ggaaaacaga attccaaatt gcagaaatgt  24720 

gtgtaataga gagtaaaagt tcctggctgc ctcacccctc ttgggcatag gctcataacc  24780 

tgagccttat gaaccccaga agctgtatga atgtggattt ataacatgta catttttctg  24840 

atggatggat ttatagcttt catcagtttc tcaaagcagt ccgtgatata aatatctgtg  24900 

gaccaccaca gtaaatggcc tagtgtctgt cctcccagga ttttgtccat gtctacacaa  24960 

atatatattt catacggaaa cacacacgca caaacatgac ataatgtttc ctttttaaaa  25020 

aattaggatt ccatgtctac acaaatattt catatagaga cacacacaca cacaaatatg  25080 

acatatttct tttttaaaaa ctcggagtag gctacataca ttgttttgta gtctttctcc  25140 

tcgatactaa ttgtagaagt gtttccagga atatgtggct ttactttctt ccattgaatg  25200 

gctgcattgc tttaagcatt cttcttatga tgagtagcta gacgctgaag aactggactg  25260 

gccttcatag ttggcccggg tcctttttct ggggtgttgc tgatttaagt ctgtggttaa  25320 

ccctccctgg ctggtaggta caatgtcaca ctgaagccct cgtggaattt tagaactcag  25380 

cagctggaag ggacagtaga aatctgatcc catccacctg ctccactgaa cggagcagac  25440 

agagccccgg ggtctccttc tcaaagtctc cagctcagtg gagcagaggt acaccctctc  25500 

acctgccctc catctttcca tgctaccctg gggtgactat agatgggacg ctattttttc  25560 

tgcttatggg caatagtttt tattgtctcc caacaggaca aagattagat acaagtttta  25620 

atagaaagta atgtttgcta ttttacccat ataaattggc caaaggtcga aattgtgatg  25680 

aaactgaata ttggcaaggt tatagaggta aacagctacc ctcattaact tttggtggca  25740 

atgtaaaatc agggcggcat cctgtcagaa ttagtgttac aatttttcct aagcggttcc  25800 

acagctagga atttattctt acaaatacac ttacaaaaat ttaagtagat ctacaaaact  25860 

gtcagttttt ttgttggtaa taccctaaaa ctggaggggg agtagtttat ctaaacgtta  25920 

atagggaatt ggcataataa ataaattgta acagataatt ttttttaagt aatggctttt  25980 

atttttcttt ccagactgtt ggtgaaattg caagagctga attataattt gaaagtcaaa  26040 

gtcttatttg ataagtaaga tatctttaac attatataac atacatgtac tattgaaatg  26100 

ttgattttgt tactcaagca cagattcctc cactaaataa gcagatttct agctgtggaa  26160 

gggtctatac tgggtggaag tttgcctccc tagggcccca gggctgaacc cagagtcctg  26220 

acaatgctgc gcatgagcag agacttctag tcagatccac cagggccatc acaaatgacc  26280 

ccagcagatt taaatcagca gctcccttgg tggatgttgg gagcttcccc tggagctggt  26340 

atcaacattc ctgtgccttt ttagactata aatgaacaaa agagtgagtg tctctttgtg  26400 

catatgagat tgtaatcagc aagtctcaaa gtggctggac ttggcatgcc accccctcct  26460 

tctaggtcag ccccaaagga gaggcccctg atccaacccg aggctgaacg tccaggctgc  26520 

catgattctg tggtcatttg ggggtttttt tctggagcga ggtcagcagc atcttgggga  26580 

gactgtgtgg agcctgtggg attcactctt gtctcttctg ggcagtggga acccaggaat  26640 

gggcatctct cttgggggtc caggtggagc agcctcatcc tattttgccc tgagacccta  26700 

gacctggact ggcactaact tggccactct cattaaggaa caagatggct ccccccccac  26760 

ccctcccata tttaatgttt ttgttagctt gtgtcacata ctgttttggc aagtttcaaa  26820 

aatgatttat gatcattcag atcattttcc tctggctttt tgctttttcc cactcctata  26880 

agaatattct tcctcatagc atggtctgtg ccttgtcttc cctgccatca tctttggtat  26940 

atcggttagc ttttgcttcc taacaaacca ctcccaaaac ttagtggcta ggccaggtgc  27000 

agtggctcac acctgtaatc ccagcacttt gggaggccaa ggcaggcaga tcacatgaag  27060 

ccagagtttg agaccggcct ggccaacatg gcgaaacccc gtctctacga aaaatacaaa  27120 

aaattagcca ggtgtggtgg tgtgcaccgg taatcacagc tacttgggag gccgaggcac  27180 

gagaatcgct tgaacctggg aggcagaggt tgcagtgagc tgagatcatg ccactgcact  27240 

ccagcctggg tgacagagtg agactctgtc taaaaacaaa caaacaaaaa cttagtggct  27300 

aaaaactcaa aaactcatta tttctcatat tctgtgagtt gtaggttgtc ctggtctgag  27360 

ctagtgtggt tggagccagg tgctctagca aggccttatt acatgactgg gactcagctg  27420 

ggataaccag gtccttcttc atgcacctca aaaaggctag cctgggcttt tcccatggtg  27480 

acagtgttcc aagtgaacaa ggcctgtgag tgctagcctt ggaacatcta tagcatcact  27540 

cccttccatc ctgttgctaa aagcaaatca aggccagctg agattcgggg gatggggaag  27600 

tatatcctac ttcctaaacg gaggaagcac aagagttgtg ttgccattgc agtttaccat  27660 

cttctgcgtc agctcagctc ctctcagctg cttcgggccc ttgtgtcttt tctgtcggtt  27720 

ccagctctga cggtcttctt agctttgaca agtcagcttt tatcatacta tcccttttat  27780 

tttaaattct ttttttttcc aaacgggaaa ataggtccct tccatagcta atggtgattg  27840 

tcattcacaa aagcattgaa cttctggttg agattggaaa ggaaggggaa ctaatagatc  27900 

tgtgagtgag cccatgattt ctagtctaca tctaggacgc tcacggatac attatttggc  27960 

cagaacatga caaagtaatc actggatata gaagtgcaag gtctttcaaa attttaacct  28020 

cagacttaaa acttaaactt gccactttcc ccagataact gactttcctg gtactttcat  28080 

gagagcatta aagatcatag agaataaaaa tgagaattaa atggtcacat tggcagtcct  28140 

ctgagctttg tgactactgg gcagtgctgt tggctggatc cccagtgctt agtgcagggc  28200 

cttgcaccaa gtagctgctc aataaacaat aacgatagct taaacattta ttgagggtgt  28260 

actggtgctg gtgcttacat ttattgaggg tgtactggtg aggaattaac tagtttattc  28320 

ctcatgatgt cctatatgtt ggcactgtta gtatgttgtg caaatgaaca atgtaaggcc  28380 

cagaccattt atttgccaaa ggtcattcag tgatacttgc cagacctagt atgggtagat  28440 

ggaagcaggg agtgaatgaa aggctgcaaa tccataggtg tagatttatt ttttattcat  28500 

cattcactta cactcttatg ctcttatact cttataattt tgttaaagct tatttcaaat  28560 

tttaaatacg catatgtgat tatatatatt ttttttgcag agatgtgaat gagagaaata  28620 

cagtaaaagg gtacgtgacg tactttacgc tatactgtgc tatattttta tttatttaat  28680 

agttggaaga cttttcagca tttctttcct atattgtata gatttaggaa gttcaacatt  28740 

ttgggcacgc acacaaaagt gatgaacatg gaggagtcca ccaatggcag tctggcggct  28800 

gaatttcggc acctggtagg gacatcagtt tcctctctat gttgtctctg gtttcttagg  28860 

acttttgaca atagagcagc cctttttggc acattgccat gtggaggacg cccccctgct  28920 

ggggtttgta gactttgtgg ctggtgtctt cctgtcgcca gtacttgggg cattttggtt  28980 

tctcctcctt atcccacgac tgttcactct gttttctggg ctgtatcact tgcagtaaaa  29040 

atatttccta agcctgtgtt tacgtaaatg atttgcactt gctgcctgca tgatccccac  29100 

ggtcactcac ctacgcttgc agttttctca aaggttatgg ctcccgtgtc ctgactgtga  29160 

tccaagtgtg gtcctgtaca ccagggttca ctcctcagcc cccgtgttcc ccttcctggt  29220 

ggccctgccc acagcccctg cctccacgcc agcctgactc taccaccttt tctccagggc  29280 

agtagggctc attccccagg cagtggagac ccagcaggat gctctgtgtg ggtgcaccat  29340 

ctttcctcct ctgtgaacat gggggctgcc agttgctgtg aggctctgtg ggtgctgagt  29400 

ccgaagcttt ccctgggccc agaacctgca ggaggttaaa ggcatccttt ttcactagag  29460 

accactttgt gatccagcaa gcctcgtagc taggaagctc tacctaactc gaaagcttag  29520 

cccaactcag gtgcctgagg gtattcagag ccgccctcag ggccccagcc tgctccctcg  29580 

tcagctcagg gattgacagc tcagagattg acgtccactt gagggcaggt ggacctggag  29640 

tcactgggcc cagcctggag gactctgctg agggaaaggg gagggagaag ctgtcccgag  29700 

cctcagggca tgaaacagtt aacgaaggtg catggggaaa cattcaggga aagtttgaac  29760 

acaggagagg agcgggttgt gattccaaga gatccctcca gactggtgtc cgcaccatga  29820 

ggccactgag gaaacgtgct tctgctcatg gaccctgaac gagggccaat ttgtcccatg  29880 

ttctgcattc taaagataaa gtcacgtctg tcattctttc tagtcctaac tttgttctca  29940 

tttgtaaatg aagatagcaa ctttgaccat taccatggtg tttactaaga tgattatttg  30000 

ttttatagca attgaaagaa cagaaaaatg ctggcaccag aacgaatgag gtgagagtcc  30060 

ctttatgttg tgaatgggcc caaatcaggc aggtctgtct agcaaggaca ggtcagttgg  30120 

tggctgggga cacctcacag aggagttcag acccatcagg gaagagcagc aaaggagctg  30180 

ggtatttggt atatctgtga caacagtaaa aacaccacat ttgtgcttta ctattgtcaa  30240 

acgattttgc acacaaattc attagacact catggacaac tcagtggagt agctgttgtg  30300 

ggcttttgta ttcttcgcat cttacgggtg aagaaattga ctcacagaag tttgcaggat  30360 

ttagctaagg gtgcacagag ttactgtcag agttggaacg agaggccact ttgatgactg  30420 

ccttgctgtg tgccaagagg gcttttcggc cattgcatcc cctaccaaaa gcattgcctt  30480 

tgcatcagtt tccttcctac agcgtgatcc ttaaggagtt gggtttgtga tgcattcaga  30540 

aatgaactat ggacaaagga tgaaatatct ggagttgttt tggttgagaa cactttcgtc  30600 

atttatgcac acacagtgtg cagtccttcc acaccaggac cagtggaggt cacatggagc  30660 

agcgggacaa acacaggccc aggcagtgca aagatgcctt ctagccctgg tgtgcgcctg  30720 

aactatgttt tgacctccct gggtctagct gttgcacctc tctagttcta agttgtgagg  30780 

ctcacatgag gtaatgctta tgaaggcact ttgaaatcta tccatttcta ccgaaaccaa  30840 

gacaacttgt gacctgccaa gttgatgtga gtcagccctt ccggtaggca gtagacaatc  30900 

acaagtatgc acttactccc agtcctctca ccttctcccc acccatctgc attgagatgt  30960 

gcagccacac tgggcaggca gacagagctg agggtcgaga agccttaggt cacaaagcca  31020 

ttagttgctc tttccccatt gacaatcatt gctctgaatc tataaccctt gccgtaactt  31080 

cagacactta tctttcagag cacactgaga tactaagaat cattaaatct gggagagatt  31140 

gaatattgag tcttcagact tgccactgat tgggcccagg atctctgtgg cctgttgcaa  31200 

tgttaattgg gcttttgaaa gttttaggat ctgtgaataa ttatattctt ttcttctttc  31260 

cttttctctt ccaagggtcc tctcatcgtt actgaagagc ttcactccct tagttttgaa  31320 

acccaattgt gccagcctgg tttggtaatt gacctcgagg taagactttt atggtcccga  31380 

gttggaaaac ttcattattt ttacttttaa ttgttacatg tatttttatt tctcagtgtt  31440 

tgttctggag gtatttacct aattgtttta aaatgatctt tttcagctaa tactatctta  31500 

accttaattt atggtccaga aagctagaca attgaattaa ccttcaaatt caataactta  31560 

aaagttccag ttgagatcgc tgatgttaat tttttttttt tacattaagc attgggactt  31620 

atttattttg ctctgccctc cccccacatt ttatgttttc attgtgacag ttttcatctt  31680 

ctcatattgc atatccctta attaattatt gtagctatta ttatttttag ggacttattt  31740 

cttaactgaa agatacatca aagacaagtt aaactggaca gaagaacaga acttaacact  31800 

tctcattttc aagtattaaa tatgtacaaa tttacaaata atcccctgct tttagttgag  31860 

gctaagcagt tatctgaagg tgacatttct gtgattcaga tgatttcaag gtctttgtca  31920 

tcctttagac gacctctctg cccgttgtgg tgatctccaa cgtcagccag ctcccgagcg  31980 

gttgggcctc catcctttgg tacaacatgc tggtggcgga acccagggta tggaaaacac  32040 

atttgctttg gtcccagggt taagcagaga ccccacgctc tcactgctgc atctctgaaa  32100 

tagccccaat ggccagttgt taagggagaa gtgacaaagc tcctgtggta tttctaaggc  32160 

ttttgagtaa atctgggaat atgaagcatg gatatgtatt tgaaatgaga attacgtttt  32220 

ttctcccatg tgggtaagct agggcagtca gtgattccat accactttat tgttattatt  32280 

tttaaaatgt atacatctac taaacaagca tgctccattc ttagagtgtg gatactcact  32340 

gttaattaat tacagtcatt tataattatt caaagtactt atgtttttta aagtcaccag  32400 

gaacacttaa ctagcaaata atgaattgtt gctcctaagg gaactacgag gctaggttcc  32460 

ttcaagcctc tggtcacaac atttttgtca acccatcaat gcataacatt gtttatgtgt  32520 

gtttctgttt aaagacacct tatctaatac acttgttgag gcattaacat tacacctata  32580 

gctaggatca ctgtaactca tgcctgagtg aagcttgcct cacacatgta ttttctccat  32640 

aaggcacatc ccagccttcc catgcttagg aacagtagac cacactgtgg tactatgctc  32700 

gggcaccatt ttaaacaggg atcaccaaca gcagtcacaa aatgcaaaaa acatggcgct  32760 

aaataggctt caaaaaggac acgtttttct tatgagaggt gaaacaggaa gcgagtgtca  32820 

ttttgtttgg cttcagttgg aagcgtgtta agagactcga attctttgct gctgtgtgct  32880 

gctgtgtgtg cacgggtgtg tcttcaaatg accccaaaga tgccatgtat ttagattttg  32940 

gagttacaag taaattttag tgagtaaaca agttcacaaa tgtgcatctg taaataatga  33000 

aaattgactg tatttctctt cccctactgt gaaagcacct gtgtgtcata taaactagaa  33060 

ttgaactttg ggatggacat atgttttagt gccacacttg tgactggtgt ctctgtagta  33120 

acccttagat tttgggtgtt ttctctctag aatctgtcct tcttcctgac tccaccatgt  33180 

gcacgatggg ctcagctttc agaagtgctg agttggcagt tttcttctgt caccaaaaga  33240 

ggtctcaatg tggaccagct gaacatgttg ggagagaagc ttcttggtat atgcatatta  33300 

acttgttatg tttataaaaa ttgaaattca taaaaatatc tctctaattg ctcttttccc  33360 

ctctgctatt ttgttaaagg taaaaaagta ctaaaatctg tcagcttttc aagctatagt  33420 

ttattatagc taagtgagaa tcatatgtca ccttagaaag aaatatagac ctgataacat  33480 

ttaaatgaat ccgttcctca ttgtctcata ttaagatttc tgagatgaat tcccaaggga  33540 

aagttttatg aattatgtaa aatatattat ttgtggctga aataagttcc tggatgagaa  33600 

agatgaaggc ctattaacca ttaaccttgg ggagaaaaaa ggaagtccct gacatgatga  33660 

gaaataaagt cctggggctt gcccactgca gcataaaccc agggcccttg cctcaggtgg  33720 

ggcccaggta gcagaggatg ccgtccccac aagaggatgc ctcttttgat ctgcagtttt  33780 

gcctgattgg acagagtcta gacagtggta gataattccc tttcagtggt taatgtcagt  33840 

gcatttatta gtccttcata ctaaacaaat caaagatcca ctagtctcaa ggtcagatct  33900 

ttcagtgagt gaggctatct gaactatttc tttaaaaatc cgtggacaac ttgtatccag  33960 

atttgctttc tgtagagcag cactgcattt actccaaaca gcagtaggct cttcctaact  34020 

cagtgacttt caacagtgcc ttcttctctt ggatatgcag ctgtcacagt ggtggtcttg  34080 

tgctgtttaa tacaagtaca aaatcccctt attccatttc tttatttggt gagagttaag  34140 

aaggatatct tttctagctg taatggctca tttgtagttc tctttgaaga ccgataatgg  34200 

gcttaccgcc tcttctcgcc caggctgctt taatttgtgt agctaataac cccgggcttc  34260 

ttcctctcgt ttctctcact tggtttttga tttgcatcag actgcctgag cccatgtcct  34320 

atttggagga aagctgaggg aaatgcataa ataatgtcta aaataaactt gtaaagtcaa  34380 

gtatcagtta cattgaagat ctaggggtga tcttgggcac ctaggttgaa ctttgaaaaa  34440 

aattctgtag gatccttggg agagggggct ctgctgagca gcagaggaag gcagtgggaa  34500 

gaggcactgg atagggaggc ttctctagga ggcccagcag aacacctttg atttagtgca  34560 

aaaggcaaag tcagctgatg gtcttggaag gaagtgtaac atgaagaggc attaaaagga  34620 

ctgtcctctc tttagaggag ggagggaaaa ggggggctgg ggagggagag agcagatggt  34680 

aaggctttgt gatgtttccg ctttccctaa aaactctcat atcgtggtgc ccaggaatct  34740 

gccatccatt cctttctgcc attaaactca aaccaacata tacatatttt agaccattta  34800 

aattccgccc tcttacagca cgtattataa ttgctcagtt aatttggcat taaggtatcc  34860 

gaaaatgtca actgtgtaca tacaatcttt gttgggtgtt tggctctttt ccttgagagc  34920 

aaatacaaga gccatccgtc caaccaaagc tttagaatca gtttctgttt gctgaaaaat  34980 

ctggttttta tttgtttagg tcctaacgcc agccccgatg gtctcattcc gtggacgagg  35040 

ttttgtaagg tgaggactgt ctgggtttta tgctctctaa gtcctttttg ctcttggttg  35100 

aggttctcat gtttcttgct gtgatagtta cttaactgtt tgcttatttt ggcgtgttct  35160 

tctctggcca aggagcccct cgggcaagtt gtttactttt cttcagtgca ggaggttggt  35220 

ctccgagcag gagtctgaca ggaagcacct gcctgagtga taacattctt tcatatctga  35280 

aatgaggctt ctgttcactc acgctggacc cctgcccagc agtgttttag ccatttctgt  35340 

gtgaaatgta tgttgtgggc tgggtggcgc agcctgacca gccagacgag tttaacggcc  35400 

tgctcgagca gaggggccca ctgcggggaa catttagatt ctgctggagt caccagacag  35460 

tgcggaccag cttgcactgc tggattttgc tagtgtgggg cagcttgtgt ttccgtgagt  35520 

cacgctggag gtcaccccca ggctagcttg ctactgtttg gaaagggaag cagagcccgg  35580 

cccagggaag tggagagctc tggagagctc ctggtctgaa tgctgataag agcggggagg  35640 

gaacttgata ttccctgcca ggggagggat cccattgctg tgctgaatgg gacagttcca  35700 

tccttggcac ctggggagtc tttgcaaatg atggtgggag gggccatggt aagtcattgt  35760 

tttagatttt tgttcataat gtctgcattt gtatactttc aggaaaatat aaatgataaa  35820 

aattttccct tctggctttg gattgaaagc atcctagaac tcattaaaaa acacctgctc  35880 

cctctctgga atgatgggta agggccaccg atagatgtat tttgaaacat atttttagtg  35940 

ctgcgaggtt gagacaaggc ctgagtccag cttcagtatt tgactaggcg tgtgtgaatc  36000 

tcacaggagt gcgcatttat aggctgcccc ttcagggctt cagaagagta gaacctcata  36060 

acattaacct tacactttct tagtacaagt atatgtattg gcaaagctaa attaaaaaaa  36120 

aaaaacaact cttgagtgcc attcatgcat tcattcactc aacaaaaatt ttttgagcac  36180 

ctactatgtg gcccctagga aggaagaaaa acccttttaa ctgggtaaat catttctatc  36240 

ttgacttatt gccaggatcg gctgatattt caacagaagc tttaacttga tcagaagggc  36300 

taaaatatga aaatatattg cctaggtctt cctctccagt aaaaaagatg tatacattca  36360 

gacattgtta tggcctttaa aataataaag gaacaataaa aggctcatta atcccacacg  36420 

tgttattgag catctgtagc atcctactga caccatgttc atatcgcttt ttttaaaacc  36480 

ttagtatata catgtataag tgaaatgctt ccttaagtgt caactgaaca gattttaaaa  36540 

tagtctacgt tttttaccgt aggttttttt tctcacattc cagccatttt cttggtttgt  36600 

aagttagcat gaaaatggaa atcatgattg aactaaaagc ccatccgtcc atctcttcga  36660 

tatccaggtg catcatgggc ttcatcagca aggagcgaga gcgtgccctg ttgaaggacc  36720 

agcagccggg gaccttcctg ctgcggttca gtgagagctc ccgggaaggg gccatcacat  36780 

tcacatgggt ggagcggtcc cagaacggag gcggtgagtg ggagtttgag cacatagtcc  36840 

caccattcca tgtgtgtgaa gtccctcttc atcgccgcca gtcagctcat acagatagtg  36900 

cgtttgggac atgaaattgc agatcttata cttaccccaa atgagccccc gataggattt  36960 

aataaaccac cacaaatgtt tgtggttgtg gttcactctc tggaacatat tgtggaaatg  37020 

atggttttga aaagacaatg ccaattcttt cctttgcatc aggtttgtta tctgcagatc  37080 

aaggatgtga gtcaatgtaa tctgcaaccc gttcttggaa ggaatcacat ttcccacagg  37140 

agtaagcatc cacattctct tagggtcatc ccagagtaga gtgtgcagat gagacaggct  37200 

tcgggagaag acttcataca catcatcagc cagggagttg atgcaggacc tgttgggacg  37260 

agtggcagcc tctaccctgc cttcaaacag agcagctgca aatttatctt tatcatatat  37320 

ggggtttcta tttaatcaaa tgagtccatg gctaaaagac tccagaaacc ttggtgttat  37380 

gaagcaggtt tctgagggtg aagctcaaca gcgtaaatga aaaaatcata aagaccaact  37440 

gattactttt cactgttgtt tagagtttgg tctgtttgaa gggcaggagg aaaaactgat  37500 

ttgttttagt aatttttata ctttcagtgg gtagacacaa aatggaatac tcttcttgtt  37560 

ctatttattt ttctgaaata ttcctcttct cctgtttttc tggaatactc ttattctatt  37620 

tgtttttctg ccatactctt cttgttctgt ttgtttttct ggaatactct tctatttttt  37680 

ttttctggaa cggtattctt tcttgttctg tttatttttc aggaatactc ttcttgttct  37740 

atttgttttt ctggaatact tttcttgttc tatttttctt ctgaggaaaa agcagggagg  37800 

ctgagatgag agtctggcgt gggcagtagt cattagaacc tgcccttttc ctcttatcct  37860 

gttcccttcc tgaaccagct gagagacaga tatagtagac caagcagaga tgaagaaaac  37920 

ccttggcaac caggaaaggt caagcaggac actaccagcc aggaagggtt aggatggcag  37980 

catagtctca tgttccacag caggctcccc agaacacctg aaccgtggcc tctggaaggt  38040 

cgacacccaa agggagattc tgtatcagct aggaagaagg atctgaattc agtctgggaa  38100 

taagaacggg agtaaattaa accatggttt attacgttaa gggttacagg gaaatgtagc  38160 

aaataggatc atttttaatt tgaaatgttg ttttctatat atcataattt attttaataa  38220 

aaaattagat ttttcaatat gaaaagaaaa acaaagtgag tttatctcta gtaatttaaa  38280 

tctgtggctt aagccaagat gatccactta gtagtaagtt atttatgtgc ctatgggcag  38340 

cacatcattg aagatgcagg cttttaaagg tttttaataa atcttttttg cccagattta  38400 

taaatctaag caaataatgg caaatagaat gttattgaga ataatacctc cctttatttt  38460 

ccctgagaca gtattataat ttatttagaa ataaagcagt ctactgtata tgttataatg  38520 

cttagattgt gattagttct tatgaggttc actcaaatcc atcaactctg cagtgtttta  38580 

tttctctgtc ctctttcatt ttgggatgtt ctatgggatt tacttagctt ttctcctttt  38640 

tagaacctga cttccatgcg gttgaaccct acacgaagaa agaactttct gctgttactt  38700 

tccctgacat cattcgcaat tacaaagtca tggctgctga gaatattcct gagaatcccc  38760 

tgaagtatct gtatccaaat attgacaaag accatgcctt tggaaagtat tactccaggc  38820 

caaaggaagg taagtggggc aagcaggtgg taacagcgtg gcacagtctt tcctgatgag  38880 

ggggtgatta ttctgaaact ccacccatgc agtgttttgc tttcgaattg gtaagagtag  38940 

gctttcaaaa gatggcataa actcagtgca ggtgaaacat aatgcattat ttggcctgat  39000 

acagctagta agaaatgaat ggacaatttc ctattttagg aatgttggaa aagtccattg  39060 

ctggtcttgt gtatttgtta ctatcactgt ttccacatga aaagggttta aataaggaac  39120 

tgtggtaaac caacagaagg catcttgctc acgttaaagt tgaggaaact gaggcctggg  39180 

agaagtgaag tggttcattt agggtggccc aattacttac tagatcctat ttttattaga  39240 

taattattaa gaggaggcct catctgagaa cacaggtctc ttgcttctca gttgtcaatg  39300 

aattgcattt gagaaggttc gaattagacc tgttttgttt ggaacacacc aacaaagctg  39360 

tttttcagaa tcagaaatct caatattagg acaattactt tggaatgaag atgtccccag  39420 

cttaccttga cagctgtgaa tattcattag ccaggctaat gccaataaac tggatagaac  39480 

tttgcatatt ttgggctcag acttcttgta agatttcaag ttgtgtaaag agaaagctcc  39540 

tagctagtgt cctgctgaac actagcttat ttccagactg aattcagatc cttcttctcg  39600 

ttgtttctgc attcccacta gaattttagg tgactcaggc agggaggtta tagctcctta  39660 

aagttttagg aggctaagct gtctagaaac acagtagaac tttaatcccc ccgaaaagtt  39720 

gatgttgtat tctgatggaa tttcggttga tggaaagcgt acacaatgtg tttatttcta  39780 

gcaccagagc caatggaact tgatggccct aaaggaactg gatatatcaa gactgagttg  39840 

atttctgtgt ctgaagtgta agtgaacaca gaagagtgac atgtttacaa acctcaagcc  39900 

agccttgctc ctggctgggg cctgttgaag atgcttgtat tttacttttc cattgtaatt  39960 

gctatcgcca tcacagctga acttgttgag atccccgtgt tactgcctat cagcatttta  40020 

ctactttaaa aaaaaaaaaa aagccaaaaa ccaaatttgt atttaaggta tataaatttt  40080 

cccaaaactg ataccctttg aaaaagtata aataaaatga gcaaaagttg atcagagtgg  40140 

gaaagtagtt cttttcaatc tagaaaaggc caaagtaatg attgagatac actgtctcca  40200 

cttgctttga ttttgttgtt tcattttata aaaggtagaa aaaattttgg aaatgtcatt  40260 

gtcagttatt tggcctgcag cactgtcttg gggtgaatgg atgtagcctt catgtaaaaa  40320 

cactgtgtgg agcagcttta tctgcattca aacctcaagg tagggatgag ggactcccca  40380 

gacatttctc tggtgctttt ctgtccaggg taagccacga ggcattgtca tctcagggta  40440 

gtgcaccgca catgctcaca cctaggctta cccaggcaga agcggcacag attccagtct  40500 

ggctattgct tatcacatcc ctggagtgtg aaacatttcc caaggggctg ggctggggac  40560 

atgagcctcc catgagctgt acttttgcaa atgtatttta caagtgtatg cacttaccca  40620 

aaataaaaat ataacgtgat aattcaatta cagacaaaag ataaagtgtg taagtatgca  40680 

ctagtgcttt catcactcag atgttgacat tgctcttttc tttttcccca ttttagtcac  40740 

ccttctagac ttcagaccac agacaacctg ctccccatgt ctcctgagga gtttgacgag  40800 

gtgtctcgga tagtgggctc tgtagaattc gacagtatgg tgagtaccac ggctggcctg  40860 

tgtgtagctc tcaataagtg tgtgtgctca gaggcaggga gcgcaccatg gcagatcccg  40920 

ggcctgtctg cgggagggag ccctggcgga gccaaggaga gtgcagtgct cagatgagcc  40980 

atgacaagtt ggagctgctg gatttaaacc acctacatca tcagtgggat ttttattcct  41040 

cgcattcagc accttccaaa aaacaagtga catttctaat attcaggttt cctcctctcc  41100 

cctttaaagt tgtccatgta gaaatttcat atattaagga actaagattt ctttgataag  41160 

caaatgtttt tcttcggaat gcgatttcat cactgtgtct aggggaggga gtgttatttt  41220 

tagaaaggga gggactaacg cttggtagtt acagtaatta gagagaatta tactttagca  41280 

gcaatgagat tacttcatct gccttatatt tgagagctaa tttgtacaag tagctcctgg  41340 

ggctgtgaag ggcttgccaa gagtaaaagg ttcaaggagt gaaatagtta atgagattcg  41400 

tgatagaaat gggaatatga ttgtccacaa aagggaacat cttccttttg gagggtgctt  41460 

ttttagtata tcaactagta ttgtttgcct ttcagcctaa aatccttcct cttaaagatt  41520 

gtgcttgctt ggctggattt ttgctgatgc tgtttaattt taagctcttt tccacatgga  41580 

gctattccag ctcattttta aaaatttatt taatgcttcc aaaaaatatc ctgagttatt  41640 

actggccttt cttccttact gtatacccgg tgcctggcaa aaagtaggtg ctcaacaaag  41700 

agaggaaggc agggagggga aaggtgagcg agaatgagag ggcgtcactc ttcagacatt  41760 

tggggaatgc gatgataagg cctcagtaag atctgcctgg attcaggttt ctcatgtgta  41820 

attttttgac ttttttcctc accttaagca cgggctacaa tcattagaga ttgtaccttc  41880 

ctacactttc ctgattgttg ttgacgaaat aggccattta gaaaaacagt tagctattgt  41940 

ggcagcgaaa tagtttcatt cctattggca agtgtaaaat gaaccttttc tgcatgtaag  42000 

aaggatcctg ctagagtttc gctcccaaca ctgctatagg cctggcacaa aaatggaata  42060 

aatataaagc cctattcaga aaatatgtta catcacagga actgtaaaat ggggggattt  42120 

tttatacctc ctttgttgtt cctgagcaaa ttacatcatc atcaccacca ccattaatag  42180 

gtgacattaa gcacctacag tataccatgt acttcacagg tagtaactca ttcagtcctc  42240 

acagccacct tctgagttag gtgttattgt tattcctact ttgacagatg tgggaacaga  42300 

gcccctcctc cccacacagc cccttttatg taacttgact aagatcaagc agttagtaaa  42360 

tggtagaaag aatatttgaa tctacctagt gagtctctag tgcatgcttt tgtccggtat  42420 

cctggaaagc ctcccacaaa aagctaatct ttgccccatt caaaacatgc accctgaaga  42480 

agctgtttgt acaggattgg gtttattctg ttattaagac aaaggcatca tggcctttgg  42540 

gtgagaggcc cgtatgtgtt tgggatttgg caatcagcat tccatctctg tcatcaccat  42600 

tattgagaaa atagatggat tggttccctc tctgcagtcc tgtggagcag ttggactgct  42660 

ctctctgctc tcaggatgat actgtgagaa caatttaaat atgctaagca catgtcagga  42720 

aacagttttg tggtctttgg acactcgctg tagccattcc gttccatttc aggtgatttt  42780 

attcatttca tttgtagaat aaaataaatc catttcacac acacacacac acacacacac  42840 

acacacacac accctctata caccactaaa gcctcccatt aaacccatag aagacttaaa  42900 

gagctaaaag aggctataat ataaaaaaaa aaaaagaaaa gaaaagaaaa agagttcata  42960 

atataggcac ggcactggtt agacaacggt tgaatcagtc tctaaattgg gttgacttca  43020 

caatggtttg ctgtcctgcc ccttggccat gacttccatc cagcatctac tcttagaatc  43080 

agagtgtata ctctgaaaca gaagcagctc tccccaagct ggctgtcttc tgccaagtct  43140 

ggaggttgca gctctgaaag agggtggtac caggggccaa cgcctggtgc agcctcagga  43200 

gcaacaggac cataggctcc tgaggaaatt gtccaagaga gccagagcac cagtgttctc  43260 

tgcagtatga cttgggcatt tgtttagtct ggatttcctg tgtaatcagt gttttcctct  43320 

gggactgtaa tagaaccagt ctactctcca agaggccttt agccaaagct cataatgata  43380 

ggactagcta ttaattgtct actactttct gtctactata ctaaacattt tacgtatgcc  43440 

atctaattta acaagcctat ctgctaggta ctattaatcc ccttttgata gatgaagact  43500 

ttgagattta gagaggttaa gccacttgcc ctggtcacgc agtgacagga tctgaatctg  43560 

ggcttcctct cttcctctat ctatctatct ccccctccct ccctctcttt acccccacgg  43620 

tccaccctat taagcacaca tttgaaacat cagccaggcc gggcgcggtg gctcacgcct  43680 

gtaatctcaa cattttggga ggccggggca ggtggatcac ctgcgatcag gagtttgaga  43740 

ccagcctggc caacacggtg aaaccccatc tctactaaaa atacaaaaat tagccagcca  43800 

tggtggtgtg tgcctgtaat cccagctact aggggggctg aggcaggagg atcgcttgaa  43860 

cctggaaggt ggaggttgca gtgagccaag atggtgccac tgcactccag tctgggcaac  43920 

agaggaagac tccgtctcca atttgaaaaa aaaaaagaaa gaaaaagaaa agagaaacat  43980 

aagctagact gaaacatagg ggcaacactt tcactgtgct tttcaatcca aaataatttt  44040 

cctaagagcc cactatgtac cacacactat tgagcacttg ggattaaaaa aaagttctcc  44100 

agaggctttc cagtctagtg ccatgtgtct cagtcttggt gctactggta ttcggggctt  44160 

cagaattatt tgttggggat tggtggggtg gggagctttc ctgtgcttcg taggaggttc  44220 

agcaacacct ctggcttcta ccaaatagat accagtaagc acatgcaccc tcacagccag  44280 

atgggacaat cagcaatgtc ttcagccatt gccaggggac agaatcatcc tcgattgaaa  44340 

gcatctggtc taaggggaaa gaccagtgtg gaagtcagac aggaagtcac tggcaggcta  44400 

gtatcctttg gatgaacagt ttacttacta taagttgtcc tggtcaaggc agctgggcag  44460 

aaggcactgg tggggaaggt gaaggggttc ttagtaattc tgcttcttta gtctgtacct  44520 

gaaacacagt cattgatttt acccactgag tggcctttag gggaagtgta tcatttccct  44580 

ctccttcctc tctccctctg gcatatggct cagcatttcc tcacccgttt cacatgagca  44640 

gatttgacac gttgctctgt ccccagactt gcataacagc aaaaagcttt atgcagtctg  44700 

gatggattca caaaattaag ccagtgggca gcagagtaat aatccgtcag cttaggtgat  44760 

ataaaaggtc ttgattagcc caggataata catagtacat gctgaaggcc ctcttattcc  44820 

acggtattta tttgccattg caagtatctt cctactactt cattctagaa tagacaagca  44880 

atgtttaatg tatgagtctg catttcacaa gatcagtgta ataaacttaa ccacattttg  44940 

tctttttaca gatgaacaca gtatagagca tgaatttttt tcatcttctc tggcgacagt  45000 

tttccttctc atctgtgatt ccctcctgct actctgttcc ttcacatcct gtgtttctag  45060 

ggaaatgaaa gaaaggccag caaattcgct gcaacctgtt gatagcaagt gaatttttct  45120 

ctaactcaga aacatcagtt actctgaagg gcatcatgca tcttactgaa ggtaaaattg  45180 

aaaggcattc tctgaagagt gggtttcaca agtgaaaaac atccagatac acccaaagta  45240 

tcaggacgag aatgagggtc ctttgggaaa ggagaagtta agcaacatct agcaaatgtt  45300 

atgcataaag tcagtgccca actgttatag gttgttggat aaatcagtgg ttatttaggg  45360 

aactgcttga cgtaggaacg gtaaatttct gtgggagaat tcttacatgt tttctttgct  45420 

ttaagtgtaa ctggcagttt tccattggtt tacctgtgaa atagttcaaa gccaagttta  45480 

tatacaatta tatcagtcct ctttcaaagg tagccatcat ggatctggta gggggaaaat  45540 

gtgtatttta ttacatcttt cacattggct atttaaagac aaagacaaat tctgtttctt  45600 

gagaagagaa tattagcttt actgtttgtt atggcttaat gacactagct aatatcaata  45660 

gaaggatgta catttccaaa ttcacaagtt gtgtttgata tccaaagctg aatacattct  45720 

gctttcatct tggtcacata caattatttt tacagttctc ccaagggagt taggctattc  45780 

acaaccactc attcaaaagt tgaaattaac catagatgta gataaactca gaaatttaat  45840 

tcatgtttct taaatgggct actttgtcct ttttgttatt agggtggtat ttagtctatt  45900 

agccacaaaa ttgggaaagg agtagaaaaa gcagtaactg acaacttgaa taatacacca  45960 

gagataatat gagaatcaga tcatttcaaa actcatttcc tatgtaactg cattgagaac  46020 

tgcatatgtt tcgctgatat atgtgttttt cacatttgcg aatggttcca ttctctctcc  46080 

tgtacttttt ccagacactt ttttgagtgg atgatgtttc gtgaagtata ctgtattttt  46140 

acctttttcc ttccttatca ctgacacaaa aagtagatta agagatgggt ttgacaaggt  46200 

tcttcccttt tacatactgc tgtctatgtg gctgtatctt gtttttccac tactgctacc  46260 

acaactatat tatcatgcaa atgctgtatt cttctttggt ggagataaag atttcttgag  46320 

ttttgtttta aaattaaagc taaagtatct gtattgcatt aaatataata tgcacacagt  46380 

gctttccgtg gcactgcata caatctgagg cctcctctct cagtttttat atagatggcg  46440 

agaacctaag tttcagttga ttttacaatt gaaatgacta aaaaacaaag aagacaacat  46500 

taaaacaata ttgtttctaa ttgctgaggt ttagctgtca gttctttttg ccctttggga  46560 

attcggcatg gtttcatttt actgcactag ccaagagact ttacttttaa gaagtattaa  46620 

aattctaaaa ttctattaat ctctcattaa tagtatttaa tataaagatt cttaaaatta  46680 

ctgacgttat gaattggttt gatgcttttc tcatgtacct caatccttat tttaaaataa  46740 

gatcaataac actattttcc taaatgttgt ccttacctgc cctacacata ttcagaattg  46800 

ctatggcaga ttaagacatg tcaatggaag aggtcagagg agtaatatta ttggcaacag  46860 

ctgtaatagg tgccataaaa agcaaacaaa caaaagtatt ttggttcttt gcgaccacag  46920 

ctgtccccaa atatacagat gattcagcac ttattttaaa atgaatctgg gtattgctaa  46980 

tacccccaaa ttagcagttt ttaattttaa aatacatgag aaatgggact ttgtcttgtc  47040 

tccaaagcag tcatctaaaa tctacacccc cacgattaga tgagttatta ctgagaagtt  47100 

attgcaccca caaaaaagct gccatttttt tccaaagatg tcaaaagcta gaaggccagg  47160 

tcttctcaaa gtaaaataca ctgtgtattg gggaaaaaag ggtaagaggc ataattacca  47220 

agttaggcat agtctgtcaa gttgtattta gctattatca tggaatagtg ttattccctg  47280 

ataatgaatg ttggcatcat aaccagaatg attattctca tctccatatc ttcgtattta  47340 

catctaggaa atataaagct tatttatagt gaacactgag agtggtctct ctccaaggag  47400 

taaagtaaat atgccctggc taactagtgt aagtttgtat tctacataat taaccattat  47460 

aagaagtcac tgagtagatc ctaacttaag ggatatttgt ttgtgtttga gtatttctcg  47520 

tgtggtgttt ctaagtttga aaagtgtttt ataagcatag agcttatgtg tgctactggg  47580 

gacaaatgtc tcattttaaa ggaaagaggg ttttctgaga tggcatgaaa tgagtgaaat  47640 

ctatttattg cctgaaagct aaagtggaat atgaaggcaa gtctttctga acagagcagt  47700 

cctgtcactg actaacccag ggaaaggaca ggaaaaagct agaaagtgtt ttgaaaactc  47760 

ttctgcttac cttttgaatt gggacattaa caaagtaagg accatttatg tgactggctt  47820 

cctttggtta gttatgattc attcattaat taattcatca gatttatata gagcacctgc  47880 

catgagccag gcattatact agatgtttgg gaaacattgg taaacaaaag caaagatccc  47940 

tgcttttatg gaacttaaga tattctgaga cactggatca tacattctag tgcagtcacc  48000 

ttattaaaac ttaagattt                                               48019 

 
           
             13  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            13 

ttgccacacc attggtctcg                                                 20 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

aggcatggtc tttgtcaata                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

cactgagaca tcctgccacc                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

caggaatttt gagtcaagct                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

tgtagcaaga agttattctc                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

atcctgaaga ttacgcttgc                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

ccgactgagc ctgattaaat                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

ctttcaattg caggtgccga                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

acctcttttg gtgacagaag                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

ccatcattcc agagagggag                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

cacccatgtg aatgtgatgg                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

aagtcaggtt cgcctccgtt                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

agaagggtga acttcagaca                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

cagagcccac tatccgagac                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

attcatgctc tatactgtgt                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

taagaattct cccacagaaa                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

attgtatata aacttggctt                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

gaaacaatat tgtttttaat                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

gcaggaaagc gacctcgtgc                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

cctccgcaga ctctgcgcag                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

ggtgcagccg agcccctccg                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

atgaaacttt tctgcgcgca                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

gtgttcactt acacttcaga                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

agccaggagc aaggctggct                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

gggatctcaa caagttcagc                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

aaatgctgat aggcagtaac                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

ttttcaagta gggcatggaa                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

caggtcatac ctgaagatta                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

tcccaaaacc taatagggac                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

cacaaacgag ctgcaaatac                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

caccaacagt ctggaaagaa                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

tttgtcactt ctcccttaac                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

caagaagagt attcctgaaa                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

gttcacttac acttcagaca                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

tagaagggtg actaaaatgg                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

tggtactcac catactgtcg                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

ctgtgttcat ctgtaaaaag                                                 20 

 
           
             50  
             20  
             DNA  
             H. sapiens  
             
 
           
            50 

cgagaccaat ggtgtggcaa                                                 20 

 
           
             51  
             20  
             DNA  
             H. sapiens  
             
 
           
            51 

tattgacaaa gaccatgcct                                                 20 

 
           
             52  
             20  
             DNA  
             H. sapiens  
             
 
           
            52 

ggtggcagga tgtctcagtg                                                 20 

 
           
             53  
             20  
             DNA  
             H. sapiens  
             
 
           
            53 

agcttgactc aaaattcctg                                                 20 

 
           
             54  
             20  
             DNA  
             H. sapiens  
             
 
           
            54 

gagaataact tcttgctaca                                                 20 

 
           
             55  
             20  
             DNA  
             H. sapiens  
             
 
           
            55 

gcaagcgtaa tcttcaggat                                                 20 

 
           
             56  
             20  
             DNA  
             H. sapiens  
             
 
           
            56 

atttaatcag gctcagtcgg                                                 20 

 
           
             57  
             20  
             DNA  
             H. sapiens  
             
 
           
            57 

tcggcacctg caattgaaag                                                 20 

 
           
             58  
             20  
             DNA  
             H. sapiens  
             
 
           
            58 

cttctgtcac caaaagaggt                                                 20 

 
           
             59  
             20  
             DNA  
             H. sapiens  
             
 
           
            59 

ctccctctct ggaatgatgg                                                 20 

 
           
             60  
             20  
             DNA  
             H. sapiens  
             
 
           
            60 

ccatcacatt cacatgggtg                                                 20 

 
           
             61  
             20  
             DNA  
             H. sapiens  
             
 
           
            61 

gtctcggata gtgggctctg                                                 20 

 
           
             62  
             20  
             DNA  
             H. sapiens  
             
 
           
            62 

acacagtata gagcatgaat                                                 20 

 
           
             63  
             20  
             DNA  
             H. sapiens  
             
 
           
            63 

tttctgtggg agaattctta                                                 20 

 
           
             64  
             20  
             DNA  
             H. sapiens  
             
 
           
            64 

aagccaagtt tatatacaat                                                 20 

 
           
             65  
             20  
             DNA  
             H. sapiens  
             
 
           
            65 

ctgcgcagag tctgcggagg                                                 20 

 
           
             66  
             20  
             DNA  
             H. sapiens  
             
 
           
            66 

cggaggggct cggctgcacc                                                 20 

 
           
             67  
             20  
             DNA  
             H. sapiens  
             
 
           
            67 

tgcgcgcaga aaagtttcat                                                 20 

 
           
             68  
             20  
             DNA  
             H. sapiens  
             
 
           
            68 

tctgaagtgt aagtgaacac                                                 20 

 
           
             69  
             20  
             DNA  
             H. sapiens  
             
 
           
            69 

agccagcctt gctcctggct                                                 20 

 
           
             70  
             20  
             DNA  
             H. sapiens  
             
 
           
            70 

gctgaacttg ttgagatccc                                                 20 

 
           
             71  
             20  
             DNA  
             H. sapiens  
             
 
           
            71 

gttactgcct atcagcattt                                                 20 

 
           
             72  
             20  
             DNA  
             H. sapiens  
             
 
           
            72 

ttccatgccc tacttgaaaa                                                 20 

 
           
             73  
             20  
             DNA  
             H. sapiens  
             
 
           
            73 

tgtctgaagt gtaagtgaac                                                 20