Patent Publication Number: US-2003232437-A1

Title: Antisense modulation of VEGF-C expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of VEGF-C. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding VEGF-C. Such compounds have been shown to modulate the expression of VEGF-C.  
       BACKGROUND OF THE INVENTION  
       [0002] All vessels of the circulatory system are lined with endothelial cells. This endothelial cell lining is formed by two processes: vasculogenesis, the de novo formation of new endothelial channels from differentiating angioblasts, and angiogenesis, the sprouting or splitting of capillaries from pre-existing vessels. These two processes are regulated by polypeptide growth factors and their receptors. Adult vasculature is normally quiescent, but it can become activated to form new capillaries as a part of wound healing or tumorigenesis. During tumorigenesis, the balance between angiogenesis inhibitors, such as endostatin and thrombospondin-1, and angiogenesis inducers, such as vascular endothelial growth factor (VEGF), is shifted and rapid vessel ingrowth occurs, supporting tumor expansion (Olofsson et al.,  Curr. Opin. Biotechnol.,  1999, 10, 528-535).  
       [0003] Aberrant regulation of endothelial cell growth and proliferation contributes to tumor formation, cardiovascular disease and atherosclerosis, and diseases such as psoriasis and rheumatoid arthritis (Enholm et al.,  Trends Cardiovasc. Med.,  1998, 8, 292-297). During embryonic vasculogenesis, VEGF is an important regulator of endothelial cell proliferation, chemotaxis, migration and vascular permeability, as well as of normal and pathological angiogenesis. A critical role of VEGF in embryogenesis is demonstrated by the unprecedented finding that inactivation of even a single VEGF allele results in embryonic lethality (Joukov et al.,  J. Cell Physiol.,  1997, 173, 211-215).  
       [0004] A family of VEGF-related molecules has recently been characterized, and consists of at least five members: VEGF/VEGF-A, VEGF-B, VEGF-2/VEGF-C, VEGF-D and placenta growth factor (PlGF). Within the VEGF family of growth factors, VEGF-C and its closest relative, VEGF-D constitute a subgroup characterized by the presence of unique amino- and carboxy-terminal extensions flanking the common VEGF-homology domain. VEGF family members transmit their signals by binding to the protein tyrosine kinase receptors VEGFR-1/FLT1, VEGFR-2/KDR/FLK1, and VEGFR-3/FLT4, which are structurally related to the PDGF family of class III transmembrane receptors. Upon ligand binding, the receptors auto- or trans-phosphorylate specific cytoplasmic tyrosine residues to initiate an intracellular cascade of signaling that ultimately reaches cytoskeletal proteins and nuclear transcription factor effectors (Olofsson et al.,  Curr. Opin. Biotechnol.,  1999, 10, 528-535; Wang et al.,  Blood,  1997, 90, 3507-3515).  
       [0005] The receptor tyrosine kinase VEGFR-3/(FLT4) is expressed mainly on lymphatic endothelia and originally was considered an orphan receptor, as its ligand was unknown and it was found not to bind VEGF. The ligand of VEGFR-3/FLT4 was purified by receptor-affinity chromatography from medium conditioned by PC-3 prostatic adenocarcinoma cells, a partial amino acid sequence was obtained, and the cDNA encoding the ligand was cloned using a degenerate PCR-based strategy. This ligand specific for the VEGFR-3/FLT4 receptor was vascular endothelial growth factor-C (VEGF-C; also known as VEGFC, vascular endothelial growth factor c precursor, FLT4 ligand, VEGF-2, vascular endothelial growth factor related protein, and VRP) (Joukov et al.,  EMBO J.,  1996, 15, 290-298).  
       [0006] Independently, a sequence was identified in the EST database as homologous to VEGF, and using the EST clone as a probe, a full length cDNA encoding this VEGF-related protein (VRP/VEGF-C) was isolated from a cDNA library made from the human glioma cell line G61. Additionally, two VEGF-C cDNA clones containing 152 and 557 base pair deletions when compared with the full-length clone were also identified, presumably generated by alternative splicing. The predicted proteins encoded by these two deleted cDNAs contain either only part or none of the VEGF-homology domain (Lee et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1996, 93, 1988-1992).  
       [0007] The genomic organization of the human and mouse VEGF-C genes was characterized, and both genes comprise over 40 kilobase pairs of genomic DNA and consist of seven coding exons. Alternative splicing leads to one mRNA transcript lacking exon 4 and another putative mRNA form lacking exons 2-4, corresponding to the 152 and 557 nucleotide deletions, respectively, described by Lee, et al. (Chilov et al.,  J. Biol. Chem.,  1997, 272, 25176-25183).  
       [0008] Antibodies recognizing two different regions of the VEGF-C precursor were generated and allowed the characterization of the VEGF-C polypeptide and its variously processed forms. VEGF-C is synthesized as a precursor protein that undergoes proteolytic processing in which the carboxy-terminal domain is cleaved upon secretion but remains bound to the amino-terminal domain by disulfide bonds. Thus, the major secreted form of VEGF-C is a homodimer formed through the disulfide linkage of the C-terminal propeptide of one polypeptide chain to the N-terminal part of the other chain. Further proteolytic processing of the N-terminal propeptide then releases the mature, biologically active, non-disulfide bonded, soluble form of VEGF-C. This mature form consists essentially of a dimer of two polypeptide chains corresponding to the VEGF-homology domain and possesses VEGF-like effects on endothelial cells, stimulating their proliferation and migration, as well as increasing permeability of blood vessels in vivo (Enholm et al.,  Trends Cardiovasc. Med.,  1998, 8, 292-297; Joukov et al.,  EMBO J.,  1997, 16, 3898-3911). The mature form of VEGF-C also has receptor specificity; although both the full-length and mature forms of VEGF-C bind to VEGFR-3/FLT4, only mature VEGF-C can bind and activate VEGFR-2/KDR (Joukov et al.,  EMBO J.,  1997, 16, 3898-3911).  
       [0009] Northern blot analyses detect 2.0 and 2.4 kb RNA species from many embryonal and adult tissues. In adult humans, the VEGF-C mRNA is expressed most prominently in hear, placenta, ovary, small intestine, and the thyroid gland, and tumor cells express almost exclusively the 2.4 kb form (Joukov et al.,  EMBO J.,  1996, 15, 290-298).  
       [0010] By in situ hybridization, VEGF-C mRNA was found to be expressed in mesenchymal cells of postimplantation mouse embryos, particularly in regions where the lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary and jugular regions, and in the developing mesenterium, which is rich in lymphatic vessels. In adult mice, the expression of VEGF-C decreases but mRNA can still be observed in the lung, heart, liver and kidney. The pattern of expression of VEGF-C in relation to its receptor VEGFR-3/FLT4 suggests a paracrine mode of action (Kukk et al.,  Development,  1996, 122, 3829-3837).  
       [0011] VEGF-C also has VEGF like properties, including stimulation of blood vascular endothelial cell proliferation and migration, as well as increasing vascular permeability (Joukov et al.,  EMBO J.,  1996, 15, 290-298; Joukov et al.,  EMBO J.,  1997, 16, 3898-3911; Lee et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1996, 93, 1988-1992). VEGF-C is also a potent inducer of lymphangiogenesis. Transgenic mice overexpressing VEGF-C directed to basal keratinocytes of the skin exhibited superficial lymphatic, but not vascular, endothelial proliferation and vessel enlargement. Thus, VEGF-C induces selective hyperplasia of the lymphatic vasculature, and VEGF-C is the first growth factor shown to specifically induce lymphangiogenesis (Jeltsch et al.,  Science,  1997, 276, 1423-1425).  
       [0012] VEGF-C is highly chemoattractive for lymphatic endothelial cells. The lymphangiogenic potency of VEGF-C was further demonstrated when exogenously added, recombinant, mature VEGF-C was found to induce proliferation of lymphatic endothelial cells and development of new lymphatic sinuses in differentiated avian chorioallantoic membrane (Oh et al.,  Dev. Biol.,  1997, 188, 96-109).  
       [0013] The lymphatic system is a low-flow, low-pressure system in intimate contact with extracellular matrix and lymph is actively exchanged with interstitial tissue fluid. Cellular trafficking in the lymphatic system is important for immunosurveillance and for pathological processes such as the metastatic spread of tumors. Aberrant function of the lymphatic system is implicated in many disease conditions such as edema, ascites, inflammation, infectious and immune diseases, fibrosis, and tumors such as Kaposi&#39;s sarcoma (KS) and lymphangioma/lymphangiomatosis (Enholm et al.,  Trends Cardiovasc. Med.,  1998, 8, 292-297).  
       [0014] A number of cytokines have been postulated to have a role in the pathogenesis of KS. The proliferative effects of basic fibroblast growth factor (bFGF) and oncostatin M (OSM) may occur via activation of the c-Jun N-terminal kinase (JNK) signaling pathway in KS cells. Serum, several growth factors such as platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF-β), and proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-1 alpha and beta (IL-1α and IL-1β) have also been demonstrated to increase the steady state of VEGF-C mRNA but not VEGF-B mRNA in human lung fibroblast IMR-90 cells (Enholm et al.,  Oncogene,  1997, 14, 2475-2483; Ristimaki et al.,  J. Biol. Chem.,  1998, 273, 8413-8418). Furthermore, the VEGFR-3/FLT4 receptor is robustly expressed in KS cells, and stimulation of KS cells with VEGF or VEGF-C resulted in an increase in JNK activity (Liu et al.,  J. Clin. Invest.,  1997, 99, 1798-1804). Thus, VEGF-C may play a role in the pathogenesis of KS.  
       [0015] Co-expression of VEGF-C and its receptor VEGFR-3/FLT4 was also detected in most samples from human thyroid adenomas and adenocarcinomas. VEGF-C mRNA was found in different types of thyroid disorder, including benign as well as malignant tumors (Shushanov et al.,  Int. J. Cancer,  2000, 86, 47-52). Furthermore, in one half of 36 human tumor tissues tested, VEGF-C mRNA was detected and, notably, all lymphomas contained low levels of VEGF-C mRNA, possibly reflecting the cell-specific pattern of expression of the gene in the corresponding normal cells. VEGF-C might also be involved in metastasis, as upregulated expression of VEGF-C was detected in prostatic adenocarcinoma PC-3 cells and was correlated with tumor dissemination into lymph nodes. Thus, cancer cells that produce VEGF-C and metastasize to lymph nodes may have a growth advantage because of their capacity to stimulate both hematic and lymphatic endothelia (Salven et al.,  Am. J. Pathol.,  1998, 153, 103-108).  
       [0016] Expression of VEGF-C and VEGFR-3/FLT4 are associated with angiogenesis and perhaps lymphangiogenesis in breast cancer. Affinity purified, polyclonal antibodies were produced against VEGF-C and used to stain and demonstrate the presence of VEGF-C in eight intraductal carcinoma and invasive breast carcinoma cell lines. The presence of VEGF-C in intraductal carcinoma cells as well as the VEGFR-3 positive capillaries surrounding the affected ducts suggest that VEGF-C secreted by the cancer cells acts predominantly as an angiogenic growth factor for blood vessels, is involved in paracrine signaling between cancer cells and the endothelium, and may be involved in modifying the permeabilities of both blood and lymphatic vessels during tumor metastasis into the axillary lymph nodes (Valtola et al.,  Am. J. Pathol.,  1999, 154, 1381-1390).  
       [0017] Immunohistochemistry was used to assess VEGF-C expression in 228 primarily surgically treated cases of postmenopausal uterine endometrial carcinoma and evaluate the correlation with established histopathologic risk factors and clinical outcome, such as vascular invasion, depth of invasion (myometrial vs. serosal-parametrial), lymphatic vessel invasion, lymph node metastasis, and 5- and 10-year disease-free survival rates. VEGF-C expression was highly correlated with all of these these histopathologic features, which bore prognostic significance and are thus predictive of the clinical outcome of postmenopausal uterine endometrial carcinoma (Hirai et al.,  Gynecol. Oncol.,  2001, 80, 181-188).  
       [0018] Because angiogenesis is suggested to be a rate limiting step in tumor development, and because of the selective nature of the VEGF-C ligand, which potently induces lymphangiogenesis as well as angiogenesis, VEGF-C is an ideal target for therapeutic modulation of growth factor signaling in pathologic conditions such as tumor growth, metastasis, and diabetic retinopathy.  
       [0019] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of VEGF-C and, to date, investigative strategies aimed at modulating VEGF-C function have involved the use of a VEGF-C mutant protein and a soluble VEGFR-3 fusion protein inhibitor.  
       [0020] A selective agonist mutant which binds and activates only VEGFR-3 but neither binds nor activates VEGFR-2, and neither induces vascular permeability in vivo nor stimulates migration of endothelial cells in culture (Joukov et al.,  J. Biol. Chem.,  1998, 273, 6599-6602).  
       [0021] VEGF-C overexpression in MCF-7 mammary tumors strongly and specifically induces the growth of tumor-associated lymphatic vessels and significantly increases tumor growth. These effects of VEGF-C are inhibited by a soluble VEGFR-3 fusion protein, suggesting that the VEGF-C facilitated tumor metastasis via the lymphatic vessels can be inhibited by blocking the interaction between VEGF-C and its receptor (Karpanen et al.,  Cancer Res.,  2001, 61, 1786-1790).  
       [0022] Consequently, there remains a long felt need for agents capable of effectively inhibiting VEGF-C function.  
       [0023] Disclosed and claimed in U.S. Pat. No. 6,130,071 is a purified and isolated mutant encoding a VEGF-C protein which is capable of binding to the VEGFR-3/FLT4 receptor but fails to bind to human KDR/VEGFR-2, a vector comprising a nucleic acid encoding said mutant, a host cell transformed or transfected with said nucleic acid, sequences complementary to and which hybridize with VEGF-C, and a method of making a polypeptide that binds to VEGFR-3/FLT4 (Alitalo and Joukov, 2000).  
       [0024] Disclosed and claimed in U.S. Pat. Nos. 6,221,839 and 6,245,530 are a purified and isolated VEGF-C polypeptide, cDNAs and vectors encoding said polypeptide, a pharmaceutical composition comprising said polypeptide, a method of modulating the activity of human VEGFR-3/FLT4 receptor tyrosine kinase comprising administering to a person in need said pharmaceutical composition, and generally disclosed are inhibitors, including antibodies, antisense oligonucleotides, and peptides which block the VEGFR-3 receptor, which may be used to control endothelial cell proliferation and lymphangiomas, to arrest metastatic growth, or to control other aspects of endothelial cell expression and growth (Alitalo and Joukov, 2001; Alitalo and Joukov, 2001).  
       [0025] Disclosed and claimed in PCT Publication WO 99/08522 is a method of stimulating angiogenesis in endothelial cells comprising co-administering to said cells at least two cytokines selected from the group consisting of VEGF, VEGF-B, VEGF-C, and FGF, a method of inhibiting endothelial cell permeation, invasion and/or metastasis in a patient comprising administering to said patient an effective endothelial cell proliferation inhibiting amount of a VEGF-C antagonist and a method of modulating angiogenic activity of endothelial cells comprising transfecting or transforming the cells with a vector containing an antisense nucleic acid for VEGF-C (Pepper et al., 1999).  
       [0026] Disclosed and claimed in PCT Publication WO 01/52904 is a composition comprising one or more antisense oligonucleotides directed against vascular endothelial growth factor (VEGF) wherein said antisense oligonucleotide inhibits proliferation-of cells exhibiting autocrine VEGF activity, wherein said one or more antisense oligonucleotide is selected from a group of fragments encoding a portion of the VEGF protein. One antisense oligonucleotide resulting in decreased VEGF-C mRNA levels is disclosed (Gill and Masood, 2001).  
       [0027] Disclosed and claimed in PCT Publication WO 00/45835 is a method for treating injury or degeneration of photoreceptors, comprising administering to a subject suffering from such photoreceptor injury or degeneration a therapeutically effective amount of VEGF-C, and a composition comprising an isolated antibody, wherein said antibody specially binds the VEGF-C polypeptide. Also generally disclosed as a potential VEGF-C antagonist is an antisense DNA oligonucleotide as well as small molecule inhibitors (Rosen et al., 2000).  
       [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 VEGF-C expression.  
       [0029] The present invention provides compositions and methods for modulating VEGF-C expression, including modulation of the alternatively spliced forms of VEGF-C.  
       SUMMARY OF THE INVENTION  
       [0030] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding VEGF-C, and which modulate the expression of VEGF-C. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of VEGF-C 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 VEGF-C 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 VEGF-C, ultimately modulating the amount of VEGF-C produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding VEGF-C. As used herein, the terms “target nucleic acid” and “nucleic acid encoding VEGF-C” encompass DNA encoding VEGF-C, 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 VEGF-C. 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 VEGF-C. 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 1 -GUG, 5 1 -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 VEGF-C, 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 “texons” 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. 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.  
       [0045] 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.  
       [0046] 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.  
       [0047] 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.  
       [0048] 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).  
       [0049] 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.  
       [0050] 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.  
       [0051] 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.  
       [0052] 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.  
       [0053] 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.  
       [0054] 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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.  
       [0060] 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.  
       [0061] 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.  
       [0062] 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.  
       [0063] 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.  
       [0064] 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 1 -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.  
       [0065] 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.  
       [0066] 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.  
       [0067] 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.  
       [0068] 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, fluores-ceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 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.  
       [0069] 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.  
       [0070] 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.  
       [0071] 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.  
       [0072] 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.  
       [0073] 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.  
       [0074] 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.  
       [0075] 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.  
       [0076] 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.  
       [0077] 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.  
       [0078] 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.  
       [0079] 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 VEGF-C 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.  
       [0080] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding VEGF-C, 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 VEGF-C 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 VEGF-C in a sample may also be prepared.  
       [0081] 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.  
       [0082] 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.  
       [0083] 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. applications Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0084] 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.  
       [0085] 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.  
       [0086] 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.  
       [0087] 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.  
       [0088] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention. Emulsions The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in  Remington&#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.  
       [0089] 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).  
       [0090] 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).  
       [0091] 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.  
       [0092] 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).  
       [0093] 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.  
       [0094] 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.  
       [0095] 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.  
       [0096] 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).  
       [0097] 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.  
       [0098] 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 (PO 310 ), hexaglycerol pentaoleate (PO 500 ), 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 (C 8 -C 12 ) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C 8 -C 10  glycerides, vegetable oils and silicone oil.  
       [0099] 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.  
       [0100] 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.  
       [0101] Liposomes  
       [0102] 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.  
       [0103] 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.  
       [0104] 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.  
       [0105] 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.  
       [0106] 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.  
       [0107] 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.  
       [0108] 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.  
       [0109] 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).  
       [0110] 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).  
       [0111] 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.  
       [0112] 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).  
       [0113] 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).  
       [0114] 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).  
       [0115] 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.).  
       [0116] 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 Bi). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0117] 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.  
       [0118] 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.  
       [0119] 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).  
       [0120] 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.  
       [0121] 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.  
       [0122] 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.  
       [0123] 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.  
       [0124] 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).  
       [0125] Penetration Enhancers  
       [0126] 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.  
       [0127] 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.  
       [0128] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,  J. Pharm. Pharmacol.,  1988, 40, 252).  
       [0129] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1-10  alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; El Hariri et al.,  J. Pharm. Pharmacol.,  1992, 44, 651-654).  
       [0130] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman &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).  
       [0131] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett,  J. Chromatogr.,  1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; Buur et al.,  J. Control Rel.,  1990, 14, 43-51).  
       [0132] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,  J. Pharm. Pharmacol.,  1987, 39, 621-626).  
       [0133] 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.  
       [0134] 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.  
       [0135] Carriers  
       [0136] 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).  
       [0137] Excipients  
       [0138] 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.).  
       [0139] 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.  
       [0140] 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.  
       [0141] 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.  
       [0142] Other Components  
       [0143] 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.  
       [0144] 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.  
       [0145] 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.  
       [0146] 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.  
       [0147] 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.  
       [0148] 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  
     [0149] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0150] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham MA 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.  
     [0151] 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).  
     [0152] 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 MA) or prepared as follows:  
     [0153] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0154] 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.  
     [0155] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0156] 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).  
     [0157] 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.  
     [0158] 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.  
     [0159] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0160] 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.  
     [0161] 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.  
     [0162] [5′-O— (4,4′-Dimethoxytriphenylmethyl)-2 1 -deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0163] 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%).  
     [0164] 2′-Fluoro Amidites  
     [0165] 2′-Fluorodeoxyadenosine Amidites  
     [0166] 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.  
     [0167] 2′-Fluorodeoxyguanosine  
     [0168] 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.  
     [0169] 2′-Fluorouridine  
     [0170] 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.  
     [0171] 2′-Fluorodeoxycytidine  
     [0172] 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.  
     [0173] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0174] 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).  
     [0175] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0176] 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% diner (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.  
     [0177] 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.).  
     [0178] 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.  
     [0179] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0180] 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.  
     [0181] 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 fractions 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.  
     [0182] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0183] 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%).  
     [0184] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0185] 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  
     [0186] 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.  
     [0187] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0188] 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%.  
     [0189] 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)  
     [0190] 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%).  
     [0191] 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)  
     [0192] 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%).  
     [0193] 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)  
     [0194] 5′-(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%).  
     [0195] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(Dimethylaminooxyethyl) Nucleoside Amidites  
     [0196] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0197] 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.  
     [0198] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0199] 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 (3×2 00 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.  
     [0200] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0201] 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.  
     [0202] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0203] 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 P205 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.  
     [0204] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0205] 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.  
     [0206] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0207] 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.  
     [0208] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0209] 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.  
     [0210] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0211] 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.  
     [0212] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0213] 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.  
     [0214] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0215] 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.  
     [0216] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0217] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2 1 -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].  
     [0218] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0219] 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.  
     [0220] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0221] 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.  
     [0222] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0223] 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-C1, 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.  
     [0224] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0225] 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  
     [0226] Oligonucleotide Synthesis  
     [0227] 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.  
     [0228] 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.  
     [0229] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0230] 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.  
     [0231] 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.  
     [0232] 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.  
     [0233] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0234] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0235] 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  
     [0236] Oligonucleoside Synthesis  
     [0237] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligo-nucleosides, also identified as amide-4 linked oligonucleo-sides, 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.  
     [0238] 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.  
     [0239] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0240] PNA Synthesis  
     [0241] 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 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  
     [0242] Synthesis of Chimeric Oligonucleotides  
     [0243] 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”.  
     [0244] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0245] 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.  
     [0246] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0247] [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.  
     [0248] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0249] [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.  
     [0250] 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  
     [0251] Oligonucleotide Isolation  
     [0252] 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  
     [0253] Oligonucleotide Synthesis—96 Well Plate Format  
     [0254] 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.  
     [0255] 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  
     [0256] Oligonucleotide Analysis—96-Well Plate Format  
     [0257] 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  
     [0258] 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  
     [0272] Analysis of Oligonucleotide Inhibition of VEGF-C Expression  
     [0273] Antisense modulation of VEGF-C expression can be assayed in a variety of ways known in the art. For example, VEGF-C 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.  
     [0274] Protein levels of VEGF-C 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 VEGF-C 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).  
     [0275] 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  
     [0276] Poly(A)+ mRNA Isolation  
     [0277] 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.  
     [0278] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0279] Total RNA Isolation  
     [0280] 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.  
     [0281] 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  
     [0282] Real-Time Quantitative PCR Analysis of VEGF-C mRNA Levels  
     [0283] Quantitation of VEGF-C 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.  
     [0284] 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.  
     [0285] 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).  
     [0286] 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).  
     [0287] 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.  
     [0288] Probes and primers to human VEGF-C were designed to hybridize to a human VEGF-C sequence, using published sequence information (GenBank accession number NM — 005429.1, incorporated herein as SEQ ID NO:4). For human VEGF-C the PCR primers were: forward primer: TCAGGCAGCGAACAAGACCT (SEQ ID NO: 5) reverse primer: TTCCTGAGCCAGGCATCTG (SEQ ID NO: 6) and the PCR probe was: FAM-CCCCACCAATTACATGTGGAATAATCACATCT-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) 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  
     [0289] Northern Blot Analysis of VEGF-C 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 VEGF-C, a human VEGF-C specific probe was prepared by PCR using the forward primer TCAGGCAGCGAACAAGACCT (SEQ ID NO: 5) and the reverse primer TTCCTGAGCCAGGCATCTG (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  
     [0293] Antisense Inhibition of Human VEGF-C Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap  
     [0294] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human VEGF-C RNA, using published sequences (GenBank accession number NM — 005429.1, incorporated herein as SEQ ID NO: 4, GenBank accession number AF020393.1, incorporated herein as SEQ ID NO: 11, GenBank accession number AI342741.1, incorporated herein as SEQ ID NO: 12, and residues 852000-911000 of GenBank accession number NT — 006118.4, incorporated herein as SEQ ID NO: 13). 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 VEGF-C mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which T-24 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 VEGF-C mRNA levels by chimeric           phosphorothioate oligonucleotides having 2′-MOE wings and a       deoxy gap                                                         TARGET               SEQ   CONTROL                   SEQ ID   TARGET       %   ID   SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   NO   NO                                                         158101   Coding   4   1257   gcaggccgaagccccgctct   51   14   2                   158102   Coding   4   1597   tcttttccaatatgaaggga   52   15   2               158103   5′UTR   4   194   ggctccgcgttcccaacttt   60   16   2               158104   3′UTR   4   1787   cttttgcagatgagctccag   80   17   2               158105   Coding   4   513   ttgcttgcataagccgtggc   72   18   2               158106   Coding   4   1203   ttgtttggtccacagatgtc   70   19   2               158107   Coding   4   1028   taataatggaatgaacttgt   51   20   2               158108   Coding   4   533   gtaactgctcctccagatct   69   21   2               158109   3′UTR   4   1773   ctccagtccatttctgtaaa   42   22   2               158110   5′UTR   4   30   ggtgtagctttttggagagg   51   23   2               158111   Coding   4   1520   tcgtacatggccgtctgtaa   79   24   2               158112   Coding   4   1602   tgtggtcttttccaatatga   57   25   2               158113   Coding   4   510   cttgcataagccgtggcctc   69   26   2               158114   Coding   4   1184   catggaatccatctgttgag   38   27   2               158115   3′UTR   4   1825   gtttggtcattggcagaaaa   65   28   2               158116   Coding   4   1532   ccttctggcggttcgtacat   46   29   2               158117   Coding   4   1319   gtttgtttttacagacacac   67   30   2               158118   Coding   4   1071   ttcgctgcctgacactgtgg   52   31   2               158119   Coding   4   1576   acaacgacacacttottcac   25   32   2               158120   Coding   4   1010   gtctgtaaacatccagttta   74   33   2               158121   3′UTR   4   1972   acatattttgcatgatataa   50   34   2               158122   3′UTR   4   1938   gtgagttttaccaattgttg   73   35   2               158123   3′UTR   4   1699   caagggtctctctgttcaca   54   36   2               158124   5′UTR   4   127   gtaaaagcctcacaggaaac   61   37   2               158125   Coding   4   1555   atatgaaaatcctggctcac   15   38   2               158126   Coding   4   825   gacacacatggaggtttaaa   72   39   2               158127   Coding   4   743   attgagtctttctccactca   75   40   2               158128   Coding   4   1136   aatcttcctgagccaggcat   35   41   2               158129   3′UTR   4   1780   agatgagctccagtccattt   34   42   2               158130   3′UTR   4   1831   ttggctgtttggtcattggc   74   43   2               158131   Start   4   362   gcaagtgcatggtggaagga   81   44   2           Codon               158132   3′UTR   4   1905   gaatgcagaaacaatatttt   25   45   2               158133   3′UTR   4   1867   tagtcattcttttaaagaaa   34   46   2               158134   3′UTR   4   1832   cttggctgtttggtcattgg   75   47   2               158135   3′UTR   4   1840   aggaaaatcttggctgtttg   55   48   2               158136   Coding   4   615   cctttccttagctgacactt   45   49   2               158137   Coding   4   1005   taaacatccagtttagacat   32   50   2               196823   5′UTR   4   138   gcgggtgtcaggtaaaagcc   70   51   2               196824   5′UTR   4   282   aggccgcgggcccctcctgg   62   52   2               196825   Start   4   372   aagaagcccagcaagtgcat   78   53   2           Codon               196826   Coding   4   548   cactggacacagaccgtaac   88   54   2               196827   Coding   4   663   tctgtccttgagttgaggtt   73   55   2               196828   Coding   4   717   atacttttcaagatctctgt   79   56   2               196829   Coding   4   723   ttatcaatacttttcaagat   38   57   2               196830   Coding   4   856   actattgcagcaacccccac   78   58   2               196831   Coding   4   1049   gtgttgctggcagggaacgt   78   59   2               196832   Coding   4   1163   catctccagcatccgaggaa   11   60   2               196833   Coding   4   1215   tcatccagctccttgtttgg   33   61   2               196834   Coding   4   1268   gtccacagctggcaggccga   62   62   2               196835   Coding   4   1388   ttcttttacatacacactgg   66   63   2               196836   Coding   4   1438   acattcacaggcacattttc   74   64   2               196837   Coding   4   1487   ggtggtggaacttctttcct   77   65   2               196838   Stop   4   1620   gtacaatcttagctcatttg   80   66   2           Codon               196839   3′UTR   4   1683   cacagacagttctactgtgg   76   67   2               196840   3′UTR   4   1878   aataaattatatagtcattc   0   68   2               196841   3′UTR   4   1926   aattgttgttgctataaaaa   2   69   2               196842   5′UTR   11   3   agttgcctgatgatccaaga   27   70   2               196843   5′UTR   11   156   tttatcctcggccactcccg   32   71   2               196844   5′UTR   11   233   tttagaggtgatgcgaccac   9   72   2               196845   5′UTR   11   389   ctccctggagctccccgttt   55   73   2               196846   5′UTR   11   408   actctccctcggaagccgtc   0   74   2               196847   5′UTR   11   684   ccttccccgaagtgagagga   68   75   2               196848   intron   12   2   tgacgaaattgttaaaaggt   11   76   2               196849   intron   12   33   atttcagactgaaatacaat   22   77   2               196850   exon:   13   2359   gcagacctaccgtggcctcg   82   78   2           intron                                   junction                                       196851   exon:   13   12230   ccatacttacttttcaagat   9   79   2           intron                                   junction                                       196852   exon:   13   13985   caatacccaccgtcttgctg   76   80   2           intron                                   junction                                       196853   intron   13   16405   acacattttgtacaggtatc   89   81   2               196854   intron   13   16859   aggaaacacgatgatgccca   70   82   2               196855   intron   13   22515   ggagaactttgaagcagttt   48   83   2               196856   exon:   13   30058   tcatactcactgtggtagtg   45   84   2           intron           junction               196857   intron   13   41999   attctttcattgtcagagct   66   85   2                  
 
     [0295] As shown in Table 1, SEQ ID NOs 16, 17, 18, 19, 21, 24, 25, 26, 28, 30, 33, 35, 37, 39, 40, 43, 44, 47, 48, 51, 52, 53, 54, 55, 56, 58, 59, 62, 63, 64, 65, 66, 67, 73, 75, 78, 80, 81, 82 and 85 demonstrated at least 55% inhibition of human EGF-C 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           n VEGF-C.                                                 TARGET           REV COMP                   SITE   SEQ ID   TARGET       OF SEQ       SEQ ID       ID   NO   SITE   SEQUENCE   ID   ACTIVE IN   NO                                                     73604   4   194   aaagttgggaacgcggagcc   16     H. sapiens     86                   73605   4   1787   ctggagctcatctgcaaaag   17     H. sapiens     87               73606   4   513   gccacggcttatgcaagcaa   18     H. sapiens     88               73607   4   1203   gacatctgtggaccaaacaa   19     H. sapiens     89               73609   4   533   agatctggaggagcagttac   21     H. sapiens     90               73612   4   1520   ttacagacggccatgtacga   24     H. sapiens     91               73613   4   1602   tcatattggaaaagaccaca   25     H. sapiens     92               73614   4   510   gaggccacggcttatgcaag   26     H. sapiens     93               73616   4   1825   ttttctgccaatgaccaaac   28     H. sapiens     94               73618   4   1319   gtgtgtctgtaaaaacaaac   30     H. sapiens     95               73621   4   1010   taaactggatgtttacagac   33     H. sapiens     96               73623   4   1938   caacaattggtaaaactcac   35     H. sapiens     97               73625   4   127   gtttcctgtgaggcttttac   37     H. sapiens     98               73627   4   825   tttaaacctccatgtgtgtc   39     H. sapiens     99               73628   4   743   tgagtggagaaagactcaat   40     H. sapiens     100               73631   4   1831   gccaatgaccaaacagccaa   43     H. sapiens     101               73632   4   362   tccttccaccatgcacttgc   44     H. sapiens     102               73635   4   1832   ccaatgaccaaacagccaag   47     H. sapiens     103               73636   4   1840   caaacagccaagattttcct   48     H. sapiens     104               114955   4   138   ggcttttacctgacacccgc   51     H. sapiens     105               114956   4   282   ccaggaggggcccgcggcct   52     H. sapiens     106               114957   4   372   atgcacttgctgggcttctt   53     H. sapiens     107               114958   4   548   gttacggtctgtgtccagtg   54     H. sapiens     108               114959   4   663   aacctcaactcaaggacaga   55     H. sapiens     109               114960   4   717   acagagatcttgaaaagtat   56     H. sapiens     110               114962   4   856   gtgggggttgctgcaatagt   58     H. sapiens     111               114963   4   1049   acgttccctgccagcaacac   59     H. sapiens     112               114966   4   1268   tcggcctgccagctgtggac   62     H. sapiens     113               114967   4   1388   ccagtgtgtatgtaaaagaa   63     H. sapiens     114               114968   4   1438   gaaaatgtgcctgtgaatgt   64     H. sapiens     115               114969   4   1487   aggaaagaagttccaccacc   65     H. sapiens     116               114970   4   1620   caaatgagctaagattgtac   66     H. sapiens     117               114971   4   1683   ccacagtagaactgtctgtg   67     H. sapiens     118               114977   11   389   aaacggggagctccagggag   73     H. sapiens     119               114979   11   684   tcctctcacttcggggaagg   75     H. sapiens     120               114982   13   2359   cgaggccacggtaggtctgc   78     H. sapiens     121               114984   13   13985   cagcaagacggtgggtattg   80     H. sapiens     122               114985   13   16405   gatacctgtacaaaatgtgt   81     H. sapiens     123               114986   13   16859   tgggcatcatcgtgtttcct   82     H. sapiens     124               114989   13   41999   agctctgacaatgaaagaat   85     H. sapiens     125                  
 
     [0296] 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 VEGF-C.  
     [0297] 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 VEGF-C 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 VEGF-C 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 VEGF-C. Once it is shown that the candidate antisense compound or compounds are capable of inhibiting the expression of a nucleic acid molecule encoding VEGF-C, the candidate antisense compound may be employed as an antisense compound in accordance with the present invention.  
     [0298] 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  
     [0299] Western Blot Analysis of VEGF-C Protein Levels  
     [0300] 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 VEGF-C 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.).  
    
     
       
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tccgtcatcg ctcctcaggg                                                 20 

 
           
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gtgcgcgcga gcccgaaatc                                                 20 

 
           
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atgcattctg cccccaagga                                                 20 

 
           
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             H. sapiens  
             
 
             
               CDS  
               (372)...(1631)  
             
           
            4 

cgcggggtgt tctggtgtcc cccgccccgc ctctccaaaa agctacaccg acgcggaccg     60 

cggcggcgtc ctccctcgcc ctcgcttcac ctcgcgggct ccgaatgcgg ggagctcgga    120 

tgtccggttt cctgtgaggc ttttacctga cacccgccgc ctttccccgg cactggctgg    180 

gagggcgccc tgcaaagttg ggaacgcgga gccccggacc cgctcccgcc gcctccggct    240 

cgcccagggg gggtcgccgg gaggagcccg ggggagaggg accaggaggg gcccgcggcc    300 

tcgcaggggc gcccgcgccc ccacccctgc ccccgccagc ggaccggtcc cccacccccg    360 

gtccttccac c atg cac ttg ctg ggc ttc ttc tct gtg gcg tgt tct ctg     410 
             Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu 
               1               5                  10 

ctc gcc gct gcg ctg ctc ccg ggt cct cgc gag gcg ccc gcc gcc gcc      458 
Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala 
     15                  20                  25 

gcc gcc ttc gag tcc gga ctc gac ctc tcg gac gcg gag ccc gac gcg      506 
Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala 
 30                  35                  40                  45 

ggc gag gcc acg gct tat gca agc aaa gat ctg gag gag cag tta cgg      554 
Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln Leu Arg 
                 50                  55                  60 

tct gtg tcc agt gta gat gaa ctc atg act gta ctc tac cca gaa tat      602 
Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro Glu Tyr 
             65                  70                  75 

tgg aaa atg tac aag tgt cag cta agg aaa gga ggc tgg caa cat aac      650 
Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn 
         80                  85                  90 

aga gaa cag gcc aac ctc aac tca agg aca gaa gag act ata aaa ttt      698 
Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe 
     95                 100                 105 

gct gca gca cat tat aat aca gag atc ttg aaa agt att gat aat gag      746 
Ala Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu 
110                 115                 120                 125 

tgg aga aag act caa tgc atg cca cgg gag gtg tgt ata gat gtg ggg      794 
Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly 
                130                 135                 140 

aag gag ttt gga gtc gcg aca aac acc ttc ttt aaa cct cca tgt gtg      842 
Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val 
            145                 150                 155 

tcc gtc tac aga tgt ggg ggt tgc tgc aat agt gag ggg ctg cag tgc      890 
Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys 
        160                 165                 170 

atg aac acc agc acg agc tac ctc agc aag acg tta ttt gaa att aca      938 
Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr 
    175                 180                 185 

gtg cct ctc tct caa ggc ccc aaa cca gta aca atc agt ttt gcc aat      986 
Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn 
190                 195                 200                 205 

cac act tcc tgc cga tgc atg tct aaa ctg gat gtt tac aga caa gtt     1034 
His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val 
                210                 215                 220 

cat tcc att att aga cgt tcc ctg cca gca aca cta cca cag tgt cag     1082 
His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln 
            225                 230                 235 

gca gcg aac aag acc tgc ccc acc aat tac atg tgg aat aat cac atc     1130 
Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile 
        240                 245                 250 

tgc aga tgc ctg gct cag gaa gat ttt atg ttt tcc tcg gat gct gga     1178 
Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly 
    255                 260                 265 

gat gac tca aca gat gga ttc cat gac atc tgt gga cca aac aag gag     1226 
Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu 
270                 275                 280                 285 

ctg gat gaa gag acc tgt cag tgt gtc tgc aga gcg ggg ctt cgg cct     1274 
Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro 
                290                 295                 300 

gcc agc tgt gga ccc cac aaa gaa cta gac aga aac tca tgc cag tgt     1322 
Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln Cys 
            305                 310                 315 

gtc tgt aaa aac aaa ctc ttc ccc agc caa tgt ggg gcc aac cga gaa     1370 
Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn Arg Glu 
        320                 325                 330 

ttt gat gaa aac aca tgc cag tgt gta tgt aaa aga acc tgc ccc aga     1418 
Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg 
    335                 340                 345 

aat caa ccc cta aat cct gga aaa tgt gcc tgt gaa tgt aca gaa agt     1466 
Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser 
350                 355                 360                 365 

cca cag aaa tgc ttg tta aaa gga aag aag ttc cac cac caa aca tgc     1514 
Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys 
                370                 375                 380 

agc tgt tac aga cgg cca tgt acg aac cgc cag aag gct tgt gag cca     1562 
Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro 
            385                 390                 395 

gga ttt tca tat agt gaa gaa gtg tgt cgt tgt gtc cct tca tat tgg     1610 
Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp 
        400                 405                 410 

aaa aga cca caa atg agc taa gattgtactg ttttccagtt catcgatttt        1661 
Lys Arg Pro Gln Met Ser 
    415 

ctattatgga aaactgtgtt gccacagtag aactgtctgt gaacagagag acccttgtgg   1721 

gtccatgcta acaaagacaa aagtctgtct ttcctgaacc atgtggataa ctttacagaa   1781 

atggactgga gctcatctgc aaaaggcctc ttgtaaagac tggttttctg ccaatgacca   1841 

aacagccaag attttcctct tgtgatttct ttaaaagaat gactatataa tttatttcca   1901 

ctaaaaatat tgtttctgca ttcattttta tagcaacaac aattggtaaa actcactgtg   1961 

atcaatattt ttatatcatg caaaatatgt ttaaaataaa atgaaaattg tatt         2015 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

tcaggcagcg aacaagacct                                                 20 

 
           
             6  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

ttcctgagcc aggcatctg                                                  19 

 
           
             7  
             32  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

ccccaccaat tacatgtgga ataatcacat ct                                   32 

 
           
             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  
             1127  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (1125)...(1127)  
             
           
            11 

gttcttggat catcaggcaa ctttcaacta cacagaccaa gggagagagg ggacccctcc     60 

gaggtcccat agggttctct gacatagtga tgaccttttt ccaaactttg agcagggcgc    120 

tgggggccag gcgtgcggga gggaggacaa gaactcggga gtggccgagg ataaagcggg    180 

ggctccctcc accccacggt gcccagtttc tccccgctgc acgtggtcca gggtggtcgc    240 

atcacctcta aagccggtcc cgccaaccgc cagccccggg actgaacttg cccctccggc    300 

cgcccgctcc ccgcagggga caggggcggg gagggagaga tccagagggg ggctggggga    360 

ggtggggccg ccggggagga ggcgagggaa acggggagct ccagggagac ggcttccgag    420 

ggagagtgag aggggagggc agcccgggct cggcacgctc cctccctcgg ccgctttctc    480 

tcacataagc gcaggcagag ggcgcgtcag tcatgccctg cccctgcgcc cgccgccgcc    540 

gccgccgccg ctcagcccgg cgcgctctgg aggatcctgc gccgcggcgc tcccgggccc    600 

cgccgccgcc agccgccccg gcggccctcc tcccgccccc ggcaccgccg ccagcgcccc    660 

cgccgcagcg cccgcggccc ggctcctctc acttcgggga aggggaggga ggagggggac    720 

gagggctctg gcgggtttgg aggggctgaa catcgcgggg tgttctggtg tcccccgccc    780 

cgcctctcca aaaagctaca ccgacgcgga ccgcggcggc gtcctccctc gccctcgctt    840 

cacctcgcgg gctccgaatg cggggagctc ggatgtccgg tttcctgtga ggcttttacc    900 

tgacacccgc cgcctttccc cggcactggc tgggagggcg ccctgcaaag ttgggaacgc    960 

ggagccccgg acccgctccc gccgcctccg gctcgcccag ggggggtcgc cgggaggagc   1020 

ccgggggaga gggaccagga ggggcccgcg gcctcgcagg ggcgcccgcg cccccacccc   1080 

tgcccccgcc agcggaccgg tcccccaccc ccggtccttc caccatg                 1127 

 
           
             12  
             409  
             DNA  
             H. sapiens  
             
 
           
            12 

aaccttttaa caatttcgtc ataaaatgag taattgtatt tcagtctgaa atttaaaaac     60 

acagaaatac cttggtagca tgatgaatcc attgccttga tctttaacgt aagtgtgttc    120 

ttgtttgttc tcctagctgt tacagacggc catgtacgaa ccgccagaag gcttgtgagc    180 

caggattttc atatagtgaa gaagtgtgtc gttgtgtccc ttcatattgg aaaagaccac    240 

aaatgagcta agattgtact gttttccagt tcatcgattt tctattatgg aaaactgtgt    300 

tgccacagta gaactgtctg tgaacagaga gacccttgtg ggtccatgct aacaaagaca    360 

aaagtctgtc tttcctgaac catgtggata actttacaga aatggactg                409 

 
           
             13  
             59001  
             DNA  
             Homo sapiens  
             
               misc_feature  
               10057-10156  
               n = A,T,C or G  
             
           
            13 

gagtctgatg ggatggaatt tcataaagat acataaaaaa gcatcttgga tacagtaaac     60 

ttaactccac aaatacaggg gaatttagac gtgactaagt agcagtacat atgaaaaatt    120 

attgaggaat tttgttgact ttaagggtag tgtgagtcaa cactgtgatt tggctgccag    180 

aaaataaact caatccaagg ctgtatcaac aaaggcatac tgtccattct gcatgctcat    240 

tacagcacta agtaccgagc catgttctca accgcatact tcatgaacat ggaaagctaa    300 

cagtatggtt aaggggggaa actggaactg tcatcttggg gaataaaagg gatatttagc    360 

caggagtaaa gttagcttag ggagaccatg ataaatattt tcaaaatatt tgaaggactc    420 

agttgtggaa gtgagattag atttattgtg taaaactcca ggagtcaaaa gcaatagaga    480 

gatagaagga aatgcttttc agcagtgttg ctcatcaata aagggagtga acagccacac    540 

agaatggaag gttccctgtc ctttgagata tttaagcctt caagtaaatt atgggtgagg    600 

agtttcaaat ctagagttga accagataag aaagtctctt cttccggtaa gatattatgg    660 

acctataaca tctgtgtact taaaagtaga ttgggagtga aaggcagact tttgatgttc    720 

tgtacactgt tgaaacccct tagcgtggtc ctctgtaacc tgctcaccct gccccaagga    780 

ggcagctagc caatgccacc agcccaacgg aaaccccagt gcttttccaa tggggaaatg    840 

cagtcacttt tctttggatg ctacacatcc tttctggaat atgtctcaca cacatctctc    900 

tttatcaccc cctttttcaa gtaaaccaac ttcttgcaga agctgacaat gtgtctcttt    960 

actctccacg aagattctgg cccttctctt cacctgtcag aagtttagga ttccaaaggg   1020 

atcattagca tccatcccaa cagcctgcac tgcatcctga gaactgcggt tcttggatca   1080 

tcaggcaact ttcaactaca cagaccaagg gagagagggg acccctccga ggtcccatag   1140 

ggttctctga catagtgatg accttttctt ggaactttta caacccccag gacatttcca   1200 

aactttgagc agggctctgg gggccaggcg tgcgggaggg aggacaagaa ctcgggagtg   1260 

gccgaggata aagcgggggc tccctccacc ccacggtgcc cagtttctcc ccgctgcacg   1320 

tggtccaggg tggtcgcatc acctctaaag ccggtcccgc caaccgccag ccccgggact   1380 

gaacttgccc ctccggccgc ccgctccccg caggggacag gggcggggag ggagagatcc   1440 

agaggggggc cgggggaggt ggggccgccg gggaggaggc gagggaaacg gggagctcca   1500 

gggagacggc ttccgaggga gagtgagagg ggagggcagc ccgggctcgg cacgctccct   1560 

ccctcggccg ctttctctca cataagcgca ggcagagggc gcgtcagtca tgccctgccc   1620 

ctgcgcccgc cgccgccgcc gccgccgctc agcccggcgc gctctggagg atcctgcgcc   1680 

gcggcgctcc cgggccccgc cgccgccagc cgccccgccg ccctcctccc gcccccggca   1740 

ccgccgccag cgcccccgcc gcagcgcccg cggcccggct cctctcactt cggggaaggg   1800 

gagggaggag ggggacgagg gctctggcgg gtttggaggg gctgaacatc gcggggtgtt   1860 

ctggtgtccc ccgccccgcc tctccaaaaa gctacaccga cgcggaccgc ggcggcgtcc   1920 

tccctcgccc tcgcttcacc tcgcgggctc cgaatgcggg gagctcggat gtccggtttc   1980 

ctgtgaggct tttacctgac acccgccgcc tttccccggc actggctggg agggcgccct   2040 

gcaaagttgg gaacgcggag ccccggaccc gctcccgccg cctccggctc gcccaggggg   2100 

gggtcgccgg gaggagcccg ggggagaggg accaggaggg gcccgcggcc tcgcaggggc   2160 

gcccgcgccc ccacccctgc ccccgccagc ggaccggtcc cccacccccg gtccttccac   2220 

catgcacttg ctgggcttct tctctgtggc gtgttctctg ctcgccgctg cgctgctccc   2280 

gggtcctcgc gaggcgcccg ccgccgccgc cgccttcgag tccggactcg acctctcgga   2340 

cgcggagccc gacgcgggcg aggccacggt aggtctgcgt tagggtttgc ggagaacccg   2400 

agagtttgcg ttagggtctg cggcggaccc gagaacctgc gcgggggaaa gtgtgtgtgc   2460 

tttaagcttg tgtacgtggg atccaaagtt actgagctca gtgcacgctg ctttggagaa   2520 

aaatcttctt ctttttaaat agaaagttgt tactagagag gcaagcaagt tacacgagtg   2580 

aagggcccgg agaggtgccc agtgagagat cccgaaatct atttcagact ggtttcctct   2640 

ggggcaacca aggggtcttg aaccctgccc agtcagcggg gctctggaga gtatgagttc   2700 

attttggtcg ggaaatgctc gtttctttcc ccagctgatt catgggactc caaacagatt   2760 

ctgggacact ggtgatcagt caacccagcc ttactttcct ggagtgttca tagtctgcag   2820 

agcaccaggc gctgtgagcg actttagaaa aaaagtgtca gggactttag taaccaggct   2880 

ccagagcttt cagagttcac ttgaagttgg tcagacttga gatgtaacgg gagattagag   2940 

tcagttagat cacaatcaag caatgcaagg cttgtttcaa ctttataagc tcttcattcc   3000 

taaaatctgg tcatgagata ctgcaaacta agtttttttt tttcttgttt gggttttttg   3060 

tttgttttgt tttcttttaa ataagaggtg tttaatcctt tgcctgaaaa tgttgccaaa   3120 

atacttttga ggcatcctga ttttgaaaaa ggatttgtgt gtgttcctac ttcttactgg   3180 

ctcttccaaa agagtatatc tttatctaaa aagttcatac ctgccataac catatagagt   3240 

atgcttaaga gagttcctaa agaagttata ttcgacattt cagttcaaac acttgcagta   3300 

ccccttgctg gaactatact gcggtagttt atttcaactg gtggcaagtg gagaggtacc   3360 

tgtggtgtgt tacagtcaac tttaatttga ctgttgatta acacatacac aatgtgtaga   3420 

aaatagcctt atattttgaa atattttatt tatatttata ttttgaaata cacttaaaga   3480 

ttgagaagac taatttttgg aaatcaaact acctttgcat ttaaattttg ggaaaacatt   3540 

aaaatgttgg aatgtgtaat aatttaatat aggggttaaa ggaatgcctc ttgagtaaaa   3600 

acaatataca tgaaatagaa cagactgcat tctgtgaatc acaaaaataa ttttcagtgc   3660 

ctatacttac attgccgtaa ctatagtgat gaaaatatct ttctgttctt aaataccgca   3720 

gaaatgtaat aatcggctca ataacgcttc tgataatttg agtccttgat tttgcagtac   3780 

ctatttgcta tttctgcaaa gtcaaaacta gtaaaagtat gatttagtaa ggcaacatct   3840 

ctgtattggt tacggtcact tactttggtt tacagtaaga aaatgtgcag tagagagtaa   3900 

aatgagaaac attccctgat aagccaatag cctgtatgag agtcactcac tgctgggtga   3960 

gtctagaatt aatgtacctt gagtgtgtgt gtgtgtgtgt ttacgtgtgt tatgtatagg   4020 

tatgggattg gaggattaaa aaagttaact tatttttaac tttcaaaagt gtcttcgttg   4080 

gtgatgtaaa atttgccatt ggtgatatgg caaattttac cattgctgtg aaaatcacta   4140 

tgcatttttg tttgcaaatg tcgcttttca tatttaacca tttttatatt ctgtatggtg   4200 

aatatgggtt agtattctgc atgtgatgtg ggttcctttc ctatgtgatg aaacatcatt   4260 

tcccatatga aacatcatct ctgagttcca tagaccacaa agcagtattt cattgctcca   4320 

ttaaagtgtc gttccccagt gggtgaatag agccttgaca gggcatgtgt cattaatgca   4380 

ttcagtgaaa ctgctgtgtc cttgctatga aacatgtcac acaactgcca agaaactgat   4440 

atgaagactc tatgtctctc cctagtggcc tgtacaagca ccacatttag atttaagcac   4500 

tagagaagaa ctggggagaa atcaattgtt aattttctat aactacatga accccaagaa   4560 

aatatttcag aaggtaaaca gaagatgcta aatatctata atcccaatag atgcaatgtt   4620 

ttcataactt tcacctagga acttttttaa tatttaaaaa atcaaaaata gcacacaaac   4680 

tatgaaccaa ggttagccat ccagcccttc agcccacccg taaagccatg caaaagattg   4740 

taggtaaaaa tcacaaatag aatgaagatc tccttattcc tcgacttata tttgcattta   4800 

ctggattcct atttctagtt atttgtggtt gcatgactta ctgaaaagtc ttaaaatgtt   4860 

tctctgaagg atgttaacac aaacaagaca gtgtcaaaaa agtaaggcag gcaaaagtaa   4920 

aattaactgg acatgatggg aacaaaagag aatagcaagt ctgaagctgt tctatttaca   4980 

atttttctta tagtatatga ttatgtttgt caacatctac tcttataaga gatttctgac   5040 

attcctctga gtgatgctac ttatatgagg ttttagatat agaaagttca attcatcttt   5100 

cattttgatt tccactccat tatagtcttc tgaactatga atatctgatt gatagctatt   5160 

tattcacaag acagttatca aaattgcttc atagaaagca gttaattact gatcttagtg   5220 

gcatatgtct aagacccgta gatctctaat cctgtccttt tcagttgagt tgagaaagac   5280 

ggagggtagt ggatttagat ttcattttgg acttccagtt ctggttccgg ttcctcttga   5340 

gaggaaggtc ctggcctcct agttatctcc atgtgtccaa cccagaatgc tgtttctcag   5400 

ctcttgctgg ggttttcacc aacagaggaa ctggcgtgag actaatgaag gtttcccgaa   5460 

tccctgggac tgtgcttgcc tgcttgcctt tggcagtcct ggggttgtta ttgagagtag   5520 

gagtttataa tcattatctg tgatgttcat aacgtgtgtt aagcacttcg caaagagctt   5580 

tgcaagttag ttttgttttt cactacacct ccataatatg gacattattt tatttcatta   5640 

aggatgatga acccaagatc tgaatgactt aaccaggaaa aagaatagat gtttttttcc   5700 

tttttttctt ttaaaaaaag ttttatggca tataccaaca gtccccgact tagaatggtt   5760 

ctatttagga cttttggact ttacaatggt gcaaagtcga tagacattca gtagaaacca   5820 

tacttcaagg tttgaatttt gatcttttcc tgggctaata atatgcagta aaatactttc   5880 

tctggatgct ggacaatgga agtgagctgt gaccagtcag ccatgtgatc gtgagagtaa   5940 

acaacaggta ctctactgta tattaaatat attttcaact caggaggaat ttgtcaggat   6000 

gtagccccat cgtaaatcga gaagcatgta tgttttagag aaatgtggta atgggtttgg   6060 

gcctttgggc atgtgattca gactcctttc agcccctctg ctggtctcct ttggcacact   6120 

atagcagcgt gcaggaaggc agagaaaagc actgcgtgtg actttgcagt taggggcacc   6180 

ttgttgcatt tatttaggtc ctaattcccc acaacgggac aaacggaaat aaacccagac   6240 

tttcttccag gctttaccac atggtgttcg tttgactaaa gataaattat aaatgaccta   6300 

atccatggta atattataag aacgactgct taactggagg cttaacaaga ttttctgaca   6360 

aacggttttt caaagttgtg ctcaagtaac ttcagtgaaa gattgtgatt ttacctctgc   6420 

caaatgacct ttcacaaata agtggatttg gaaagaacaa atttaagttg gcagaagcgt   6480 

tatacttcct ctgacttctg agcatatatt gtttcacctt tcaccagtta cattattcct   6540 

tgtagatgtg cattttataa atactatatg ggtctacctt ttgttacaaa cacatatttg   6600 

tatgtacaga tttgtataaa ttgtggatta agttatcatt gatttttctc attagaaaaa   6660 

acagatatag cagagaactt taaacaagga gagatgctga tgagtttgag ttatagtgta   6720 

attataaaat ttactataaa tattttaggt ttgtgtaatt gtttttctga aacttactag   6780 

tgtaaatttt tggggtggct tctttcctcg gttgattaat tttttttttc agagtttatg   6840 

tttaaccatg agaaagcaga gactgttttt atttaatttg ctttattggg catccatcat   6900 

ggaattaatc aaattgtaca ttaaatgctt tgtcagtgga ttagtactgt ttccttatac   6960 

tgctcgcgcc ctagtggctt tcaggtgtta ggagttatcc acgatagttt tttttccaat   7020 

tttgaaatcc aaaaactcat tttctttttc tttctttcct tttttttttt ttttttgaga   7080 

cagggtcatg ctctattgcc caggctggag tgcagtggca tgatgttggc tcactgcaac   7140 

ctctgcctcg tgggttcaag cgattctcgt gcctcagcct cccaagtagc tggaattaca   7200 

ggcatgtgcc actagagcag gctaattttt gtatttttca tagagagagg gttttgtctt   7260 

gtaggcgaag ctggtttcaa actcctgact tcaggtgatc cacttgatct cctaaagtgc   7320 

tgggattaca ggcatgagcc actgcgcccg gccccaaaag ctaattttct taaattattt   7380 

ggtgctataa tcctgatctg aactgacata aggctattta tagtcattat tatctaattt   7440 

aaatatttct aatcttcact gggaaaatag taatgtattt atttacatga tgctgccatc   7500 

agtcctgctt gtttttgggg tgtgagtatt aatatgtcta tgcacttggt ttccagaaaa   7560 

attccaggtt cagagactct tgcagtctcc aggcattttg gataagcaga tggattgtag   7620 

ccctgttcca caggccttcc tccattgctt tattgccttg gggctgcctg gaagaggcag   7680 

ggaaaggtaa tccaggaatt caagaaagaa gccatgtgga tgctcctaat tcctttttac   7740 

ccattagtac caggtttatt tgctctttaa tctacttcaa ctcatgttaa tcagttctta   7800 

ctagtggcag cttcagttct aagcaggaaa ttacagttca gtgtgtgatg caaaaaatgt   7860 

agagtgagtg atcttgtcta taacaagctt ataatcattt tggagaaaat aaatttactc   7920 

attaaaaata ctacaagtga attaaactat aaatccagac catgtaagtg gttttgtaag   7980 

agagtcttgt gagttaccac cagttgttgg ctagccttct gggagttgca tgggcaggcc   8040 

tgggaaaggt ggaatgatat gagacccaat gttaaaaatg agtagatgct aattacagaa   8100 

atgggggaga gagggatgtt ttgaatattg aagaatgaca gcaaagtaca gaggaagtaa   8160 

aggtcaggga tatttgggga actagaagca aaccctttgg tgcaaaagtc aagagcttta   8220 

agggaaactg aagggagcat ggtgggaagt gtgaaaacca aagagaaagt ctgagtttga   8280 

gttgaaagca gcaggagcca atgaagattc tttagtggta tgctgaaagt gaggttttta   8340 

aggagcttaa cctggcattg ttgcacagaa gtgtctaaga aagctgacca gaagcttctc   8400 

agaagcttct cagaagccat tttcaactgc agggtttctt aaagtgtctg gcagtgactc   8460 

tgcctttctc attacagtgg ccacccatat atggtattcg aactcctcta tatgctcctg   8520 

attgctggcg atgatagtgg tgattacttt tgacttactg taggcaccca tcattgtgct   8580 

atctggatac atttacaaaa gtatagaaat agacatctct gccattatgg tttgcattcc   8640 

aaacgaagca agcagtgtaa acagtggtca tgcttagata cacatacaca gaacccccca   8700 

cacacatatt catgcataca ccgcttaata gttcttcaga ctgccaagtg tgacttacgt   8760 

gttctttctt gcatggttaa gaatttgtag acagttcatt gaaatagaat atttagacaa   8820 

tcagaagtgt acaagattag aatctctata tttgcacaca catttatttg taggtcagtg   8880 

ggcaattgaa aaaaaagaga aggagagaac cctaaagtgt cctggaaatt cctgcttttt   8940 

aaaatctgct aaaaatgaga ccagaaaggt gggagtgggg atgtgaggag gtgggtaaac   9000 

taccaaataa acaatataaa tactttggct gttcttataa aaggtttttt taaatatggt   9060 

ggaataattt actcaaactc aagagatgcc acctactaga gaaaggacat actgaaagag   9120 

gaaatattca aatgcacaac tttgtgtaaa aggtaatact tacaagttta aaagactcac   9180 

ttctaaagaa gtttggcttc aacctgtcct catttggaca ggtggaaaga tgtatatttt   9240 

ggggtagacc agaaagatgt ggtttgactt ctatagttga aaggtttcta tttagacatt   9300 

tattttgtaa tttaatttac ctaaaacttc atccctaaat taccattttc tcttactttt   9360 

atgcaatagt aaagtatgca gtcataagtg ataaaggctg ggatcaaagc tagctatttt   9420 

gctaactgca gaacctatgc actctgttta cactgcctgt gatagcgcct ggaagaactc   9480 

actgccaata tttctgcttg tttcagtcac ataactgttg cttgtgtcag tgacgtaact   9540 

tgtttagaga tgcagtgcag tggataagag cacagggtat ggacatgcct ttctgtattc   9600 

atgtcctgag gatgcctctt agtacctgtg tgaccttgga caagttactt agtttctctc   9660 

tgcttctttg tagcacaatg agtagatgct aatttttaat ttttaatttt ttctttttct   9720 

ttgtgtctgt tttgtgtatt tagttacttt atcttttttt tttttttttt ttttttgaga   9780 

cagactctgg ctctgtagcc caggctggat ggaatgcagt ggtgctatct cagctcactg   9840 

caagctccgc ctcccaggtt cacgccattc tcctgcctca gcctcctgag tagctggaac   9900 

tacaggtgcc caccaccaca cccagctaat ttttttttta tattttttta gtagagacgg   9960 

ggtttgacca tgttagccag aatggtctca atctcctgac ctcgtgatct gcccgcctcg  10020 

gcctcccaaa gtgctgggat tacaggtgtg aaccacnnnn nnnnnnnnnn nnnnnnnnnn  10080 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  10140 

nnnnnnnnnn nnnnnncaat cacacacagg tccctattgc tccttttctt ctagggctat  10200 

tcaacaagca gagacaaaca aaaactgatc tgtcacattt tactgcaggt aacagtgacc  10260 

acaaatacac ctgtacactg ttttttttcc aacattattg aggtgtgact gacaaataaa  10320 

aatggtatat atttaaggta tgtgatgcga tgttttgctg tacatgtaca ttgtgaaatg  10380 

attatcataa tcatgttaac atatccatca cctcacatat tgttattttt ataatcactt  10440 

ttataaattt tcccttcagt agtaaccttt tgagattctg aaacactttt gagttttgaa  10500 

tgttgtgtca taagtgagtt tttttggcaa atattttata aacatctaat tgggaaactg  10560 

tgagaggatg ttttgtaact ttcatttaaa ctaagatctc ctctccacct ccacccttcc  10620 

catattcagt agattttcta tggctgtatg gtgtatcagg aatcatccca tatacttcag  10680 

aaagtaatgg aataatataa atatttttct aacatagacc tacagagatt aagtgatata  10740 

ctcaaatcat accataaatg gtagtgaaag ggtaaactca acattcacag ccaacttcat  10800 

tatctaatag ggaacaacac atttcatatt aataattgac ttttattttt aggaggctct  10860 

gttataatga attaaaaaat gagattagat tatgtttctc aatatatggt acaaaaatca  10920 

cttacataga ataacttcga ttattaaaaa tagaagtgaa aaatactttt cagattaaat  10980 

aagatttcaa ttttaaggca aatatgttca ttttgtgctc aaaaattaga caggaaaact  11040 

ataatctgtt aaaatgtagg ttcttgatcc acccaagaaa ccatggagag gtgggggaca  11100 

gttacctgca tttctgacag ttagttcatt gactcttatt cgactaaact ttgagaacca  11160 

gtgtccaaag tatttactta catatttata tttaaaagct gggataatca tgacaatata  11220 

aaataaacaa ttgatgttgt attttacttg gcagatagat aaagctatac aagtttcctt  11280 

tttcctaatt ctaaagcata attagaaaaa aattgtattt atttcttaga tatggtaact  11340 

actaaaatat aatttggaat gttctactta aatgagtata cttatgtatg cagttagccg  11400 

tcaataagat ttagtgttct ctgaactatt tcatttctat gaaggtggta ttaatcttga  11460 

tgataaaatt ttcttttaaa tattgagaga ttctgccaaa atataaatct tactgtgaca  11520 

atgttgttga aattcctttc ttcatatgat tatttgttct aaaatagctc ccatgaaagt  11580 

aaaattccta ctctcaagca tcctgcttaa gcatattgta ttgttgctgg taactaaaaa  11640 

taaagcaaag cattagggtg gatttcagtg taattttatc tgtttagtag agtgatacaa  11700 

atgaataaat tttcaaacat acaaattgac caattatttg aaagagctat gaataaaata  11760 

aataagctac cttaaaatta ttttaaaatt atatcacaaa atttttttcc taacaacaaa  11820 

atatgtattt ttaattgttt tggttcatac aaattaaaat acaaatataa ttttaccaag  11880 

gaaagtgaag taatttagga gtaattaata atatttgtgt tagggaacgg agcatagata  11940 

ctattaaacc taaccgtttt atagtgcatt tgtttctaaa tcccttaagc tggtaagtca  12000 

ttgatcttgc atttttcttt gacaggctta tgcaagcaaa gatctggagg agcagttacg  12060 

gtctgtgtcc agtgtagatg aactcatgac tgtactctac ccagaatatt ggaaaatgta  12120 

caagtgtcag ctaaggaaag gaggctggca acataacaga gaacaggcca acctcaactc  12180 

aaggacagaa gagactataa aatttgctgc agcacattat aatacagaga tcttgaaaag  12240 

taagtatggg aaataaaatg tatagtaagc cttatatatc aaaccagtag aagttgccag  12300 

cctggttctt taatttcagt gtttctatca ccgaatacac ctttagggcg gaaagcaaat  12360 

agaaaataaa gaattacttc aaaatacaac attttatgat ttcactttga gtattaagat  12420 

gacttcataa aatacaggat agataggtcc atgcataaac aacacacatt tattttttca  12480 

ggtgatagcc attggaaaat ttttaaaaca gatggctata ttttacatac tatatctgtt  12540 

taataactta aacattcttt atcttatcaa ataattatac cttaatttcc tatctgattt  12600 

ggattgagaa atgcttcatt tgaaatcacc ttttcagatg tcacttgttg ctttgctgct  12660 

tcacagataa ctggctcaga gcgggactta agctgatgag gtatctagca taaagcttca  12720 

caaaaaatga tcatgttgca taaaatgtca tagcatcaat tggtggggta tgactgggca  12780 

cagaggttca tgtctgtaat ccgagaccat tgggaggcca aagtgggagg attgcttgaa  12840 

gccagagtca gagaccagcc tgaacaacac agcaagacct catctctatt aaaaaaaatt  12900 

aaaaagtggt ggagtaattt aaaaacatat ttgccttcag ttggtagctc ttaatcgcta  12960 

taaaagtttg atattggaaa gtattttgag aacagaggca aacaaaatgg cttcatggtt  13020 

gtataaccaa tagtttatgt ccttttccaa aaattctatt ttaggggaaa tatgggataa  13080 

gtaaactagt atacttagga agtacattat ttgtaatgca gaatttaaaa agtcaacagt  13140 

attataggta agctactaac tcatcagaaa caaaattcca gaggagagtt aaaagggata  13200 

ggaaatattg actttgctga agaaagttca gaatgagtcc cagttggtga aaaatagcct  13260 

tgtattttac gtagctgcgg ccacgacatt actcaaggtt gccaaatagt ccgcctgaaa  13320 

aggacgtttg acttgaatct aaaaccatag ctgtcttctc atatgcaact cagttttaaa  13380 

gaaaaataaa attatattta tatttgcatt gagagcacaa aaatggccca tactatagag  13440 

aatgaacatg actctctctg cacatcttat aaacattcat atattaaatt accattttcc  13500 

taagaaagaa aaaccaaaca ttagtgttgt gggatattgg tgtactttta ataaagcata  13560 

atgctaattt ttcttattgc ctagctatag aaatgagttt aagaaatatc cttagcttgt  13620 

cttttcaatg ttatatcatt ctcattcaca agtcaattta ggatcccatc agctgtctct  13680 

ttagttatat attgaactta aatgttaaaa tctgaaatga atgtgagtga tttgcagctg  13740 

ctgtgggctt tttttatctt ggtggtttcc gtaaaactta ctgatttccc tatgacttga  13800 

caggtattga taatgagtgg agaaagactc aatgcatgcc acgggaggtg tgtatagatg  13860 

tggggaagga gtttggagtc gcgacaaaca ccttctttaa acctccatgt gtgtccgtct  13920 

acagatgtgg gggttgctgc aatagtgagg ggctgcagtg catgaacacc agcacgagct  13980 

acctcagcaa gacggtgggt attgcccatt cctgctacct gcgagagccc ccttaatcag  14040 

aagctttaac tttggggatg atgtgtttaa acttctggtt gctttaaact aacttggttc  14100 

aactcagggt ctcttatatt ttctttaggg gaatgaagtc agtttttcag tgttttgtgt  14160 

gtttgacaac acagtgtcac aaagaaagta attgatgctt tattactttg caaactcttc  14220 

atatagtgga gtcaaagcct catgcagaaa acaataaata tacatatgta tgtgtgtaca  14280 

tgtacgtgtg tatgtgtgtg tgttgatctt caagaataaa catgttcatt tggcaataaa  14340 

atacttttaa atgggtgagt tttcagatgg tggagataac agtgcaaaca gtattcaaag  14400 

tgtaaaatta aatgcgtgtg ttctgtgaaa cagtggtata gtattgaaat aagccatctc  14460 

actgaagaaa tttattgtct tcctgtcgaa agcaaaggaa ataactctga cactaaatct  14520 

cagatgtttt gaataaactg ggaagtttat ctcattaaat ggtaagcttc tgataaccag  14580 

gaaatgtctc ttaaggttgg ctttggatca gggattttca gaccagacac ataaacccgc  14640 

tttgtgggcc ttacgtggta gaggaggctg gctgtggaac acgcgtatgt gctggcgata  14700 

accatgaaag tgttgtttga aattgtgtgc acctctggcc tctgaacaga aatgtttgga  14760 

gaaatccagt cccgtgggct ggactgaagc aaacttctcc cgaaagacaa aactgtggtt  14820 

gtcctaatga cctttctaag ctatttttgg agttagactc acacacgtaa atctggctaa  14880 

attcagaact gttctttgac ctggagaatg aaaaatgaaa ttgaaagatg tggtgttaga  14940 

tttgatttgt atgatgtggt atgacagaaa attcacattt taattttatg caaactttag  15000 

ctgatctaca tggttttcag taataaaacc tactcttatt tcaatatagt attaatagaa  15060 

atcagagtta agattactat ctcaattcca tgaactacat tttttcagct agctgaatat  15120 

ttccaaaatt tttactgtta cgatcatttg gcctctgagt ttggagacat tgtcttatta  15180 

tacaacccaa attaattgct ttcttttcaa aaattgagtt tctttatatt gctcacccac  15240 

ttgataaaga aacaagccat tttagattaa gacttgcaag aagaaacaaa aaataaatta  15300 

taaagctaaa ttttctattc agcttgcctc tgcctcaact ctaaaggtta aaaaaaaatt  15360 

aaaaggtgat gctttggggc ctgctatcat ttcttttagt atcatagtta gttaagctta  15420 

gccagatgca attcattctg attatttcct aaaagcacag caaaataatg taggggtcgt  15480 

tccaacatgt gttctcaagg aatacaatgt gactctaaac atagtcctgc atttagaaac  15540 

tttaccagaa aaatgtacag ttgtaggaac atgacaatat ttcagtggtt gccaaaatta  15600 

taatattttt gatagactaa catataatta tactattgga catagcaaaa gttatcctta  15660 

gttcatttat cctccctgag tctttatcaa acaacaataa acaataagac gttatagttt  15720 

tgtacttact attctaaata aattaatact acttgaacag gttttattgg tattatgtat  15780 

tttatcataa ccctgaaata aacaggattc tttaagatcc tctaaattct agaactgcat  15840 

actgtaaaat aaattttgga atgccgttaa tgatttatat tttaaaataa ttcagttaag  15900 

caatatgatg tattctagat tatattccta atatggtatt tgatttaaat caaatagtaa  15960 

accaaggtac tttggatact gtttccaact aataagataa ccttctctct gctcttcctc  16020 

cttccatttc ctttcttcat aatcttttct ataaattact ttcaagcata ttttttaaaa  16080 

acatagatcc aaatctatgg ctaggcatgg tggctcacgc ctgtggtctc agctgctgag  16140 

gaggcagagg caggaggatc gctggagccc aggagttcag ggctgcagtg tgctaggatt  16200 

gcaccactgc actcccagcc taggtgacag agcatgaccc tgtttcaaaa acaaaaaaac  16260 

cccacagagc tgatgtcagt ctccagcata aaggcctttc agtgatttcc cattgcttgt  16320 

aaaataagac aaaaatcttt tttgaaggtt atatttattg acatatcata gctgtacata  16380 

ttttggggtc atgtgttata tgttgatacc tgtacaaaat gtgtaatgat caaatcaggg  16440 

taattgggat atccatcatc tcaaacattt atcttttctt tctattggga acaatacagt  16500 

acttctcttg cagcttttta aaaaatatac aatatatcgt tattcactcc aatttccctg  16560 

atgtactatt gaacactaaa acttactcct tctatttaac tgtgcttttg tacccctcaa  16620 

ccatcctctc tccatcctcc ctacccttgc ccctcccagc ctctgataat cagtattctt  16680 

ctctctactt ccatgagatc cactcgttta gctcccacat aaatgagaac atgcaggatt  16740 

ttcaagacca catctttatc acaacctgca atctctgccc catagcccct gttcagcatc  16800 

atctaatgta atgattcttg atcttgtgct tccagtgctc tagactttct ccaattcctg  16860 

ggcatcatcg tgtttcctct gctcctgggc ccttgcacac acacctttag gttaacctgg  16920 

ccttcctgtc tcctctttcc tgaggtgcta gctcttcaga tcttaatttg ggtgtcaagt  16980 

ggctttggca aggcctcagg caagactttc tgacctattt ggattagacc caatatttgt  17040 

agtaaatgct ttatagtttc ccatattttt aaaatagtat ttattacagc ctataatcac  17100 

atagtttata gttatgtagt cattgttact tgattaatgt ctccctcctc cacaaaggca  17160 

aaaacagatc cgttcactac ttatcccagt gcctaccaca gaatcagtgc acaaatctct  17220 

tttagtatca cataaaaact gtggaccctt tcatgagaac aaatgaaaat atagacacat  17280 

ccacagcatt tccccccaca gttttaggga gtttgttccc tgacccctag ccttttccct  17340 

ttaaaccagg ctttaaaact gctgccttga agattattaa taataaaata gctgattaac  17400 

acacttgtgg aattttcaaa taattggtct ttttgctaaa caaacgttta tgctatcaat  17460 

ttagcttgat cttttaaaaa atatctgttt ctggagagga acagaaaagg taactattgg  17520 

gtactgggct taattcctgg gtgatgaaat aatctgtgca acaaccccca tgacatgagt  17580 

ttacctatgt cacaaacctg cacatgcacc cccaaaccta aaataaaagt ttacctattt  17640 

aacaaacctg cacatggacc cccaaaccta aaataaaagt ttacctatgt aacaaacctg  17700 

cacatggacc cccaaaccta aaataaaacc ccaaacctaa aataaaagtt tatgtcacaa  17760 

acctgcacat gcacccccaa acctaaaata aaaccccaaa cctaaaataa aagtttacct  17820 

atgtcacaaa cctgcacatg cacccccaaa cctaaaataa aagtttacct gtgtcgcaaa  17880 

tctgcatatg caccgcgaaa cctaaaataa aagtttacct atgtcacaaa tctgcacatg  17940 

gacccccaaa cctaaaataa aagtttacct atgtcacaaa cctgcacatg gacccccaaa  18000 

cctaaaataa aaccccaaac ttaaaataaa agtttaccta tgtcacaaac ctgcacatgg  18060 

acccacaaac ctgcacatgg acccccaaac ctaaaataaa accccaaacc taaaataaaa  18120 

gtttacctat gtcacaaacc tgcacatgca cccccaaacc taaaataaaa gtttacgtat  18180 

gtaacaaacc tgcacatgga cccccaaacc taaatataaa ctttatatat gtatgtatat  18240 

atatacatat gtatatacag atatacatat atgtatctgt aaaagaaaca tatctatcta  18300 

tatctatatc tagatatcca gatgactaga tgagatatag atatctgttt ctcttacaga  18360 

taaggaagaa aattcttctt tattctctca ttcattagtt aatatgagca aaagccaaca  18420 

ttatatcttt cttactattg gtcaaccaaa taaatgcata ctttacatat gggccaaata  18480 

gatacctaag cataccttag gtaaaatcta aacttggaaa acagaaaatc agtacattgt  18540 

tagtaagcaa agtaaataaa tttagtcttc tattaaccat ttgagttttt tctgtgacca  18600 

ttgcatattc attttctgca ttggtctaat taggtgtcac attcacccag tgggtattgg  18660 

agtgaaagta tttattcaag ggtaggtgtg tatgctcagt agcataaaaa ttgctctatc  18720 

aagagggagg tgttggagag gtttaggctt tagaaattgc aaggcttcgc tgggcactgt  18780 

ggcttacacc tgtaatccca gcactttggg atgccaagac aggcagatct cttgagctta  18840 

ggaattggag accagcctgg gcaacatagt gaaaccctgt ctccccaaaa aagtataaaa  18900 

attagccagg catggtgttg tgcacctgtg gtcccagcta cgcgggaggt agctgaggtg  18960 

ggaggattgc tggatcctgg aaggcagagg ctgcagtgag taaagatcgc agcactgcac  19020 

tccagcctgg gtgacagagc cagaccctgc ctcaaaaaaa aaaaaaacaa aaaaaaaaca  19080 

aaagaaaaaa attacaaggg gccaggggta agatgagcat ttagtgctaa gtgtaagctg  19140 

catataggtg aaataatttt gcccagtcta cttagtactt aaacatcaga tatttgctat  19200 

aagaaaattg tctactttag atcacatgga atcttttctt gctgtatttc atctctttca  19260 

cctcccagct ttgcagaatt taggcttact tttataatag atttacaatg ttgatatgag  19320 

ttttgtcaac ttcttcccaa tcatttgctt gtgtgaaaaa tcaggacttg cccatgaaat  19380 

ttctgaagtt tgtctgcttt tttgttattg gttgattttt ttttcagttt tttttctatc  19440 

ctattggtgg gctatatatt tacaataatc acaatattta tacattctga tctgtaatgc  19500 

agtcatattt taaaatttat atagcattag tttttatttt gtcattttaa ttttttaaag  19560 

atttgaagat tttgagaatt aaaaatttgt agttcatcaa aaatatggta tttctcctga  19620 

gaattctgac ctaagacacc ttctctgcag actgtaactc tccttgtatt ttctcatcta  19680 

ctttttttta cttttcccaa acctttccct ccaattcagg cttctttcct gatttctata  19740 

ctgtccactc aaccttgtga cttggctgtc cctcagacag gtcagtctgt ctcacttaac  19800 

agttagaacc tattccctac caccttgccc aaataagcaa gtaagtgaat cctattcttt  19860 

ttccagtgtt ctgtgtctta gtaaatgaca tcatcattca gccagtcggt caagccaagt  19920 

tattcttcct ttcaccaaaa ctaattgcta ttcaatctac cttgcaaaca tctctaggac  19980 

ctatccagtg tctccacgct gctaatcccg ctctgatcta ggtcacattt accccttacc  20040 

tagaccattc ctcttggaca agcctaataa ctggtctcct acctccattc ttcctttcct  20100 

ccaatctatt tgccactttt tagcagtgat ttttttttct tttcttaatg ctaagtcaga  20160 

tactgctaaa aactccttga taacattgcc ttattgggat ggagccagca ttcaactagt  20220 

gtggtgataa gcttatcttt tctaattggg ttgcttaggc cagcatctgg tcagattgat  20280 

tggtacaaat gttctaagtt gttagtgttt tgaatggaac tcttctttca gacaaaatcc  20340 

cttaaacata atttcctggg ccctcaatga tctggatgct acctaagtgt ccagcctcaa  20400 

atgtcaatgc ttttccccac aaacactact taccactaag acccaaattc ttttcacttc  20460 

ttgggttatg tcaatatctg tcacctccag tattgcaatg ctgtactctg tgtttgaaac  20520 

atctccctac acatgatccc gtatctcgat gtatctagtt aatgtctact catccttagg  20580 

cctaaacata aatgtgactt ccacgacatt ctgttatgtg cttccacagc atccattgtt  20640 

gctgtatctt agcatcatta ccttatctgt cagattgttt tatctttgtt taattcccta  20700 

ggtttttttt tttttttttt tttaaaaggg agtctaactt ttgttgccca ggggggggag  20760 

tgcaggggca ggaactaagc taactgggac ctttgcctcc tgggttaaag ggattatttt  20820 

gcctcaccct cccaagtacc tgggattgca ggtgtgcacc accacacctg gctaattttt  20880 

tgtatttgaa gaaaaaaggg ggtttcacca tgttacccag gctggtttaa aaactcctga  20940 

cctcagggga tcctcccacc tttgcctccc aaagggctgg gattacaggg gtgacccact  21000 

gcccccagtt ttattccata ggttctaaac tccataaaaa caaaaattgc gtttgtttgt  21060 

tacactgaac aataccacag tagttggccc ataccaaaca cttaaaagtg aataaaatga  21120 

atttttgaat aaaaaaaaac aaatttgggg tcaaaggtca aattttgaat ttgctgattt  21180 

aaaaaagtct gaatggtgtt tagggggaaa taccaaaagg tttgatgtac aattctaaaa  21240 

ttttaaaaca ataactgggt ctttaaatat agatttggga ggaagcatca ttctgataaa  21300 

tgtcatttga agcctggtgt gcttattaat tcttagggta ttgatgtatc acaagagaag  21360 

agcaaacatc agtaccaagg gaaagccaac attcatcatt agctgtgccc tgcatttccc  21420 

tctttgattt tgtattatca tgcccttttc ctatccaaag gattcgctta tatttttgtt  21480 

tatagtataa ggaatttttt tttttttgaa acggagtctc actcttgtca cccaggctgg  21540 

agtgcaatgg tgtgatcttg gctcactgca acctccacct cctgggttca agcgattctc  21600 

ctgcctcagc ctcccgagta gctgggatta caggtgtcca ccaccacgcc cagctgattt  21660 

tgtattttaa tagagatagg ttttcaccac gttggccagg ctggtctcga actcctgact  21720 

tcaagtgatc cactcgcctc ggccacccaa tgtgctggga ttacaggcat gagccaccgc  21780 

acccggcaga tagttttaat ttaaattcag ttagacaagt gcatgtggta cacccattat  21840 

gtgacgcttc atattatgta ctaaaagcat gctatgcaca aagtgtaggt gtacagtgat  21900 

ggatagatcc tgccctatgg aactaacaaa ttagcaagcg aaatcaacat ttatcaagta  21960 

attaaaagta gtttaagtgt tgtgaaggaa aagtacaaag gcttatggaa gtgcatatcc  22020 

aggatgttta taaaccaagg gaggtcgagg cataaaattt ctgaaagcaa gaatgcagtg  22080 

ttcctacatt tttactaata ttccaagttc tgaaaacgtg tacttaagta gcagcttttc  22140 

ctgagtttat tcaatttatt gcattatcag tgtagtatga atgggagtgt gtgatgattt  22200 

ccttcaaata taaaaggaaa tttcttaatg ctgtgttttc ttaaggtaaa aaaaacaaaa  22260 

aaaagacttt tcgttttgtt aaaagagttc caggcacatt ttgtagcaaa gccaccaatt  22320 

ggcatgatgt aaattgtaag gacaatgtac cttaggtaat gttaaactca agttttttat  22380 

agatacttca tagaaatgct tttttaaaat gggactcaat gaaggggagg ttatcagggc  22440 

gtgtgaggaa gccagctgtt cattgaaaag taaaagcata tgcactgtgc attccaaagt  22500 

atactaatgg ataaaaactg cttcaaagtt ctccacacaa gtatctaagt ggctttgaaa  22560 

agttaagtag attggatgtc ttttcaacct ctttcataaa catacacttt gagtatattt  22620 

tcttatgaga agttgtatgt ttaagagata taaccaaaaa gtagaaaatg aattttgcag  22680 

ggggtaaatt atttggtgat gaaagcagaa gcagaagaaa gtcatctgct tttgaggcac  22740 

acatttggat taaaagttac ttgaatgtta gtatttacct tatatatttt actaaatcta  22800 

ggaaagaata aagaaaaaca gctcaatctc tagagtcttc ctgaaaatgg tggggttaaa  22860 

tcaagctcaa ggaactagaa gttctttgct gtgttcatta gtgccagact aaagaagcca  22920 

gtttagttaa gaagctagct tgctatgagc taattctgat acatagaagc agcattattt  22980 

taaattctac attgccatct taaaaacaag cagctacttc tttttcatta ttttataaag  23040 

caataatatg agcaatcaat aatatgagca tgtatattaa caaagacatg ctcgattata  23100 

tttaagttgg atgtttcata aaattttaca acaaaaaata tttaaaccta agtaaaatat  23160 

gcaactatat aatgaaatta gaggtattat tttgcactgc agtcagagat atttcttcca  23220 

ttaattaaac agaatactgc agcagtctgg aagattaatt tattacattc ctttgtattc  23280 

agcaagtgga acgtgatttg tatctttagc aagttaatat ttggcaagca cagtttttgg  23340 

aaatcaggtt ttatcctgat gtggaaaaag aactctatgc cagtaccagt tatcagaagt  23400 

ttctttaaga ataacatttg ccttaagaac ataattaatg catctgtgaa aaataccact  23460 

tttttccaac ccaaggtaca atgagattca cgtattttat tcagtatttc taaaactctt  23520 

caattgaggt ttttatttta attgtggtac tgtgtatact ttttatgcat aacaatatgt  23580 

taaaattaat tcagtaccag atttaaatct gttaggctat ctgttattcc attttctttc  23640 

ccaaacccaa tttaagttcc acaggccttt tatagctggc catgtggttg caatgctaag  23700 

tcaaaatgct aagacctttt cccattttgg ggcattattt ctttaaattc gttgtagttt  23760 

taaaatctga ttttatagtt attaatcata ttcttctggg ggtacatata tctgtactta  23820 

atggactaaa gtttggaata tttaaattta aattaaatat ggtagtgaga tttgttctga  23880 

agagaaactg gttatattta ggatataatg gtgttttaaa ggaaggcatt ttttgtgtca  23940 

tatttgtatc cacacaaaca ttacttataa tttagtacta aattataact caagatcctc  24000 

tcagtaagga agttctacac ctttttcttg aatagccagc aagataagta ctattaggaa  24060 

ttgttgcttt atagccgttg ctatgctgct gtaaacaacc tattgtttca aaatgtggct  24120 

ttatttgaag tccattcttg ttggtgagaa ctgactatta aatattccta ttaaagaatg  24180 

ccagtagatt acaactggga acctctcagt tggaaacact tgccatttcc tcttgtttat  24240 

tatagtttta cagaatatac agtcagattt ttttttcagt ttccaacctt gcttaaatgc  24300 

ctttttttat atgtcctttt gatcttctcc aaaaccaaca actggtatta tatgcctaaa  24360 

gtatatgtca ggcttcataa tgaattgtat tattacaact ttttttgtgt cctacattgg  24420 

acacaagtta atattactta ttggttcaga acaattcact ttattgtata aaactgtttt  24480 

agaaaaatca taattttcaa aatatcaaat tataatagat acattatcag ttaaatacat  24540 

catcaatcag tatttactaa ataaacatac ttggagctat tagctagtgc tgaataatac  24600 

ataaatttta tgtgctatga acttaaatgt agaacagaaa atactattat atacacaata  24660 

aagcacaaaa tagtctgaca atagtatcat gattcatatt ttcttaaaat aatctactta  24720 

ctgtttcaaa cattgatatg tttggataga tctaaactct tttgagagga aattctagtt  24780 

actatgttag ttcagatcat tattaatggc aagaacaaat tgcataaggg aattaaactc  24840 

cagtgtttac ggtaagctat ttggtatttc agaaattcta ccatggtatc tctctctgtg  24900 

tatatgtatg ttctcattca ccctcaccgt gaggtctctt tttttttctt tttctttttt  24960 

tttttttttt tttttttcgg agagggagtc cacctctgtt ggccaggctg gagtgcaggt  25020 

gcagtggtgc aatctgggct cactgcaacc tctgcctccc gggttcaaga aattctcctg  25080 

cctcagcctc ccaagtagct gggattacag gcatccgcca cgccacccag ctaattttag  25140 

tatttttagt agaaacggga tttcaccatg ttgggccggc tggtcttgaa ctcctgacct  25200 

caagtgatcc tcccacctca gcctcccaaa gtgctggggt tacaggcgtg agtcaccatg  25260 

ccccgccctc attgttaggt ttctgatcag catagaggca aggtcattac tgatgactgt  25320 

tatcctgaga ttctagcaag gtgcttggta tcatgtaggt actgaataaa tagttactga  25380 

aaactaaaca aggaaagatt ttaaataaaa attttaaaat ttagaatttc tttaacacaa  25440 

atttaactaa atcttggctg atgtgaatga tgataaagaa caagagaaag aagaggagga  25500 

agagaaatcc aaacattaca ctgaaatcac cattattatt atccctattt tataggtaat  25560 

gaaatggagg cttggggaag ttaggtaatt tttccaagtc tcagaagtag taaattgaag  25620 

atccaggact ctaattactg tctgtttctc aagactaagt aaactcttgt ttattcttac  25680 

atacttcgtg gagtagggaa gttgccagtg atttgaaaat gttatgtatt tattttggga  25740 

gggcgccatg caagtttata atgcacttct aattattatt ttttcagttc aaaaaatgta  25800 

tattttctaa gcacatacta tctaggaaaa aagcttacgg aaattcttag taacgtaggg  25860 

attaaatata gctatgtaaa aattcacaaa gtggtctgta aacaagaaaa tagaatttac  25920 

tatatttatt gaaaaggcag tagaaactgt aatcatgtct aactgataag agtaagtcag  25980 

tcaatataca cacatgagac cagcacagaa agaattagaa aaattaaaga ggcctcttta  26040 

gtatgtgaca agagctggta taggtacaat tatatcttaa tacataccca gatggcatat  26100 

tcccagtaga tattactaat tttcctgaag ttgatagata aataaaactc aattatcaag  26160 

actctttcta cttttaatat tatcaaaggt atctttctct ggactcttaa aattttttca  26220 

cactggtatc atcaagtagc aggtcaaaat ttagggatag aggatgtgtt tcaagagaag  26280 

aacaaaattt tttaacttta atttttcaat ctctgttcct tttgacaaaa ttaactgaat  26340 

tcagcctgcc aattgctact tagacacaaa aaagagaaat gagacattag ttattaagat  26400 

tttgattttg attcctttat gttgtgattt cctttatttc ctttaagtag ttaaactgca  26460 

acaaagattt ttttctctcc aactgtaaaa aagtcttcca ttttaccact tataaaagtc  26520 

ttcaacttag aggaaaagac agttctctaa acagtgattt ggttttagtg ctgttttacc  26580 

tgtctcctta ttcttttagt cagtgtattc aaaaacacat ttcttgcagc ctttgaaagc  26640 

atagagccac acttagtttt ccaggaagat gctttcttaa tgtgaattag ctattcttca  26700 

tctcaccaga acattcttca aagctgctgt tcctgaattc atgcttgata cccatgggtg  26760 

gatcctgatt tgtgaacatt atctacctag gcatggatga ggtctctcca tgatcactga  26820 

tactgctcac ggcaatctgt ctaccatttg gaaaacaggc tggggcagaa tctggaaggg  26880 

cagccatcag tgggagcagg aatgatgctg ccaagggtta ggggtgggaa aacataaact  26940 

cttctacaat agctgttagt tatctcaaat agtgccttac cttcactatc aggctctgtc  27000 

ctgcctggtt tcactggaga caatgccaaa tcctgctgtg cttacttatt actttgtttt  27060 

tgcattgcta taaaggaata cctaagactg ggtagtttat aaagaaaata ggcttaattg  27120 

gcttatggtt ctgcaggctg tacaggaagc atgatgctgg catttgctca ccttctggtg  27180 

atgcctcagg aagcttgcaa tcttggtgga agggggcgct ggtgtgtcat atggccagag  27240 

tgtgagcaag agagggaagg gagaggtctc agactccttt aaacaaccag ctctagcatg  27300 

aactaacagg aaaatctcac ttcttaccga aggatggtgc taagccattc atgagggatc  27360 

tggtcccatg acccattcat ctcccgccag ctcccacctc taacattggg aatcacattt  27420 

caacatgaga tttggaggag acacagatcc aaaccatatc agattacaaa cactgtgcgt  27480 

gtaagcaaaa ttgtttctgc tggtctgagc cacttgagcc ctttcccact agtcacatgg  27540 

agcacctctt cttgtccccc gtctccactg ctgcttcctt cctacagccc ctgccctgcc  27600 

acagcccttg gaaccacatc gggtgctggt tatctcctgt caattcttgt tacatttgac  27660 

ttcccgaaga catgtggccc tgtgaccaca cacacctgtt tgaagctctc ccctctttct  27720 

ctggcacccg ttggcttgac agtcagtctc ccctcctgat gctccatcat tgttatgctt  27780 

cttcccagac atattcacta gttcctttgc ctcttcccac ctcatagatg ttcctgtgcc  27840 

ccaggtttct attcttagac attttctctt ttcatttaga gtgctcatca agtaatctca  27900 

tcaatgtcca agactccaat tacagcttaa atgttcatga cttttcaatg gctctcctaa  27960 

actctagaca cacatatgtg actgcctact agacctaaat tcttaggtac accttcctac  28020 

catctacatt attgcagagc ttaaataatc aaattataaa ttatacaaat ataaaaaata  28080 

agtaatttta taaatttatt aaaaattgct ttttaaaacc actttgtatg tcttctgtag  28140 

tgctagttct taatatagat tccatagatg tcttaaaaga tagaaaaatt gtagcttatg  28200 

aagtcactgc agaattcctc tcttaaaata ggtatatata gtgcaaagga ttactgccat  28260 

tgcaatattt cacctaatca aagattttat ccaacgtgta taaacgggta tctttttaga  28320 

taagaaactg gtcaaaatca taatagtggc atatgttgag gtcacatagt gaagttatct  28380 

atgcagatca gacagtattg tggagaatac ctagaatctt gagaatttac acaaatagta  28440 

ataacttaga tctgtatagc aaataatata catatcagat tcatttagtt tatgtaatct  28500 

ttataatctg agaataatga gtcaagtgtt atccttagtt tactgattat caaagaaaaa  28560 

tacagcttgg agaagtcaaa tgaattttcc aaatttatgc tacttataaa tgggagagct  28620 

aataaaagcc acctgtctgg gtataaccct gtacctattt cttctaaagc atattgtttt  28680 

ctaaagaaag tagttacatt ttaaatgaat atataaggaa tacatcttga atgaagacaa  28740 

tatgcatttt ataatttaca tatttctgta tttcattatt cagaattcat ttaaaaagtc  28800 

tccttgttgg catggacaaa attggcagct aatcctatcc cataaatatt ttcaatggtc  28860 

ttcagctgat ggcgtgaaag ttcagcaaag gccaagtcac aatgctttga agtttgtcat  28920 

cttttttatc actgctcaga ttgtcatgaa gacaccaagt aatacatgga tcaattatag  28980 

ttaaatgact gcaagttgat cttatcaagc agttagtggg caagatttca cttttaccat  29040 

tttttctggt aatgatttag agtgtaagaa cactaggaat ttaaatctac ttagttttac  29100 

ttaatcaatg atgtatttgt ttatttaatt taaaacattg tttttggaga aggaagagaa  29160 

cacaagaagt tcttgatcca taaataaggc gttcagaagg agaccagtta aactaaattt  29220 

tgactgtaat tcagaattgt gtaaaatgcc ctactttatt ttaagctcat tattttcata  29280 

attcctttac caagatagca gaaacatggc tttatctatt aaattataaa tgtagttttt  29340 

tttgttttaa acataatcaa tttataagag atctaagaca tataaaacta cattgaggta  29400 

gaaatttaat aaaaattaac tttacatcta tgtaacttac gtctatgtat tcagtttatt  29460 

taaagtaggt cataaccaag tatttctggg ttgtgtggcc aatagctttg ctcctaaatc  29520 

acaaagatgt ttctgtgtgt gacacttttt aaatccctgg aatattttaa atgttcattt  29580 

caaataatct ataactttga aacagttctt ttttcttgaa cacattgttc tgtcacgtct  29640 

aataggataa ccaattagtt ggttatccta attctaatat gcttcataac tagtgacata  29700 

agataaacat atggagtatt tattactctg ataaaagata ctggttagga gcaagacagt  29760 

ttttactttg taatcataaa aacatagcgt cctgcgtaca ctaggcagct acttatatag  29820 

agctaatctc actctttttc ggactccaat atttaccata aaagtgcttt acagcatcta  29880 

tataattttt tcttcttttt gtcccttctt tgtagttatt tgaaattaca gtgcctctct  29940 

ctcaaggccc caaaccagta acaatcagtt ttgccaatca cacttcctgc cgatgcatgt  30000 

ctaaactgga tgtttacaga caagttcatt ccattattag acgttccctg ccagcaacac  30060 

taccacagtg agtatgaatt aaatctattt tttctgcatc ctctactaat attaaattat  30120 

tttcaagtaa acataagtta aacaaataac ctctgtgaaa tttactgtaa tatactctgc  30180 

ttcaaataca aaacaaagac aaaattttgt atagtataca atgaaaataa actgaaaatt  30240 

aaaattcagt gggaagcgta ttttagaggc aaagtaattc acaatataaa agaagtatgt  30300 

ctgtgaaata aaacacattc caccagtgtt cataaaatat cagtaaggta gcttaattga  30360 

tttaacattc ctgaaatcag gtatgctaac tcataaatca tattaaagtt caaaacattt  30420 

cagccttgat aagattttag gccaaaacaa aaagtaaata taaataattt tatatctatt  30480 

agcattcagt tgctttcttc aatctcaaac ataaaaatat atatgtgtgt gtgtgtatat  30540 

gtgtatatat gtatctccat gtttcttatt tcagcaatat ttgaatagtg aaatagtgct  30600 

taattacttg aaaaaaattt ctgttaatat catttcaaat tgacattcaa accattattt  30660 

gttattcagg tttcctttcc atcctatttt tttggagggg caaatggcag catatacctc  30720 

atgtgatcat ataggagaac ttatcacgtt ctcctgtatg atcatgttct gaccctgtga  30780 

atagctgata atgaattaat atgcattact gcctaagatc tgagagtttt ctgccccaaa  30840 

gtagggatgc aaccctttgt tccttaggat atccggtgca tatggatgaa acttacataa  30900 

ctctagcatt tgtaacaaat gtataaataa attgatcaaa atacatatat attacaaaaa  30960 

agaaaacctt acatttttaa aagactcaac aataaatgta atatgcaaac catggttttc  31020 

cccactgaat cacgttgtac tactctgctt agtgtggcag atagggggac cccttgtttg  31080 

tccagggaca gtttaggaaa acctggaaga tagcctaatt tgagtgacag agatctgtcc  31140 

ccttttatgt gagaaagcaa agatcttaca gacatttgtg aataaatgta tgctaataca  31200 

agcaagagac cactaccatc tgaactcaag aggatgccgt atgtgcaatt tcatccccgc  31260 

aggctagaga gttctgttca ccttcctcag ccctgagcag aagccctcag tgactcctcc  31320 

attctgagag ctttagccat tatctccata ctcatggctt ccaaaatgac atttgaagat  31380 

ctaaccatat ttttgactta taatatttta tctaaatcta tcttctattg ctttgcttgt  31440 

aattatatta gctgtaagta caaagagaaa gacttgttaa atcctttatt tctacattat  31500 

tcatggcagt gttttctcat tttctttgag gccttgaaat cacagagttg tcattacttc  31560 

ttcttcttct tctatcccat ctaaacacaa ttgattattt tggaggaggg ggagatatgt  31620 

gttccaagct aaacttatat ttcatttcca tcactgccat tatcatccac ttcattatgc  31680 

atgtctctga tctaatgcag tgatattaat actagttttc aagcatcaag tctttctcct  31740 

gttgtataat gtcactgttt tatgaagtaa gctttaaaag gcatcgtttc attcatctca  31800 

ccagaaacag tgactttcag ttgctcaaag aactgagcac aaatttctca ccaccctatt  31860 

ctggtgtcag ccttcctttc ttgttttgtc ttccattatt cacccagtct gtgtttcacc  31920 

aagtccgatt cccagaacgc caagctcatt tcctgctctg tatcttgttt ctattctggc  31980 

cctggcttgt gttctgcctt tccaaagccc tctccatttc tcagagttca ggtaaaggtc  32040 

tttctcctgc atttgtcttg tccgaatacc acggccaact ttgcttcttg aatacccata  32100 

gcactcactg gattaacttc atcctcacat ggctttatca gggtctgcaa atgttgcatg  32160 

tggaactgga ttatgagagt gtttccagta agtttatatt gtctgcaaca ttcagctaac  32220 

aacactcaaa aatacttgag ccattcctgt gctctcagcc cctttacctg cattgtctga  32280 

tttaaccctt aacaaaatca tctagaaggt cagcagcagc atttgcctta ttttacagat  32340 

aaaggtggaa atcacactcc aaagctgttg gctaaataat gtacataaat gcttgttaaa  32400 

taaaatgact catttcctgg atgtatttta tagcatttgc catgttttca tcattactta  32460 

ttcatataac tactatccta tacactgttt gaggtcaaga agagacagga agctacagca  32520 

aaaagagcct gagtcttaga gtaagccata cctgattctg aattcagatt tgccactaat  32580 

tcattctctg tgatcaggta ggtgttttat gtctctgaaa ctcagtttac ttatatataa  32640 

actgtaaaga tgtaaagagg attgaatgaa gtactgaatg cttatgcaat gcttagcaga  32700 

gtgtctggta tccagaaggt agtcattaaa gggcaaattt attattacca ttaatatcta  32760 

agtctgattc atttcctcac ccatgtgttt ttcagtattt ttaaaaccac tatctacagt  32820 

aagaaaaaca ttttatattc caaaccagga tgtgtggtat acacacacat agtgtgtctc  32880 

tgtgtatata tatatgtcga tattagagaa ttttaaatta gctagataac ttcacatgta  32940 

attaattatg tataagcata aaactaaaac aaattaccat ttaaaaatat tgtccttaat  33000 

tatggtgctg ctcaagcttt ctcattatca caaccccaag gatcctttta taaacatttt  33060 

ttccctaatc actttcccca taaaatttta atgtaacaaa tatactctgt ctttatacta  33120 

tatgtatctc tgtgctttat acataaaaag gaagtgtaaa gtttactttt ttgttcaatg  33180 

tagtgttaaa taacaaataa aatgtttaag ttaacattta aattaaaaat ttttaatctc  33240 

ttaacagcta acataaattt ataattgtat ttgccaggaa acttttaagg taatctgttt  33300 

acttatataa attgtaatat atcaaattac atatgcaaag taacaattga tatgaatata  33360 

taatacaaat ataataattt ataaaaacat tcaaaatcac tatttttcca gcaaaagtaa  33420 

aataattttt aaaataactt ctttaaaaat atttggagag cttaactatt aactttgctc  33480 

agtatgagaa aacttttaac ttatatagat gataaatatt aatttagata ttgtcaactt  33540 

tttcttttca tcatttgaca tgtaatggta tgactaacat ttgtagaatt aataataaaa  33600 

tataaattat tattatttaa ttctcaaagc tcaagtcaca catgaaaaca ccaaggacca  33660 

agcataagca atatccacag ggcaaccaat ggcagccaat aggcccacat aggtaccact  33720 

cagtatatgt ttgaaatcac tcagtcaatt aagagaaaat cctccagtca tagccatcta  33780 

gtcaccataa tttcatagct catatattgc aaacacttgg ggtgtgttcc gttgccaatg  33840 

ctgttcatgt aatggccaat aaagaccaaa tagaatttaa gtattaagga atattatttt  33900 

cttgggtttt ggagagccaa aaatcattga gatattaaga ttttagcccc atccaggcct  33960 

catacccaat ttttaccccc ttagagatga ttttactccc tttatgaata tttgccttat  34020 

tattacgtgt gatataccca aataattttt ctattctgtt tttctttgtg aaaatattgg  34080 

tctcaaccct ttatgaatta tggcccacaa ttcaataaaa gctacactag tgataatata  34140 

tatatgatgg acatgtgact atgttataaa attctaacat ttgttaagat ttaagaggaa  34200 

caatcaccac cacggtggct aaaattaaaa acactgacaa aaacaagtgt tagaatgtgg  34260 

tgcaaccaaa actcctatgt caccaatggg cattaaaatt gttcaatcat ttttcaaaac  34320 

agttgacagt ttcttatgaa tttaacatgt acatatttcc acacaataac ttgtgtacaa  34380 

atgttcttta ttagtcaata actggaaaca actcaaatgc ccatgaaaga tgagtggatt  34440 

aacacatctt agtatatgca tacagtggaa cattgctgag caatgtaaaa agaatgaact  34500 

actgatacac acaagaacat aaaaggatct cagagacatt aagttgagtg aaataagctt  34560 

ggctaaaaag agtacattct ctatgatttg atttacatga ggcccttaaa agacgcaaat  34620 

gtactctaga gtgagagaac gcaattcagt gtttgtttga ggatttggat caggattgac  34680 

tgacaatggg caagagagac attgtgaggt gttagaaaca ttctttattt tgattgtgac  34740 

agtagttaat gtggatacat aaacttacga aaagatgata aattcaaaat aggagcatgt  34800 

tattgtatgc aaattattcc ccaataaaat tgatttttga aaagcctaga aaagaattcc  34860 

tttattaagt acaatttttt aagtagctta ggtttcaaat agtagatttc tctttttttt  34920 

tttttttttt tttttttttt tgagacagat tctcactcta tcacccaggc tggagtacaa  34980 

tgacgtgatc tcagctcact gcaacctcct cctcctgggt tcaagcaatt ctctgcctca  35040 

tcctcccgag tagctgtgat tacagttgcc cgccaccatg cctggttaat ttttgtattt  35100 

ttagtagaga tggggtttca ccattctggc caggctggtc ttgaactcct gaccttgtga  35160 

tctacccgcc tcggcctccc aaagtgctgg gattgcaggc atgagccacc acgcccagcc  35220 

aaaatagtag attttttaaa acctttacaa taacatttta agtcaaatct ttgattaggg  35280 

agggagattt cccacagtca tccaaaaact atacgttgag tcactaagat aaagaatgct  35340 

tctagatcct attagagaaa acaaagatgc gcttggctgg ctctgaactt agagcactta  35400 

tagtcaagta agaacaacac atattatagt tacattatta ttgttttatt ttactaaata  35460 

ttacaaagca ccaagcatct gccacacatt gtctcatgtt catacaaata ctgccggtgg  35520 

ctccattcgt aatacacatc acatagtaca aggctgaaga gtgtggatgc tgtagggagc  35580 

ttccagtttt gagaatttag tctctgattt ctctttgagt ttgagtaggt tctgtaaact  35640 

ttcttgtgac acattttttt taatctacaa aagagcacat aagtaagaat gttacatata  35700 

acatcataca gaatatttat aagctaatct tcactgaaat caatctgttc aatagcatta  35760 

taccatattt gacataccat agccatgtta atctgatatt gtagaatagc atagtataat  35820 

aataataact cctaactcaa ggatgttgtg atctttataa ccagcaatcc atgttaaata  35880 

ttagcacagt gcctaaaaca tattaagcat tcaataaatg atcgctacta tttttactaa  35940 

catcctacag atttggaaat tgagtcttag aaatgttaat gtgtaaaatg ctaaagagcc  36000 

aaaaaaactg ccaggaaaat ataaaaatta aaatcatttt atttctgaaa cccatgtgtt  36060 

ccccccaata tcttctaaca tttctagtat ttacagaaaa actttcaagt ctcaatatca  36120 

gaaagtttca taaaagccag aggaagtagc aattctcttt agcagacaga gttagatacc  36180 

aattttcata ttggtgttct cacagattat tttttccaat tatttcctct catattttct  36240 

tcaaatttaa ctccatgtat ttccatgggc cctttagtag agactttctt ttcatggagg  36300 

gagtcatttg tgattaagta gcgatcccct agattttcct ccttggtagt agtgtttgtt  36360 

tattcattca tgagacattt atgtcaggtc ttctatctct ataatactgt attaggaaat  36420 

gtggggtatt tttaaatgaa taatataatt ctttagctaa aataactcat gtactagagt  36480 

acaaacaaac aatatttcta gtatagtaag tgtctaaaac actatgctgt acttcattca  36540 

ctcattcaac aaatagcatt tgtgtttctg ctagatacta ataaggtggt tagaccacag  36600 

agatagaaag atacatagaa ttgttactcc agaagaaatt ccattcttga accagacatg  36660 

taaacatatg actgaaatca gtgaaatcag tgtggtgagg aagcatcttg gagacatggc  36720 

tacagtagag aggggataca tgagtctgga gaggtcaaca gaatcacata tgcccagcca  36780 

aggatgggct gtttcttaaa gtctcttgag aactactaaa agtatatatg cagagatgtg  36840 

gtatctgcag ttttgtatta tagaaagaac agcttggtgg caagggagtg tgtggattag  36900 

aggaagcaaa acgggaggtg gaaaaattcg ggagtaatca tttgaaataa tctgagtgaa  36960 

aaataataaa gatggtggcc acagcagtgg atatggggat agaaagaaaa gaggtggaaa  37020 

agacgtttga aatacaagaa agcatatgac ttgggaaccg catatggggg atggtaaagt  37080 

gggagaagtt gaggatgaaa ctgtatggat gacattcaaa agacaaggag cacctgttca  37140 

tagtaggaac ttacctgaaa aggagaactg agtttttaac acatgttagc ttgagtgctt  37200 

gtaatatttg taagacatca gagtggtaga gtaattggaa gagatgacct caaccaaaag  37260 

aaatatagca agtaagaaga caagatgagc cacagatgaa actgtgggga aatcaatatt  37320 

taagaaacag ttatctgtag agcagacaca ataatctgga gaaacagtaa tctgtacggg  37380 

agactgagaa gaagcaatca gaacaatcgg aataacaaca gagagagaga ccacaaaagc  37440 

cagtggagac atgagttcaa ggaaggagga agtggtaaga ggtgccaaat attagaaagg  37500 

ttcaagaata actgggactt actctgttag ctgaacagca tgaaatagag atgtttattt  37560 

actaataact taccatactc tgggcacaga gagggagaat gtgttttgag aaagggggag  37620 

agaaagcaag acagtgaaag aaattcacat ttacacatga gaaactgaat cagaaagggt  37680 

aagtaaattt ccccaatgtc acacaactag aaaagtagca aagcagagat tcaaactaaa  37740 

tttgtttgga tataatccat ttcttttgag gtgtggctat gactgcattt ttattgtttt  37800 

ttagttgata cataattgta catatttatg gggtatatgt gatattttga tacatacata  37860 

taatgcgtaa tggtcagctc aggatatttt ggatatccat cacctcaaac atttatcatt  37920 

tctttgtgtt gtaaacattt taaatcttct agctattttg aaatatataa taaaatatta  37980 

ttaactattg ttaccctact gtgctgtgga atactagaac ttgtttcttc tatataactg  38040 

tactttggaa tgcattacca acctctcttc attcccccca caactgaccc acacttccca  38100 

gactctagta accatcattc tactctctac ttccgtgaga ttcacttttt ggctctcata  38160 

aatgagtgag aacatgagat atgtgtcttt ctgtgcccag cttatttcac ttaacataat  38220 

agcctccagt tccctccatg ttgatgcaaa tgacaggatt gcattctttt tatggctaaa  38280 

tagtactccg ttgtgtatat ataacagatt ttctttattt ttaacttcta taaattaaaa  38340 

aatatcaaac atcaaggaca aaacaaaaac aaaacaaaaa agttcccttc taccccaatt  38400 

tgaaatgtaa caactatttt tttgtattta ctaaagatac aacccaaatc cccagtcatc  38460 

attcttcact cttcattccc agaagtaacc accactcaaa aattgtatta ttctcactct  38520 

tctgtatttt tatggcatct ggatttatta acataccaga agatattata cagtgcagct  38580 

ttgagtgttt tgtattttaa gttaaaatgt tggcattact ttttttctct tgacattatg  38640 

ttttacagat atagtcatat tgctatctgt acacctggtg tattcattta ataactatat  38700 

agcattccat taaataaaca caagttttta tgtttttttt attctgtgac gatgtactat  38760 

agggaagtca atatacacta cagattagca agagagtaca atcaaagagg agagagtgat  38820 

gtcaaataga caaggacctt gaagttaata gtgctaaatt tattagttat ttggttagca  38880 

tggacaatgt ccctaaagat gtaataaaat aacacgttct gtgcacagtt aataaaccag  38940 

gttgatttca gtcttaagct gtgaaagcaa ttggatatta aatgaaaaca caatactgtg  39000 

attctaaagc tttgagcagc agcctaggga atttcaccta ttctgtagag gtattagggg  39060 

agcagaggtt gtttctgggt agaggaatga ttgacaagac aatgttcagc atagccaaga  39120 

aatcttgaat tatttttaat ggataataaa tatagaagat aggtattatt tcaatttgga  39180 

agacagtcgc ctcgtggtga aaaagagaaa agggatgaaa aaagatgagg aaaaaaaatg  39240 

gataaatcta gcctgaaggc taatttgcat atttatttat ttgctgtctt ttatttcttt  39300 

tattaaaatg ttgacattgt ttactcagta aaacttattt gcttgcaaga agttacactt  39360 

actagggttt atttaagaag attttattct tatgagaaca tgtcttattt ccaaggaata  39420 

gagtaagtag atttatagcc aaggaaacat ttgatttgga acttgaggtc tccgtgtttt  39480 

tggtctatta atatttaaca gatcctcagt ccaaatgcct agatgcttaa atatccataa  39540 

gttgtacttt tagccataat gcttcactct ttccttttta atttgaaggt tatacattat  39600 

tgctttacca aaagacaatg aagcaaaata gcctaataac ataaatttaa taaactgtat  39660 

cagagcagaa atggagagat agcttagctt ccagttaatt ggaccacagt ctcgtactag  39720 

atcatcagaa actactgttt ttcagcattt taaaacatgc atctttagtc tcccctgaaa  39780 

ttatcctctt tgccagttca aaatctttat gtccatatac ggttattttt gaaatcgtaa  39840 

gtcatatgtc tgttcatccc ctttcctata aagctcatta tctaaacatt gtctcacact  39900 

tcaaaggatc acctgttgat gtgaatggat ttaccagtgg aactcaggca agagattcca  39960 

ggctgtgagc gttactgttt ttgtaaaggg aaggtaactt ttatatatag tttgcctgcc  40020 

tttggacagt cttgtttcta ccttctaaaa tacgataatt gaaagcacag cgttcctatg  40080 

gcttgctgat agattgtgag aaataacagc aaaatatata cagctgagga gattgcatca  40140 

atacatctct gcaccgaaat tatggcagtg atttgcctag aattgggggc tattagatca  40200 

aagattgtcc taaagtttaa agttacattc aaccaaacac atggacaatt tttgttaata  40260 

tcatattaga ccagatttac cagttgtttt ctaaaattta atatacattt tttaaccatt  40320 

ttaggttaca gaaaaagcag aaagtataga gagttttcat ataacatcct ctttcacgag  40380 

tttcctctag ttttaacatc ttgcattagc atggtacatt tgtaacagtt gaggagccga  40440 

tactgacatg tgatcgttaa ctaaaatcca tagcttacat tagggttccc tcttttttgg  40500 

gtgtaagttc tgtggatttt gacaaaggta taaatgatgt gtcaatcatt acagtatcat  40560 

gcagaatact tttactgccc taaaagtctc cgtgttccgc ctgttcattt ctcctcccct  40620 

gtattcctgg catcaaccag ccactttgat gtcatatcaa ctcttatacc ctcataacgt  40680 

gttttttatt gtcatcattg ttttgttttt gtttttctgt taatgacgta tggctgtgac  40740 

aaggtaatga gaccagcggc tgttgttgga ggaccttttt tcttaattcc tggttactta  40800 

tccttgaatt atgtgacaag ctgataaatc caataaccat ggattttcca ttgtttcaac  40860 

ctgccaatac ttctctcaag cttgtatatc caagaaaata taaaaaatag tagagcagaa  40920 

aattacaagc ttccatactt atcctactct ccctgtgtcc ccatccccac ttctggtggc  40980 

tttccaactc agttaagtcc tgtacttagc cagttggtga ccacacaatg tgacatgttt  41040 

tatatccaag tcagtctcag actaggtgag ttttttatgt ggcagattac tgcttagttt  41100 

atactcagac aaaaaggaaa aaattaaaca tataaacacc cttttttttt tttcgagagt  41160 

ctcatgctgt tgcccaggct agagtgcagt ggcgtgatct cagctcactg catcctctgc  41220 

ctcccgggtt ccagcgattc tcctgcctca gcctcctgag taactgggat tacaagcacg  41280 

tgccaccaca cctggctaat ttgtatattt ttagtagaga tggggtttcg ccatattggc  41340 

caggcttgtc tcaaactcct gacctcaggt gatctgccca tctcgacctc ccaaaatgct  41400 

gggattacag gcgtaagcca ccgtgcccag cctaaaccca tgttttaaat taatgcaatg  41460 

attgtattag cctttcaaac attaagactg gcaaaattgt tacaactatg gagttttcat  41520 

tgattcatcc tactcaccat cttttccttt aatatctgaa caaaccccaa ctctgttcac  41580 

tgttctccct gtgagtagac taatggaaca agagcaaaca gtaacacaaa cagacgctaa  41640 

cacaagttca tgcagaatca ataaaaatca gtacaacaga agataagtct atgtgttttt  41700 

gatacatacg attaagcatg tgccttttta acaatttata taaaacctaa aatatatgta  41760 

tttctgattt ttacctgtag tactgaagaa gttacttaat aacgttgaat ataaaggcca  41820 

cttttactta accaccttcc attcactatt aactgctgtt tccaaagtgt aagcaaatca  41880 

gtctctgtgt acatagtcaa gtgtatacaa gcgtcaggcg taacaaactc aagatgaaca  41940 

tgacagttca aagatatttg ggacaaaatt gttgaagcat ttttacccag ggctctgtag  42000 

ctctgacaat gaaagaatat agttgctctt ccagctgcta ttcagacaga aagcttgggc  42060 

aagaagggtc tgtatctatg ttcttcataa tacaattaca agtttgaact tcagataaca  42120 

tcagcagttg gcatgtggaa aaccaaaacc ctattttggt atttatcaag attgttaatg  42180 

gagtcaggtt tcccttattt gtttctttaa tggggtacag aacatcctgt tggataaccc  42240 

gctgagtgac atgacgatgc tctgaaggaa tgcatgagag cttgtggtac ctgccttgaa  42300 

acatgggttt cactaatgct ggtggctcac acttcccatt gaacaagact agagatagga  42360 

aggctatttg agggacacag ctatggaacc ataggtgcca catggtaagc caaattttta  42420 

tttgttgtgt tgttgtgaaa aaccttatta aaagagcttc caatgagagt acttgattaa  42480 

taacacagtt cgtatctata gaaataattt gcttttcaag aaaatcatca tgtgctacag  42540 

ttgaattgac attaatgtta tcattcattt tgaatgatct gtgaagtatt ttaagagaga  42600 

tctggggaag taaatcaata agtagtttaa cataaatcaa ggtgcagtta tttttttcaa  42660 

ttagaaatat attacaaaga ttctgtcatt tccaacaacg tgaaataacc tggaggaaat  42720 

tgtgctgagt gaaataagcc agacacagaa agatgcatat tactgatctc ccttatatat  42780 

ggaatctaga aaagtagaat tcatagatac agaatagaag agtggtcacc acggtcttgg  42840 

gggtggggga catggggaaa tgttagagtg tgacaacttg cagttacaag gtgaatatgc  42900 

tctggagacc taacgtatag tacagcatag ttactctact caatagtatt ctcactgcac  42960 

cccccaactc ccccacacac acagtaactc tttgaggtgt ggatatgtta attagcttga  43020 

ttttggtaat catttcacag tgtatacata taccaaaaca tcactttgta taccttatat  43080 

atgtacaata tttatcaatc atacttcaat aaagctggaa aaatgcatga atattatata  43140 

tatgtatatg tatatacaaa tgtataagag attatagcag attatagctc tttgaaaaag  43200 

aataacattt cagcccagtt ctgactaaga ccaaacaaaa ggtgatagca tgttttagtt  43260 

ccttaaatgt ggatttgagg agtcaagaaa tctccaagtg taggaaaacc tccgtggcaa  43320 

agagtgtaga atatgagaat cacaaaaata gcatgcacaa aatagcctgc tgtgatgttg  43380 

aaagtataag agctagtaat tattattatt attattattt ttatttttat tattatactt  43440 

taagttttag ggtacatgtg cacattgtgc aggttagtta catatgtata catgtgccat  43500 

gctggtgtgc tgcacccact aacttgtcat ctaccattag gtatatctcc caatgctatc  43560 

cctccccctc cctccacccc acaacagtcc ccagagtgtg atgttcccct tcctgtgtcc  43620 

atgtgatctc attgttcaat tcccacctat gagtgagaat atgcggtgtt tggttttttg  43680 

ttcttgtgat agtttactga gaatgatgac ttccaatttc atccatgtcc ctacaaagga  43740 

catgaactca tcatttttta tggctgcata gtattccatg gtgtatatgt gccacatttt  43800 

cttaatccag tctatcattg ttggacattt gggttggttc caagtctttg ctatcgtgaa  43860 

taatgccgca ataaacatac gtgtgcatgt gtctttatag cagcatgatt tatagttctt  43920 

tgggtatata cccagtaatg ggatggctgg gtcaaatggt atttccagtt ctagatccct  43980 

gaggaatcgc cacactgact tccacaatgg ttgaactagt ttacagtccc accaacagtg  44040 

taaaagtgtt cctatttctc cacatcctct ccagcacctg ttgtttcctg acttttgaat  44100 

gattgccatt ctacgtggtg tgagatggta tctcattgta gttttgattt gcatttctct  44160 

gatgaccagt gatggtgagc attttttcat gtgttttttg gctgcataaa tgtcttcttt  44220 

tgagaagtgt ctgttcatgt ccttcgccca ctttttgatg gggttgtttg tttttttctt  44280 

gtaaatttgt ttgagttcat tgtagattct ggatattagc cctttgtcag atgagtaggt  44340 

tgtgaaaatt ttctcccatt ttgtaggttg cctgttcact ctgatggtag tttcttttgc  44400 

tgtgcagaag ctctttagtt taattagatc ccatttatca attttgtctt ttgttgccat  44460 

tgcttttggt gttttagacg tgaagtcctt gcccatgcct atgtcctgaa tggtaatgcc  44520 

taggttttct tctagggttt ttatggtttt aggtctaacg tttaagtctt taatccatct  44580 

tgaactgatt tttgtataag gtgtaaggaa gggatccagt ttcagctttc tacatatggc  44640 

tagccagttt tcccagcacc atttattaaa tagggcatcc tttccccatt gcttgttttt  44700 

ctcaggtttg tcaaagatca gattgttgta gatatgcggc gttatttctg agggctctgt  44760 

tctgttccat tgatctatat ctctgttttg aaccagtacc atgctgtttt ggttactgta  44820 

gccttgtggt atagtttgaa gtcaggtagg gtgatgcctc cagctttgtt cttttggctt  44880 

agggttgact tggtgatgca ggctcttttt tggttccata tgaactttaa agtagttttt  44940 

tccagttctg tgaagaaagt cattggtagc ttgatgggga tggcattgaa tctgtaaatt  45000 

accttgggca gtatggccat tttcacgata ttgattcttc ctacccatga gcatggaatg  45060 

ttcttccatt tgtttgtatc ctcttttatt tccttgagca gtggtttgta gttctccttg  45120 

aagaggtcct tcacatccct tgtaagttgg attcctaggt attttattct ctttgaagca  45180 

attgtgaatg ggagttcact catgatttgg ctctctgttt gtctgttgtt ggtgtataag  45240 

agtgcttgtg atttttgtac attgattttg tatctggaga ctttgctgaa gttgcttatc  45300 

agcttaagga gatttttggc tgagacaatg gggttttcta gatatacaat catgttgtct  45360 

gcaaacaggg acaatttgac ttcctctttt cctaattgaa taccctttat ttccttctcc  45420 

tgcctaattg ccctggccag aacttccaac actatgttga atagtagtgg tgagagaggg  45480 

catccctgtc ttgtgccagt tttcaaaggg aatgcttcca gtttttgccc attcagtatg  45540 

atattggctg tgggtttgtc atagatagct cttattattt tgaaatatgt cccatcaata  45600 

cctaatttat tgagagtttt tagcatgaag ggttgttgaa ttttgtcaaa ggctttttct  45660 

gcatctattg agataatcat gtggtttttg tctttggctc tgtttatatg ctggattaca  45720 

tttatttatt gatttgcata tattgaacca gtcttgcatc ccagggatga agcccgcttg  45780 

atcatggtgg ataagctttt tgatgtgctg ctggattcgt tttgccagta ttttattgag  45840 

gatttttgca tcaatgttca tcaaggatat tggtctaaaa ttctcttttt ttgttgtgtc  45900 

tctgcctggc tttcatatca gaatgatgct ggcctcataa aatgagttag ggaggattcc  45960 

ctctttttct attgattgga atagtttcag aaggaatggt accagttcct ccttgtacct  46020 

ctggtagaat tgggctgtga atccgtctgg tcctggactc tttttggttg gtaagctatt  46080 

gattattgcc acaatttcag atcctgttat tggtctattc agagattcaa cttcttcctg  46140 

gtttagtctt gggagagtgt atgtgtcgag gaatttattc atttcttcta gattttctag  46200 

tttatttgca tagaggtgtt tgtagtattc tctgatggta gtttgtattt ctgtgggatc  46260 

ggtggtgata tcccctttat cattttttat tgcgtctatt tgattcttct ctcttttttt  46320 

ctttattagt cttgctagca gtctatcaat tttgttgatc ccttcaaaaa accagctcct  46380 

ggattcatta atttttgaag ggttttttgt gtctctattt ccttcagttc tgctctgatt  46440 

ttagttattt cttgccttct gctagctttt gaatgtgttt gctcttgctt ttctagttct  46500 

tttaattgtg atgttagggt gtcaattttg gatctttcct gctttctctt gtgggcattt  46560 

agtgctataa atttccctct acacactgct ttgaatgcgt cccagagatt ctggtatgtt  46620 

gtgtctttgt tctcgttggt ttcaaagaac atctttattt ctgccttcat ttcgttacgt  46680 

acccagtagt cattcaggag caggttgttc agtttccatg tagttgagca gttttgagtg  46740 

agattcttaa tcctgagttc tagtttgatt gcactgtggt ctgagagata gtttgttata  46800 

atttctgttc ttttacattt gctgaggaga gctttacttc caagtatgtg gtcaattttg  46860 

gaataggtgt ggtgtggtgc tgaaaaaaat atatattcca ttgatttggg gtggagagtt  46920 

ctgtagatgt ctattaggtc cgcttggtgc agaactgagt tcaattcctg cgtatccttg  46980 

ttgactttct gtctcgttga tctgtctaat gttgacagtg ggtgttaaag tctcccatta  47040 

ttaatgtgtg ggagtctaag tctctttgta ggtcactcag gacttgcttt atgaatcttg  47100 

gtgctcctgt attgggtgca tatatattta ggatagttag ctcttcttgt tgaattgatc  47160 

cctttacgat tatgtaatgg ccttttttgt ctcttttgat ctttgttggt ttaaagtctg  47220 

ttttatcaga gactaggatt acaacccctg cctttttttg ttttccattt gcttggtaga  47280 

tcttcctcca tccttttatt ttgagcctat gtgtgtctct gcacgtgaga tgggtttcct  47340 

gaatacagca cactgatggg tcttgactct ttatccaatt tgccagtctg tgtcttttaa  47400 

ttggagcatt tagtccattg acatttaaag ttaatattgt tatgtgtgaa tttgatcctg  47460 

tcattatgat gttagctggt tattttgctc attagttgat gcagtttctt cctagtctcg  47520 

atggtcttta cattttggca tgattttgca gcggctggta ccagttgttc ctttccatat  47580 

ttagcgcttc cttcaggagc tcttttaggg caggcctggt ggtgacaaaa tctctcagca  47640 

tttgcttgtc tgtaaagtat tttatttctc cttcacttat atgaagctta ttttggctgg  47700 

atatgaaatt ctgggttgaa aattcttttc tttaagaatg ttgaatattg gcccccactc  47760 

tcttctggct tgtagggttt ctgccgagag atcctctgtt agtctgatgg gcttcccttt  47820 

gagggtaacc agacctttct ctctggctgc ccttaacatt ttttccttca tttcaacttt  47880 

ggtgaatctg acagttatgt gtcttggagt tgctcttctc gaggagtatc tttgtggcat  47940 

tctctgtatt tcctgaatct gaacgttggc ctgccttgct agattgggga agttctcctg  48000 

gataatatcc tgcagagtgt tttccaactt ggttccattc tccccatcac tttcaggtac  48060 

accaatcaga catagatttg gtcttttcac atagtcccat atttcttgga ggctttgctc  48120 

atttcttttt attctttttt ctctaaactt cccttctcgc ttcatttcat tcatttcatc  48180 

ttccattgct gatactcttt cttccagttg attgcatcgg ctcctgaggc ttctgcattc  48240 

ttcacgtagt tctcgagcct tggttttcag ctccatcagc tcctttaagc acttctctgt  48300 

attggttatt ctagttatac attcttctaa atttttttca aagttttcaa cttctttgcc  48360 

tttggtttga atgtcctcct gtagctcaga gtaatttgat cgtctgaagc cttcttctcg  48420 

cagctcgtca aagttattct ccatccagct ttgttccatt gctggtaagg aactgtgttt  48480 

ctttggagga ggagaggcac tctgcttttt agactttcca gtttttctgt tctgtttttt  48540 

ccccatcttt gtggttttat cgacttttgg tctttgatga tggtgatgta cagatgggtt  48600 

ttcggtgtgg atgtcctttc tgtttgttag ttttccttct aacagacagg accctcagct  48660 

gcaggtctgt tggaataccc tgctgtgtga ggtgtcagtg tgcccctgct ggggggtgcc  48720 

tcccagttag gctgctcggg ggtcaggggt cagggaccca cttgaggaag cagtctgccc  48780 

gttctcagat ctccagctgc gtgctgggag aaccactgct ctcttcaaag ctgtcagaca  48840 

gggacattta agtctgcaga agttacttct gtctttttgt ttgtctgtgc cctgccccca  48900 

gaggtggagc ctacagaggc aggcaggcct ccttgagctg tggtgggctc cacccagttc  48960 

gagcttcccg gctgctttgt ttacctaatc aagcctgggc aatggcgggc gtccctcccc  49020 

cagcctcatt gccgccttgc agtttgatct cagactgctg tgctagcaat cagcgagact  49080 

ccgtgggcgt aggaccctcg agccaggtgc gggtataatc tcgtggtgtg ccgttttttg  49140 

tttttttttt gtttgtttgt ttgtttgttt ttgagacgga gtctcgctct gtcgcccagg  49200 

ctggagtgca gtggcgggat ctcggctcac tgcaagctcc gcctgccggg ttcacgccat  49260 

tctcctgcct cagcctccca agtagctggg actacaggcg cccgccacta cgcccggcta  49320 

attttttgta tttttagtag agacggggtt tcaccgtttt agccgggatg gtctcgatct  49380 

cctgacctcg tgatccgccc gcctcggcct cccaaagtgc tgggattaca ggcgtgagcc  49440 

accgcgccgg gccggtgcgc cgttttttaa gcccgtcgga aaagcgcagt attcgggtgg  49500 

gagtgacccg attttccagg tgctgcccct cacccctttc tttgactagg aaagggaact  49560 

ccctgaccac ttgtgcttcc caagtgaggc aatgcctcgc cctgcttcgg ctcgcgcacg  49620 

gtgcgtgcac ccactgacct gcgcccactg tctggcactc cctagtgaga tgaacccggt  49680 

acctcagatg gaaatgcaga aatcacccgt cttctgcgtt gctcaggctg ggagctgtag  49740 

accggagctg ttcctatttg gccatcttgg ctcctcccca agagctggta attattaccc  49800 

aacgtttaga atataatgta acagaacgta tttggaatac atttatcttt taatatttca  49860 

aaggagagat aaatgcaaac taagatttta tttatcatta aattcattgt tctttagttt  49920 

gaaaaacaca tatactcata tttttgtcac gtgattttaa ccagcataaa gcaatagctg  49980 

ccacaccaaa gtcaagtgaa gctgtgaaaa taattaatga aacaattctg ataaaagcat  50040 

ctttatcctt catacaggtt ataggcagga ttgtggttgt gcctgtgtgt gtgtgtggtg  50100 

gggtcggtgg ggggatagga aagttagttt attcacatga ttctcaacat agacatacct  50160 

tgcaaataat tgtgcaattc cctgctttgc tttgcagaca gaatgtcttt gaatcaatgt  50220 

cttttataaa tacactatac aaaaaacata attataaacc tttaaagtat ttatgcatac  50280 

tttaatattc agaactagaa gagaaatttc tgtaaggaga aatgctttgg agattaaaac  50340 

gtaaaggaga taaccaaatt ttactaaacc tctatccatc atgaccacat ttcctttcat  50400 

tctaaaataa aaatattttg gaaatgttga ccccctgccc accatttttg cctgagaagt  50460 

tttaaatgcg aagaacttgc tcctcacttt tgaagcacgg tgctttcttt aggaaccaat  50520 

ctgtatcaca tgcattctat tggtttgtat tttattacat atcattctat tacattttaa  50580 

aatgcatttc tgagcccatg tcattgattt catgattcat taatgggttg caagcagcag  50640 

ctggaataac actggcttag gtaatggcag tataaatcac atgttggtaa aatgaaagat  50700 

gcctttctac caacacgtta taatggccac tcgatgccaa catggcagtg tgttacttaa  50760 

accatatatc aatcaacaca gaagcgacat aaccaaaatg tttttaccat catttataga  50820 

agattatgag tttcatgcag ccaggattaa ttgcattgct attatagaac ttaatataag  50880 

ttgtatcata tttgatggaa ggatgctaac tatcaagagg atataaattt taaaaacaat  50940 

gatactgttt tggataagtc ctgactgtct gcattttatc tagaacaata tatgttcatg  51000 

attaacctag aaaataatga caaaaatgcg caatgagtag agacttgccc ttggaattcc  51060 

taaaattatt aggaaggtac aatgttctcc aagaaatatt ggtaagtgaa aaaaacaagg  51120 

tttacaataa tgtgtaacgg atgctatctg tgtaacaaaa tgtgaaaaaa catatatgca  51180 

attttttggc aagaagatac cctgaaaaca taaaccaaaa agccaaaaaa aaaaaaagaa  51240 

acttgctgat gatacagaac ttctctgact gtacattttt ctcatagtct taactctgat  51300 

aattacatac atgtttgaca ttctcaaaag taagaacata gaagtacata atatatgcag  51360 

gataaagcaa ataacagttt atgtgaagat tgttagaagc caaggttttc atggaagtta  51420 

ttagtatgaa gtcaaagtag tacattaaaa tccagtaatg ttaattggat ttggaagtaa  51480 

cagtatgaaa ttttttctct aaaaatgtgt gcaggaatct cttgcttaag atcagtaatc  51540 

tattcccaaa aatagatttt gagagcacat ttctgttcta aatgggaaca attatagtga  51600 

atggaaggta aaccaaaaca tagctatttc ctacaaaata attgatacct aataatgcct  51660 

gtagaaaagc atagaaaagt gacaaactct cttgataaaa tctaaaatgt atattgtttc  51720 

ctaatcagca attattgatg ttgctaacat acagttgctt tgtatctagt tacatggacc  51780 

gtgtatatgc tgtatgagaa attgtcactt tgtggtatag catagcaaat aacacagcaa  51840 

tcaaaaagtt aaaagtcaaa attaaaataa ataaaagacc cataatagac tgtttttcac  51900 

cttgctaaag gagaaatgac tcttgaagaa actcaagaag ggacaaagaa aggtcaaatc  51960 

agtcattgct atttgcagaa ggtgatgggt ggatggagga caagggggca atcttcgaca  52020 

aactgtcata tgaagaggaa tgaagagacc ttccccatgt ctgtgtctag tagatgctgt  52080 

agagacttgc tggacaggaa agctttggta aatcttcaga aatgatgttc aagctgaagg  52140 

aagatgttca agctgcattt tggttgcaaa atgcagattt gcttgttttt ctgttgaaaa  52200 

attcctctat gtgatgctgt aagagttacc aggctattat taaaagaaag atgtattgtc  52260 

acactgagta tgtctgcaaa actttcttag taactggcct gtttgtttat actcaatgca  52320 

catgttaaga tggctacata ggattcttac tatgttctat caatttggaa tgtaactcaa  52380 

cattcttcca gcacataaag ttgtttacaa tatataatga gtgtgaaaga tataaataaa  52440 

tacttctatt tgatgtctac accaacatta gatgaaaaaa tgaggtagtg agaagttgat  52500 

atatatcctg taaattagca tatttaaaac caaaaatcct tctactgggt aaatattaac  52560 

taggaaaaca ttctacagga tataatgcat ttttctagct ctgccccctg aaaaggtcta  52620 

gtagtaatgt gatctgatgg cactaaacac ttctgatctc aaatgatagc ctctaaaatg  52680 

ctgttttcca acaaagagaa ttagggctcc tgaagaaatg gctgatgcca ggtgtggagg  52740 

agaaaatgag caagctgagc ttgtgatata tgttcacccc agagagggct catgtcacgt  52800 

gggcaccaac agagaggggt ctcaggtagc aacagtgggg ccattggacc atcaaaggag  52860 

caattagggt acttgactga agcctgtagg ttcataattt ttactctaaa acaaggaagg  52920 

aaggtgggaa gaaaggggaa aggaggggaa gggaaggaag aaaggaggga gggagggaag  52980 

gaaggaagta aagagctaga acaacaatga aacaaaatat ctttgataac tttttggggg  53040 

agtacagagt ggaaattttg catttctgga ggctttacaa gatggggatc tgtgcttcag  53100 

aaataagtca gtaaagatac atttacatag atgtttcctc cagtattgct tggaaaagcc  53160 

aaaatcaaag tgatgtagat agttacctaa gagatgaata aaataaataa tggtacatct  53220 

tttgtatgga atattacata gccactaaaa ataatggagc agaactctac tcaatgacat  53280 

ataaaaatgt ccatgatata ttgtataatg gaaaaaaatt ttaaagcaaa aggttttacc  53340 

ttatttattt cacaagctaa aaccctatat gattatacaa accaatgtgt ttgccttaaa  53400 

aagataaaag tctggtgtcc tctaagacca taccacaaac agtgatcacc tctgggattg  53460 

gaattttata gcagttgtgg gtagagagag tttgggagaa aacattcaca ttttatctca  53520 

taattcttat atttttaaag cttttctatg agcattattt tgatacatat gtgagcagag  53580 

atggtccctt ctaggtacca gttacaccat tgctaaacct cgtctgtttc ttccaaaggt  53640 

gtcaggcagc gaacaagacc tgccccacca attacatgtg gaataatcac atctgcagat  53700 

gcctggctca ggaagatttt atgttttcct cggatgctgg agatggtaag cagaatgatg  53760 

ctttaaagac taaacgccag gggtctatat tgtgattaac atactctaat attaaaacca  53820 

cacatataat attcatcatt cttctaaata agtgacnnnn nnnnnnnnnn nnnnnnnnnn  53880 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn  53940 

nnnnnnnnnn nnnnnngggg cttcggcctg ccagctgtgg accccacaaa gaactagaca  54000 

gaaactcatg ccagtgtgtc tgtaaaaaca aactcttccc cagccaatgt ggggccaacc  54060 

gagaatttga tgaaaacaca tgccagtgtg tatgtaaaag aacctgcccc agaaatcaac  54120 

ccctaaatcc tggaaaatgt gcctgtgaat gtacagaaag tccacagaaa tgcttgttaa  54180 

aaggaaagaa gttccaccac caaacatgca ggtaagagat cctcatgaag aatattaatt  54240 

tatcttacta gaaatatgct ctaaaccaaa agcagttttc ttaaccaaag taacaattgc  54300 

ttgttagaat gaggccaata cattctgaaa gacaaaaatc ttatattttt ttaaccaatg  54360 

ctaaacttgc atgagactgg atttgcttcc aaaaaagcaa gacaggagcc ttgaggatca  54420 

aaattagatc caagctcaga atatactcta aattagtggt tcctgtggga tgttttcaaa  54480 

atacctaaag gcaatccact tcacccccac caaaaagtag gatgccttca gacataattt  54540 

aggacaactg ttctaaatgc agacaatttt tatgcaaaac ttacaaacaa aggacaggag  54600 

agtaccactg gtatagaatt atatctggaa atgaaaatac atagctaagt atttgataga  54660 

attagagaaa taaatgaata tgttgttaaa ggtacccaag actcatgtct taaacttggt  54720 

tttgactctt ctgcatcaag aaaaaaaaaa tgaagttatt cgactcaagc acattcagtg  54780 

ttaatcccga aacctcttcc tctttaatct cagagaataa attgatccta ctatttaagc  54840 

cagacctgtc cgtttgacat tttctccaca ttccttcaac attgcttcat gccattcctt  54900 

ctttcttctg taacttgaac atcttcaact ctactttttt tccccttagt ttataaaagt  54960 

tcccaaattt cttctatttt attagcctct tactgtgaag ttgagcaaac tttagcaacc  55020 

caggtctgtt ctcttcaaag ctgctcaaaa acgaggcatt tgttttcacc tctttcacct  55080 

accatgtatt caaccaacta aaacctggtt tatggcctac agtgctatgt taaacctctg  55140 

tttaacgttt cattggcctc atagttgcca aatccaacag agaattttgt ccttaggtaa  55200 

cctgtccttt ccatggcctt tttctttaag ctcacctctt ctatgacatg acattgtact  55260 

ttcctagttg tctacttaat tttcagacca tttgttttta atcactttca ctggattctc  55320 

tttttacatc cattctttac atatgggtag ataatgttct ttttaacaga aaaaagcttg  55380 

tatttcattt atttactatc ttcactaatg ttcattatta tttttccaca gtatataacg  55440 

agttcatctt tttattctca agagttttgt gtccttattt taatgtatat ttttggtggt  55500 

tttcctaaac taatatttta gctactggat aaaaaggctt tagtggatct ggctatctca  55560 

ttgattatat ttttcttccc ttatactgca aagtacatca taggtataat tttattttga  55620 

gaaagaacta tattccaatt ttacctatat attcctactg taataaccaa gaggacattt  55680 

caaaaatgta tatgctacta ttttcagaag gatatagata ctaaatcttt ttaagcctta  55740 

ttaaactatg ttttatattg aattcttatt gagtgaattt agtatacatt gaaaaatctg  55800 

aaattcttat gtctgcatac ctacttttct acagaattat atgaacacag acaatatttc  55860 

tatcattgca tttctcctaa tactatacat tctttcaaaa agactctgtt tactgcaact  55920 

catcctagat tcatttttct aagtgtataa ttccaagctt tgtgatataa attggagcaa  55980 

aagagaaaat atcatatttt gaaaatatat attctcctca ttctctctta atatctatta  56040 

caaagtgcag taggcagagt atatattctt tggttcattg ccttaattaa cagcttttat  56100 

ggatcattaa gtatcatatc cctttttagt agaggaatac cagtagtcat aactatgaat  56160 

ttgcatttgc aaagtttaag aaaaattaac tagacatgct ttcttttcta gagccaaata  56220 

aatgaaatgt caaaaccaaa ttgtgtaggg ttttttatag cacacttttg atttccacct  56280 

caggtcatct tgcacttttc cctttacacc tattcccaca cattaggcat gctcatactc  56340 

caaaatgttt ttaaaagata gctccaattc tcaccaggcg tggtggctca cacctgtaat  56400 

cctagcactt tggaaggcca aggcgggcag atcacttgag gccaggagtt caagaccagc  56460 

ccagccaaca tggcaaaacc ctggctctat gaaaaataca gaaacttagc caggcgtggc  56520 

cacacatacc tgtaatccca gctactcggg aggctgagac acaagaatgg cttgagccca  56580 

ggaggcggag gttgcagtaa gctgagattg ccccactgcg ctccagcctg ggcgacagaa  56640 

caagactctg tctcaaaaaa tgaagcctca attcactgga gcaacagggc aggagacaca  56700 

ctgaccaatt gataatttgt cctctcaccg ctactggaaa ctggaagtct aggatgggag  56760 

gaaggggacc ctcgctgctg agtattgtcc ttctcacctc cctgccccag aatgcctctg  56820 

tttcacctgg agccaaggtg gaaaggaaga gttagtcacc gtcatgcagc cagctgccaa  56880 

gtagagacac aagagtgagg gtggggctgc tcactgagga ccacagttct gccaacatgg  56940 

ctgggtttaa ttgcctggaa gtctgggtcc catacaagtc ccagactcat gtgaattctg  57000 

ccatgcttat gtgatcacag taagtcagca gaccttcagt gttcactttc agaatttctg  57060 

ttttatgttt ctttcaccca tgaagcaaag agcacttcaa aggaactaat gcttacaata  57120 

cctcatcctt ccatttgtta tatcagcaag caagactttt attttaaggc ttttccattc  57180 

ctcttaaatg ttgtaattct aagcagaaac aaactttttt aaacctatga acaatttcgt  57240 

cataaaatta gtaattttat tccagtctga aatttaaaaa cacagaaata ccttggtagc  57300 

atgatgaatc cattgccttg atctttaacg taagtgtgtt cttgtttgtt ctcctagctg  57360 

ttacagacgg ccatgtacga accgccagaa ggcttgtgag ccaggatttt catatagtga  57420 

agaagtgtgt cgttgtgtcc cttcatattg gaaaagacca caaatgatga gctaagattg  57480 

tactgttttc cagttcatcg attttctatt atggaaaact gtgttgccac agtagaactg  57540 

tctgtgaaca gagagaccct tgtgggtcca tgctaacaaa gacaaaagtc tgtctttcct  57600 

gaaccatgtg gataacttta cagaaatgga ctggagctca tctgcaaaag gcctcttgta  57660 

aagactggtt ttctgccaat gaccaaacag ccaagatttt cctcttgtga tttctttaaa  57720 

agaatgacta tataatttat ttccactaaa aatattgttt ctgcattcat ttttatagca  57780 

acaacaattg gtaaaactca ctgtgatcaa tatttttata tcatgcaaaa tatgtttaaa  57840 

ataaaatgaa aattgtatta taagctgcta agttcagtcc attatcatct tacatgatga  57900 

acgaaaacta ctatcatgaa gacactgatc tttctctgcc cttttttgtt ctctaaccag  57960 

atgtcacata tgtattacta tgataaaaag tatgatcctg tgaaagagag tgtcagagga  58020 

caacagaatg ctattgcttc atctcttata tgtttaatga ttataaacat tttagtacat  58080 

gatacttttg aatttatgac caagtgaatc aatatgaaac atcttgtaag atagactact  58140 

tagcattgtg attaaaagtc attcagtgct ctgagaacat tcagaatctt acgttggtag  58200 

aaaatcctgc agtatatatt aaaatggctt taaatatttt ctcaaaaata atcttttcca  58260 

aatatttgac tttttctggc cagctaaaat actttttgtg agtggaagtg ctcctatcca  58320 

atacatttta aaaaaaaact aaaaataata tttaaatact catgcatgtt taaaaggaaa  58380 

catttcagcc acacaattga agtgcttgtt tcattttcaa aaaggcatta cactaaaata  58440 

cctttatctt tttttttttt tttcttcaga tagtgttgct ctgttgccca ggctggagtg  58500 

cagtggcgtg atctcagctc actgccccta acctctgcct cccggattca agtgattctc  58560 

ctgcctcagc ctcctgagta gctgggatta taggcacgtg ccggcacacc tggctaattt  58620 

ttgtattttt agtgcagatg gggtttcacc atgttggcca gtctgatcac gaactcctga  58680 

cctcaagtga tccacagacg tcggcctcaa agtgctggga ttacaggcat gaaccaccgt  58740 

gcccagccta aaataccttt tactaaaaca aagattttgc ctgcataaat aaaaccaaat  58800 

aagtggtttg ttcgttgaca gagaattata tactggtaca tcataactac tgtataaaaa  58860 

taattagtcc tgaaaagaga aaatattcct cataagcatg taaaggtagc acattaattt  58920 

aactaaaaca tttttgtttt tttgaaacgg aatctcgctc tgttgcccag gctggagtgc  58980 

agtggtgcga tctcggctca c                                            59001 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

gcaggccgaa gccccgctct                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

tcttttccaa tatgaaggga                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

ggctccgcgt tcccaacttt                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

cttttgcaga tgagctccag                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

ttgcttgcat aagccgtggc                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

ttgtttggtc cacagatgtc                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

taataatgga atgaacttgt                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

gtaactgctc ctccagatct                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

ctccagtcca tttctgtaaa                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

ggtgtagctt tttggagagg                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

tcgtacatgg ccgtctgtaa                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

tgtggtcttt tccaatatga                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

cttgcataag ccgtggcctc                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

catggaatcc atctgttgag                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

gtttggtcat tggcagaaaa                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

ccttctggcg gttcgtacat                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

gtttgttttt acagacacac                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

ttcgctgcct gacactgtgg                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

acaacgacac acttcttcac                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

gtctgtaaac atccagttta                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

acatattttg catgatataa                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

gtgagtttta ccaattgttg                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

caagggtctc tctgttcaca                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

gtaaaagcct cacaggaaac                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

atatgaaaat cctggctcac                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

gacacacatg gaggtttaaa                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

attgagtctt tctccactca                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

aatcttcctg agccaggcat                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

agatgagctc cagtccattt                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

ttggctgttt ggtcattggc                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

gcaagtgcat ggtggaagga                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

gaatgcagaa acaatatttt                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

tagtcattct tttaaagaaa                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

cttggctgtt tggtcattgg                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

aggaaaatct tggctgtttg                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

cctttcctta gctgacactt                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

taaacatcca gtttagacat                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

gcgggtgtca ggtaaaagcc                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

aggccgcggg cccctcctgg                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

aagaagccca gcaagtgcat                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

cactggacac agaccgtaac                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

tctgtccttg agttgaggtt                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

atacttttca agatctctgt                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

ttatcaatac ttttcaagat                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

actattgcag caacccccac                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

gtgttgctgg cagggaacgt                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

catctccagc atccgaggaa                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

tcatccagct ccttgtttgg                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

gtccacagct ggcaggccga                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

ttcttttaca tacacactgg                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

acattcacag gcacattttc                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

ggtggtggaa cttctttcct                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

gtacaatctt agctcatttg                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

cacagacagt tctactgtgg                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

aataaattat atagtcattc                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

aattgttgtt gctataaaaa                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

agttgcctga tgatccaaga                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

tttatcctcg gccactcccg                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

tttagaggtg atgcgaccac                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

ctccctggag ctccccgttt                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

actctccctc ggaagccgtc                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

ccttccccga agtgagagga                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

tgacgaaatt gttaaaaggt                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

atttcagact gaaatacaat                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

gcagacctac cgtggcctcg                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

ccatacttac ttttcaagat                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

caatacccac cgtcttgctg                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

acacattttg tacaggtatc                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

aggaaacacg atgatgccca                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

ggagaacttt gaagcagttt                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

tcatactcac tgtggtagtg                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

attctttcat tgtcagagct                                                 20 

 
           
             86  
             20  
             DNA  
             H. sapiens  
             
 
           
            86 

aaagttggga acgcggagcc                                                 20 

 
           
             87  
             20  
             DNA  
             H. sapiens  
             
 
           
            87 

ctggagctca tctgcaaaag                                                 20 

 
           
             88  
             20  
             DNA  
             H. sapiens  
             
 
           
            88 

gccacggctt atgcaagcaa                                                 20 

 
           
             89  
             20  
             DNA  
             H. sapiens  
             
 
           
            89 

gacatctgtg gaccaaacaa                                                 20 

 
           
             90  
             20  
             DNA  
             H. sapiens  
             
 
           
            90 

agatctggag gagcagttac                                                 20 

 
           
             91  
             20  
             DNA  
             H. sapiens  
             
 
           
            91 

ttacagacgg ccatgtacga                                                 20 

 
           
             92  
             20  
             DNA  
             H. sapiens  
             
 
           
            92 

tcatattgga aaagaccaca                                                 20 

 
           
             93  
             20  
             DNA  
             H. sapiens  
             
 
           
            93 

gaggccacgg cttatgcaag                                                 20 

 
           
             94  
             20  
             DNA  
             H. sapiens  
             
 
           
            94 

ttttctgcca atgaccaaac                                                 20 

 
           
             95  
             20  
             DNA  
             H. sapiens  
             
 
           
            95 

gtgtgtctgt aaaaacaaac                                                 20 

 
           
             96  
             20  
             DNA  
             H. sapiens  
             
 
           
            96 

taaactggat gtttacagac                                                 20 

 
           
             97  
             20  
             DNA  
             H. sapiens  
             
 
           
            97 

caacaattgg taaaactcac                                                 20 

 
           
             98  
             20  
             DNA  
             H. sapiens  
             
 
           
            98 

gtttcctgtg aggcttttac                                                 20 

 
           
             99  
             20  
             DNA  
             H. sapiens  
             
 
           
            99 

tttaaacctc catgtgtgtc                                                 20 

 
           
             100  
             20  
             DNA  
             H. sapiens  
             
 
           
            100 

tgagtggaga aagactcaat                                                 20 

 
           
             101  
             20  
             DNA  
             H. sapiens  
             
 
           
            101 

gccaatgacc aaacagccaa                                                 20 

 
           
             102  
             20  
             DNA  
             H. sapiens  
             
 
           
            102 

tccttccacc atgcacttgc                                                 20 

 
           
             103  
             20  
             DNA  
             H. sapiens  
             
 
           
            103 

ccaatgacca aacagccaag                                                 20 

 
           
             104  
             20  
             DNA  
             H. sapiens  
             
 
           
            104 

caaacagcca agattttcct                                                 20 

 
           
             105  
             20  
             DNA  
             H. sapiens  
             
 
           
            105 

ggcttttacc tgacacccgc                                                 20 

 
           
             106  
             20  
             DNA  
             H. sapiens  
             
 
           
            106 

ccaggagggg cccgcggcct                                                 20 

 
           
             107  
             20  
             DNA  
             H. sapiens  
             
 
           
            107 

atgcacttgc tgggcttctt                                                 20 

 
           
             108  
             20  
             DNA  
             H. sapiens  
             
 
           
            108 

gttacggtct gtgtccagtg                                                 20 

 
           
             109  
             20  
             DNA  
             H. sapiens  
             
 
           
            109 

aacctcaact caaggacaga                                                 20 

 
           
             110  
             20  
             DNA  
             H. sapiens  
             
 
           
            110 

acagagatct tgaaaagtat                                                 20 

 
           
             111  
             20  
             DNA  
             H. sapiens  
             
 
           
            111 

gtgggggttg ctgcaatagt                                                 20 

 
           
             112  
             20  
             DNA  
             H. sapiens  
             
 
           
            112 

acgttccctg ccagcaacac                                                 20 

 
           
             113  
             20  
             DNA  
             H. sapiens  
             
 
           
            113 

tcggcctgcc agctgtggac                                                 20 

 
           
             114  
             20  
             DNA  
             H. sapiens  
             
 
           
            114 

ccagtgtgta tgtaaaagaa                                                 20 

 
           
             115  
             20  
             DNA  
             H. sapiens  
             
 
           
            115 

gaaaatgtgc ctgtgaatgt                                                 20 

 
           
             116  
             20  
             DNA  
             H. sapiens  
             
 
           
            116 

aggaaagaag ttccaccacc                                                 20 

 
           
             117  
             20  
             DNA  
             H. sapiens  
             
 
           
            117 

caaatgagct aagattgtac                                                 20 

 
           
             118  
             20  
             DNA  
             H. sapiens  
             
 
           
            118 

ccacagtaga actgtctgtg                                                 20 

 
           
             119  
             20  
             DNA  
             H. sapiens  
             
 
           
            119 

aaacggggag ctccagggag                                                 20 

 
           
             120  
             20  
             DNA  
             H. sapiens  
             
 
           
            120 

tcctctcact tcggggaagg                                                 20 

 
           
             121  
             20  
             DNA  
             H. sapiens  
             
 
           
            121 

cgaggccacg gtaggtctgc                                                 20 

 
           
             122  
             20  
             DNA  
             H. sapiens  
             
 
           
            122 

cagcaagacg gtgggtattg                                                 20 

 
           
             123  
             20  
             DNA  
             H. sapiens  
             
 
           
            123 

gatacctgta caaaatgtgt                                                 20 

 
           
             124  
             20  
             DNA  
             H. sapiens  
             
 
           
            124 

tgggcatcat cgtgtttcct                                                 20 

 
           
             125  
             20  
             DNA  
             H. sapiens  
             
 
           
            125 

agctctgaca atgaaagaat                                                 20