Patent Publication Number: US-2003224515-A1

Title: Antisense modulation of sterol regulatory element-binding protein-1 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of sterol regulatory element-binding protein-1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding sterol regulatory element-binding protein-1. Such compounds have been shown to modulate the expression of sterol regulatory element-binding protein-1.  
       BACKGROUND OF THE INVENTION  
       [0002] Cholesterol and fatty acids are primary components of cellular membranes. Cholesterol plays several essential roles in mammalian cell biology. It modulates the properties of cell membranes and serves as the precursor for steroid hormones, bile acids, and vitamin D and is required for proper embryonic patterning. High plasma cholesterol levels contribute to atherosclerotic disease, whereas cholesterol deficit causes developmental defects, thus cholesterol levels must be carefully controlled. Fatty acid synthesis, called lipogenesis, is an energy storage system specialized to adipose tissue and the liver and is also required to support cellular growth. Lipogenesis is stimulated primarily by hormones such as insulin and the availability of carbohydrates (Shimano,  Prog. Lipid Res.,  2001, 40, 439-452).  
       [0003] The transcription of genes involved in cholesterol and fatty acid biosynthesis is controlled by the transcription factors known as sterol regulatory element-binding protein-1 and -2. These target genes include, but are not limited to: LDL receptor, HMG CoA synthase, HMG CoA reductase, farnesyl diphosphate synthase, squalene synthase, lanosterol 14a-demethylase, acetyl CoA carboxylase, fatty acid synthase, stearoly CoA desaturase-1 and -2, acetyl CoA binding protein, ATP citrate lyase, malic enzyme, PPAR gamma, Acetyl CoA synthase, glycerol-3-phosphate acyltransferase, lipoprotein lipase, and HCL receptor. The 5′ region of these genes contains the sterol regulatory element-1 (SRE-1) or E-box promoters to which the basic helix-loop-helix sterol regulatory element-binding protein-1 binds (Shimano,  Prog. Lipid Res.,  2001, 40, 439-452).  
       [0004] The gene encoding sterol regulatory element-binding protein-1 (also called SREBP-1, SREBP-1a, SREBP-1c, sterol regulatory element BP-1c, sterol regulatory element-binding transcription factor 1, and SREBF1) was cloned in 1993 and two alternatively spliced isoforms exist, termed SREBP-1a and SREBP-1c, with alternative sequences on both the 5′ and 3′ ends (Yokoyama et al.,  Cell,  1993, 75, 187-197). Both of these activate transcription of genes containing SRE-1 promoters, therefore the significance of the alternative splicing is not currently known. Disclosed and claimed in U.S. Pat. No. 5,527,690 is a nucleic acid sequence encoding sterol regulatory element-binding protein-1, as are expression vectors expressing the recombinant DNA, and host cells containing said vectors (Goldstein et al., 1996).  
       [0005] In a feedback control mechanism, the intracellular cholesterol levels serves as a regulator of transcriptional activity whereby transcription is suppressed when cholesterol levels increase. Sterol regulatory element-binding protein-1 is localized to the endoplasmic reticulum by a C-terminal hydrophobic extension. In sterol-depleted cells, sterol regulatory element-binding protein-1 is cleaved by sterol regulatory element-binding protein-1 cleavage activating protein (SCAP), a protease which is inhibited by cholesterol. The soluble form of sterol regulatory element-binding protein-1 then translocates to the nucleus. Upon accumulation of sterols in the cells, sterol regulatory element-binding protein-1 remains bound to the membrane and transcription of sterol regulated genes decreases. (Sakai and Rawson,  Curr. Opin. Lipidol.,  2001, 12, 261-266).  
       [0006] Sterol regulatory element-binding protein-1 may also play a role in repressing the transcription of some genes with SRE-1 promoters via a postulated mechanism whereby sterol regulatory element-binding protein-1 displaces a positive regulator of the those gene. Repression of caveolin transcription by sterol regulatory element-binding protein-1 has been observed and this may be another feature of sterol regulation since caveolin is involved in regulating cellular cholesterol content (Bist et al.,  Proc. Natl. Acad. Sci. U. S. A.,  1997, 94, 10693-10698).  
       [0007] Sterol regulatory element-binding protein-1c may a link cholesterol and fatty acid metabolism. The liver X receptors (LXR) are a class of transcription factors that are induced by oxysterols, which mostly arise as metabolic derivatives of cholesterol. One of the target genes transcribed by LXRs is sterol regulatory element-binding protein-1c, the upregulation of which promotes lipid synthesis to coordinate the homeostatic balance between fatty acids and sterols (Repa et al.,  Genes Dev.,  2000, 14, 2819-2830).  
       [0008] Glucose and insulin are required for the production of fatty acids via the induction of hepatic lipogenic enzymes. Sterol regulatory element-binding protein-1c is upregulated by insulin in vivo and in hepatocyte cultures (Azzout-Marniche et al.,  Biochem. J.,  2000, 350 Pt 2, 389-393.; Shimomura et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1997, 94, 12354-12359). Sterol regulatory element-binding protein-1c is also upregulated in the ob/ob mouse and a transgenic mouse model of lipodystrophy (Shimomura et al.,  Mol. Cell,  2000, 6, 77-86). The pivotal role sterol regulatory element-binding protein-1 has in lipid metabolism and the action of insulin suggests that sterol regulatory element-binding protein-1c might be involved in pathologies such as type 2 diabetes, obesity, and insulin resistance syndromes and is a potential target for pharmacological manipulation (Ferre et al.,  Biochem. Soc. Trans.,  2001, 29, 547-552).  
       [0009] Growth-factor induced activation of the sterol regulatory element-binding protein-1 pathway has been proposed as one of the mechanisms responsible for upregulation of lipogenic gene expression in a subset of cancer cells. In LNCaP prostate cancer cells, the growth factor EGF stimulates sterol regulatory element-binding protein-1 expression which then leads to upregulation of the expression of fatty acid synthase (FAS). This pathway has been suggested as a target for chemotherapeutic intervention because increased expression of FAS has been observed in certain aggressive cancers such as prostate, breast, ovary, colon, tongue, thyroid, and endometrium (Swinnen et al.,  Oncogene,  2000, 19, 5173-5181).  
       [0010] Upregulation or increase in soluble sterol regulatory element-binding protein-1 may be a side effect of antiretroviral therapy used in AIDS patients. Highly-active antiretroviral therapy (HAART) has dramatically reduced AIDS-related deaths, however long-term HAART has been associated with a unique syndrome of lipodystrophy and other metabolic complications such as hyperlipidemia, insulin resistance, and lactic acidosis. Lipodystrophy observed in AIDS patients has also been observed in a mouse model overexpressing sterol regulatory element-binding protein-1 (Shimomura et al.,  Genes Dev.,  1998, 12, 3182-3194). Thus HAART-associated lipodystrophy has been attributed overexpression or an increase in soluble sterol regulatory element-binding protein-1, which leads to perturbations in the synergistic regulation of genes involved in maintenance of cholesterol homeostasis (Nerurkar et al.,  Clin. Biochem.,  2001, 34, 519-529). Consistent with this hypothesis is the observation that sterol regulatory element-binding protein-1 is upregulated in 3T3-L1 preadipocytes undergoing differentiation enhanced by ritonavir, a protease inhibitor used in HIV therapy. The postulated mechanism involves ritonavir-stimulated inhibition of proteasomal activity, the route through which sterol regulatory element-binding protein-1 is degraded in cells (Nguyen et al.,  AIDS,  2000, 14, 2467-2473).  
       [0011] Transgenic mice overexpressing sterol regulatory element-binding protein-1 in adipose tissue exhibit many of the features of congenital generalized lipodystrophy, an autosomal recessive disorder in humans characterized by profound insulin resistance, hyperinsulinemia, hyperglycemia, a paucity of white fat, and an enlarged fatty liver (Shimomura et al.,  Genes Dev.,  1998, 12, 3182-3194).  
       [0012] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of sterol regulatory element-binding protein-1 and to date, investigative strategies aimed at modulating sterol regulatory element-binding protein-1 function have involved the use of an antisense expression vector. The decreased expression of by an antisense cDNA in HepG2 cells illustrated that sterol regulatory element-binding protein-1 is selectively involved in the signal transduction pathway of insulin and insulin-like growth factor leading to low density lipoprotein receptor gene activation (Streicher et al.,  Z Ernahrungswiss,  1998, 37, 85-87.; Streicher et al.,  J. Biol. Chem.,  1996, 271, 7128-7133).  
       [0013] A natural process in which sterol regulatory element-binding protein-1 expression is suppressed demonstrates the potential benefits of downregulating genes encoding proteins of lipid synthesis. Polyunsaturated fatty acids decrease the nuclear abundance and expression of sterol regulatory element-binding protein-1 and simultaneously upregulate the expression of genes encoding proteins involved in fatty acid oxidation. These beneficial effects associated with oxidation of fatty acids instead of storage include a reduced risk of heart disease and improvements in the metabolic syndrome such as increased insulin sensitivity (Clarke,  J. Nutr.,  2001, 131, 1129-1132).  
       [0014] Consequently, there remains a long felt need for agents capable of effectively inhibiting sterol regulatory element-binding protein-1 function.  
       [0015] 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 sterol regulatory element-binding protein-1 expression.  
       [0016] The present invention provides compositions and methods for modulating sterol regulatory element-binding protein-1 expression.  
       SUMMARY OF THE INVENTION  
       [0017] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding sterol regulatory element-binding protein-1, and which modulate the expression of sterol regulatory element-binding protein-1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of sterol regulatory element-binding protein-1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of sterol regulatory element-binding protein-1 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  
       [0018] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding sterol regulatory element-binding protein-1, ultimately modulating the amount of sterol regulatory element-binding protein-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding sterol regulatory element-binding protein-1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding sterol regulatory element-binding protein-1” encompass DNA encoding sterol regulatory element-binding protein-1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, 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 sterol regulatory element-binding protein-1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.  
       [0019] 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 sterol regulatory element-binding protein-1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding sterol regulatory element-binding protein-1, regardless of the sequence(s) of such codons.  
       [0020] 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.  
       [0021] 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.  
       [0022] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0023] 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.  
       [0024] 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.  
       [0025] 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.  
       [0026] 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.  
       [0027] 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.  
       [0028] 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).  
       [0029] 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.  
       [0030] 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.  
       [0031] 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.  
       [0032] 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.  
       [0033] 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.  
       [0034] 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.  
       [0035] 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.  
       [0036] 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).  
       [0037] 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.  
       [0038] 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.  
       [0039] 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.  
       [0040] 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.  
       [0041] 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.  
       [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 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.  
       [0043] 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.  
       [0044] 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.  
       [0045] 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.  
       [0046] 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.  
       [0047] 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.  
       [0048] 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.  
       [0049] 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.  
       [0050] 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.  
       [0051] 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.  
       [0052] Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.  
       [0053] 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.  
       [0054] 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.  
       [0055] 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.  
       [0056] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res.,  1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &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.  
       [0057] 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.  
       [0058] 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.  
       [0059] 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.  
       [0060] 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.  
       [0061] 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.  
       [0062] 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.  
       [0063] 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.  
       [0064] 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.  
       [0065] 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.  
       [0066] 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.  
       [0067] 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 sterol regulatory element-binding protein-1 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.  
       [0068] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding sterol regulatory element-binding protein-1, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding sterol regulatory element-binding protein-1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of sterol regulatory element-binding protein-1 in a sample may also be prepared.  
       [0069] 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. Oligonucleotide&#39;s with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.  
       [0070] 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.  
       [0071] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. 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.  
       [0072] 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.  
       [0073] 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.  
       [0074] 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.  
       [0075] 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.  
       [0076] 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.  
       [0077] Emulsions  
       [0078] 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.  
       [0079] 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).  
       [0080] 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).  
       [0081] 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.  
       [0082] 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).  
       [0083] 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.  
       [0084] 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.  
       [0085] 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.  
       [0086] 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).  
       [0087] 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.  
       [0088] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.  
       [0089] 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.  
       [0090] 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.  
       [0091] Liposomes  
       [0092] 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.  
       [0093] 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.  
       [0094] 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.  
       [0095] 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.  
       [0096] 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.  
       [0097] 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.  
       [0098] 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.  
       [0099] 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).  
       [0100] 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).  
       [0101] 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.  
       [0102] 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).  
       [0103] 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).  
       [0104] 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).  
       [0105] 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.).  
       [0106] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. ( Bull. Chem. Soc. Jpn.,  1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Illum et al. ( FEBS Lett.,  1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. ( FEBS Lett.,  1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. ( Biochimica et Biophysica Acta,  1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0107] 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.  
       [0108] 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.  
       [0109] 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).  
       [0110] 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.  
       [0111] 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.  
       [0112] 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.  
       [0113] 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.  
       [0114] 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).  
       [0115] Penetration Enhancers  
       [0116] 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.  
       [0117] 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.  
       [0118] 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).  
       [0119] 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).  
       [0120] 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).  
       [0121] 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).  
       [0122] 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).  
       [0123] 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.  
       [0124] 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.  
       [0125] Carriers  
       [0126] 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).  
       [0127] Excipients  
       [0128] 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.).  
       [0129] 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.  
       [0130] 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.  
       [0131] 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.  
       [0132] Other Components  
       [0133] 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.  
       [0134] 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.  
       [0135] 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.  
       [0136] 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.  
       [0137] 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.  
       [0138] 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  
     [0139] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0140] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.  
     [0141] 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).  
     [0142] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al.,  Nucleic Acids Research,  1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:  
     [0143] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0144] 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.  
     [0145] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0146] 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).  
     [0147] 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.  
     [0148] 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.  
     [0149] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0150] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DHF (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.  
     [0151] 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.  
     [0152] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0153] 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%).  
     [0154] 2′-Fluoro Amidites  
     [0155] 2′-Fluorodeoxyadenosine Amidites  
     [0156] 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.  
     [0157] 2′-Fluorodeoxyguanosine  
     [0158] 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.  
     [0159] 2′-Fluorouridine  
     [0160] 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.  
     [0161] 2′-Fluorodeoxycytidine  
     [0162] 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.  
     [0163] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0164] 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).  
     [0165] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0166] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.  
     [0167] 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.).  
     [0168] 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.  
     [0169] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0170] 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.  
     [0171] 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.  
     [0172] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0173] 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%).  
     [0174] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0175] 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  
     [0176] 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.  
     [0177] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0178] 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%.  
     [0179] 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)  
     [0180] 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%).  
     [0181] 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)  
     [0182] 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%).  
     [0183] 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)  
     [0184] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).  
     [0185] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0186] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0187] 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.  
     [0188] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0189] O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (R f  0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH 2 Cl 2  (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.  
     [0190] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0191] 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.  
     [0192] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0193] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.  
     [0194] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0195] 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.  
     [0196] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0197] 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.  
     [0198] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0199] 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.  
     [0200] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0201] 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.  
     [0202] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0203] 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.  
     [0204] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0205] 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.  
     [0206] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0207] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].  
     [0208] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0209] 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.  
     [0210] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0211] 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.  
     [0212] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0213] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers were washed with saturated NaHCO 3  solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH 2 Cl 2 /TEA) to afford the product.  
     [0214] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0215] 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  
     [0216] Oligonucleotide Synthesis  
     [0217] 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.  
     [0218] 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.  
     [0219] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0220] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0221] 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.  
     [0222] 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.  
     [0223] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0224] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0225] 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  
     [0226] Oligonucleoside Synthesis  
     [0227] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.  
     [0228] 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.  
     [0229] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0230] PNA Synthesis  
     [0231] 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  
     [0232] Synthesis of Chimeric Oligonucleotides  
     [0233] 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”.  
     [0234] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0235] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide 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.  
     [0236] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0237] [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.  
     [0238] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0239] [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.  
     [0240] 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  
     [0241] Oligonucleotide Isolation  
     [0242] 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  
     [0243] Oligonucleotide Synthesis—96 Well Plate Format  
     [0244] Oligonucleotides were synthesized via solid phase P(IIT) 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.  
     [0245] 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  
     [0246] Oligonucleotide Analysis—96-Well Plate Format  
     [0247] 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  
     [0248] Cell Culture and Oligonucleotide Treatment  
     [0249] 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.  
     [0250] T-24 Cells:  
     [0251] 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.  
     [0252] 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.  
     [0253] A549 Cells:  
     [0254] 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.  
     [0255] NHDF Cells:  
     [0256] 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.  
     [0257] HEK Cells:  
     [0258] 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.  
     [0259] b.END Cells:  
     [0260] The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). 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 3000 cells/well for use in RT-PCR analysis.  
     [0261] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.  
     [0262] Treatment with Antisense Compounds:  
     [0263] 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.  
     [0264] 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  
     [0265] Analysis of Oligonucleotide Inhibition of Sterol Regulatory Element-Binding Protein-1 Expression  
     [0266] Antisense modulation of sterol regulatory element-binding protein-1 expression can be assayed in a variety of ways known in the art. For example, sterol regulatory element-binding protein-1 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.  
     [0267] Protein levels of sterol regulatory element-binding protein-1 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 sterol regulatory element-binding protein-1 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).  
     [0268] 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  
     [0269] Poly(A)+ mRNA Isolation  
     [0270] 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.  
     [0271] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0272] Total RNA Isolation  
     [0273] 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 RWl 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.  
     [0274] 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  
     [0275] Real-Time Quantitative PCR Analysis of Sterol Regulatory Element-Binding Protein-1 mRNA Levels  
     [0276] Quantitation of sterol regulatory element-binding protein-1 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.  
     [0277] 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.  
     [0278] 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).  
     [0279] 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 RiboGreenTM (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 RiboGreenTM 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).  
     [0280] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM 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.  
     [0281] Probes and primers to human sterol regulatory element-binding protein-1 were designed to hybridize to a human sterol regulatory element-binding protein-1 sequence, using published sequence information (GenBank accession number U00968.1, incorporated herein as SEQ ID NO:4). For human sterol regulatory element-binding protein-1 the PCR primers were:  
     [0282] forward primer: GTCCTGCGTCGAAGCTTTG (SEQ ID NO: 5)  
     [0283] reverse primer: AGGTCGAACTGTGGAGGCC (SEQ ID NO: 6) and the PCR probe was: FAM-AGGCCGAAGGCAGTGCAAGAGACTC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:  
     [0284] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)  
     [0285] reverse primer: GAAGATGGTGATGGGATTTC GGGTCTCGCTCCTGGAAGAT (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.  
     [0286] Probes and primers to mouse sterol regulatory element-binding protein-1 were designed to hybridize to a mouse sterol regulatory element-binding protein-1 sequence, using published sequence information. A consensus sequence of mouse sterol regulatory element-binding protein-1 was assembled using GenBank accession numbers AI116616, BF385567, AB017337, AI552487, BF160829, BE553319, NM — 024166 and AW476364, and is incorporated herein as SEQ ID NO:11. For mouse sterol regulatory element-binding protein-1 the PCR primers were:  
     [0287] forward primer: TTGGCCACAGTACCTTTGGTT (SEQ ID NO:12)  
     [0288] reverse primer: CTGAGCCTAGGGCCTTGCT (SEQ ID NO: 13) and the PCR probe was: FAM-CATCCACCGACTCGCAGCTGG-TAMRA (SEQ ID NO: 14) where FAM is the fluorescent reporter dye and TAMRA is the quencher dye. For mouse GAPDH the PCR primers were:  
     [0289] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO:15)  
     [0290] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO:16) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 17) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     [0291] Northern Blot Analysis of Sterol Regulatory Element-Binding Protein-1 mRNA Levels  
     [0292] 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.  
     [0293] To detect human sterol regulatory element-binding protein-1, a human sterol regulatory element-binding protein-1 specific probe was prepared by PCR using the forward primer GTCCTGCGTCGAAGCTTTG (SEQ ID NO: 5) and the reverse primer AGGTCGAACTGTGGAGGCC (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.).  
     [0294] To detect mouse sterol regulatory element-binding protein-1, a mouse sterol regulatory element-binding protein-1 specific probe was prepared by PCR using the forward primer TTGGCCACAGTACCTTTGGTT (SEQ ID NO: 12) and the reverse primer CTGAGCCTAGGGCCTTGCT (SEQ ID NO: 13). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0295] 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  
     [0296] Antisense Inhibition of Human Sterol Regulatory Element-Binding Protein-1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap  
     [0297] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human sterol regulatory element-binding protein-1 RNA, using published sequences (GenBank accession number U00968.1, incorporated herein as SEQ ID NO: 4, residues 79000-10600 of GenBank accession number NT — 010657.5, incorporated herein as SEQ ID NO: 18, GenBank accession number AV704194.1, incorporated herein as SEQ ID NO: 19, and GenBank accession number NM — 004176.1, incorporated herein as SEQ ID NO: 20). 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 sterol regulatory element-binding protein-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. 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 sterol regulatory element-binding           protein-1 mRNA levels by chimeric phosphorothioate       oligonucleotides having 2′-MOE wings and a deoxy gap                                                         TARGET                   CONTROL                   SEQ ID   TARGET       %   SEQ ID   SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   NO   NO                                                         166175   Coding   4   981   tgtctgcacagtggtgccag   60   21   1                   166181   Coding   4   1521   ctccgagtcactgccactgc   75   22   1               206245   Exon: Exon   19   90   tgaagcatgtcttcgaaagt   18   23   1           Junction                   219635   Coding   20   273   gtcactgtcttggttgttga   48   24   1               219636   Coding   20   278   gggaagtcactgtcttggtt   65   25   1               219637   Coding   20   283   ggccagggaagtcactgtct   84   26   1               219642   Coding   20   893   gagtctgccttgatgaagtg   49   27   1               219644   Coding   20   1088   gccttgctgccagctgcgag   65   28   1               219647   Coding   20   1229   gcagatttattcagctttgc   59   29   1               219648   Coding   20   1234   agacagcagatttattcagc   51   30   1               219649   Coding   20   1239   gcgcaagacagcagatttat   69   31   1               219650   Coding   20   1244   gccttgcgcaagacagcaga   53   32   1               219651   Coding   20   1249   cgatggccttgcgcaagaca   55   33   1               219652   Coding   20   1254   gtagtcgatggccttgcgca   65   34   1               219657   Coding   20   1610   aggcgggagcggtccagcat   52   35   1               219667   Coding   20   2440   ggcagagccactgcatggca   65   36   1               219672   Coding   20   2780   gaggcccaccacttggccac   45   37   1               219673   Coding   20   2806   gccagtggatcaccacagct   62   38   1               219675   Coding   20   2928   ccgggcagccttgaaggagt   49   39   1               219678   Coding   20   2994   actggccttctcacagatgg   58   40   1               219679   Coding   20   3004   gcaggtacccactggccttc   80   41   1               219696   3′UTR   20   4091   ctatgaaaataaagtttgca   58   42   1               220043   Start   18   14369   gccgacttcacctgtcaagg   46   43   1           Codon               220046   Intron:   18   18250   ggagggcttcctgcagaaat   42   44   1           Exon           Junction               220047   Intron:   18   19601   atttattcagctgcacggtg   74   45   1           Exon           Junction               220048   Intron:   18   21600   gtgcttccctggaaggcaag   0   46   1           Exon           Junction               220049   Intron   18   22459   gcaccagccttggccaggag   66   47   1               220050   Intron   18   23023   ccctgtggaaggagagagct   24   48   1               220051   Intron:   18   23228   gggtctacgcctgcagaaga   17   49   1           Exon           Junction               220052   Exon:   18   24407   gggcactcaccctccgcatg   64   50   1           Intron           Junction               220053   Coding   19   31   gtccaggccgttggccctac   62   51   1               220054   Coding   19   73   agtgcaatccatggctccgc   67   52   1               220055   Exon: Exon   19   97   gataagctgaagcatgtctt   54   53   1           Junction               220056   5′UTR   20   33   gtcctgccctggcctcagag   68   54   1               220057   Start   20   159   tggctcgtccatggcgcagc   58   55   1           Codon               220058   Coding   20   174   cgcctcgctgaagggtggct   67   56   1               220059   Coding   20   249   ctgaagcatgtcttcgatgt   45   57   1               220060   Coding   20   386   ctcaatgtggcaggaggtgg   59   58   1               220061   Coding   20   597   tgggaagctctgtggcagga   62   59   1               220062   Coding   20   718   ccagtggcaggccaggcagc   67   60   1               220063   Coding   20   998   agggtcggcaaaggccctgt   61   61   1               220064   Coding   20   1074   tgcgagccggttgataggca   47   62   1               220065   Coding   20   1127   gctgtgcgcttctctccacg   64   63   1               220066   Coding   20   1204   tgcccaccaccagatccttg   67   64   1               220067   Coding   20   1310   cgcagacttaggttctcctg   74   65   1               220068   Coding   20   1330   tgcttttgtggacagcagtg   59   66   1               220069   Coding   20   1364   ctgccacaggccgacaccag   60   67   1               220070   Coding   20   1915   cagcctgcttgcgatgcctc   66   68   1               220071   Coding   20   1927   ccaggtccaggtcagcctgc   49   69   1               220072   Coding   20   2173   gcttatggtagaccagggct   46   70   1               220073   Coding   20   2204   gtgtgcttccccatggtgtg   36   71   1               220074   Coding   20   2310   cgccacatagatctcggcca   72   72   1               220075   Coding   20   2317   atgcagccgccacatagatc   51   73   1               220076   Coding   20   2584   ctcgctctaagagatgttcc   72   74   1               220077   Coding   20   2631   atcagctgacccagggctgg   67   75   1               220078   Coding   20   2655   ggcatccgagaattccttgt   32   76   1               220079   Coding   20   2754   tacgccggtggtggtggcca   32   77   1               220080   Coding   20   2966   gctggaccagactctgcctt   64   78   1               220081   Coding   20   3037   agctgctggctggtgtggta   64   79   1               220082   Coding   20   3182   cgcagctcaagggcggaagc   67   80   1               220083   Coding   20   3547   tctgctgacagtcgtgcagc   38   81   1               220084   Coding   20   3560   aggcgcatgagcatctgctg   72   82   1               220086   Coding   20   3585   ggaagtgacagtggtcccac   59   83   1               220089   Stop Codon   20   3595   gggtctagctggaagtgaca   66   84   1               220091   3′UTR   20   3643   cacgggaccaaagtggctag   80   85   1               220094   3′UTR   20   3657   caggacagaagctgcacggg   60   86   1               220096   3′UTR   20   3732   ggcacacagcagccgcaggt   75   87   1               220099   3′UTR   20   3745   cttccaccgcgaaggcacac   61   88   1               220101   3′UTR   20   3785   atggccgccggtcttagggt   24   89   1               220104   3′UTR   20   3795   cagcaccatcatggccgccg   85   90   1               220106   3′UTR   20   3874   ctaaggtgcctgcagagcaa   75   91   1               220109   3′UTR   20   923   acagggaaatgtacccctct   80   92   1               220111   3′UTR   20   3937   tggcttccgtcagcacaggg   53   93   1               220114   3′UTR   20   3953   tccgggaaagccaagttggc   57   94   1               220116   3′UTR   20   4043   tcaggaggctaagcacgctg   63   95   1               220118   3′UTR   20   4079   agtttgcaaaaggcaaagta   52   96   1               220120   3′UTR   20   4118   ttaattctctgtacaaaact   63   97   1                  
 
     [0298] As shown in Table 1, SEQ ID NOs 21, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 72, 73, 74, 75, 78, 79, 80, 82, 83, 84, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96 and 97 demonstrated at least 40% inhibition of human sterol regulatory element-binding protein-1 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 3. 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 3 is the species in which each of the preferred target regions was found.  
     Example 16  
     [0299] Antisense Inhibition of Mouse Sterol Regulatory Element-Binding Protein-1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap.  
     [0300] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse sterol regulatory element-binding protein-1 RNA, using published sequences (a consensus sequence of mouse sterol regulatory element-binding protein-1 assembled using GenBank accession numbers AI116616, BF385567, AB017337, AI552487, BF160829, BE553319, NM — 024166 and AW476364, incorporated herein as SEQ ID NO: 11, GenBank accession number AB046200.1, incorporated herein as SEQ ID NO: 98, GenBank accession number AI115845.1, incorporated herein as SEQ ID NO: 99, and a sequence assembled from orded contigs from GenBank accession number AC096624.3, incorporated herein as SEQ ID NO: 100). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 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 mouse sterol regulatory element-binding protein-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which b.END 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 2                          Inhibition of mouse sterol regulatory element-binding           protein-1 mRNA levels by chimeric phosphorothioate       oligonucleotides having 2′-MOE wings and a deoxy gap                                                         TARGET                   CONTROL                   SEQ ID   TARGET       %   SEQ ID   SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   NO   NO                                                         206241   Exon: Exon   99   78   tggagcatgtcttcaaatgt   29   101   1               Junction               219634   Start   11   61   tgtgcaatccatggctccgt   64   102   1           Codon               219638   Genomic   11   348   aagagaagctctcaggagag   11   103   1               219639   Genomic   11   396   ccttgggtcctcccaggaag   46   104   1               219640   Genomic   11   416   ggacaagggtgcaggtgtca   47   105   1               219641   Genomic   11   515   gatggtgagtggcactggct   57   106   1               219643   Coding   11   1026   ggatgggcagtttgtctgtg   88   107   1               219645   Coding   11   1090   gctgtgcgcttctcaccacg   74   108   1               219646   Coding   11   1110   gcttctcaatggcattgtgg   72   109   1               219653   Coding   11   1326   cactgccacaagctgacacc   72   110   1               219654   Coding   11   1350   ccatagacacatctgtgcct   60   111   1               219655   Coding   11   1476   gctcagagtcactgccacca   74   112   1               219656   Exon: Exon   11   1515   gggctttgacctggctatcc   61   113   1           Junction               219658   Exon: Exon   11   1711   ttagagccatctctgctctc   27   114   1           Junction               219659   Genomic   11   1731   gcagcaaccactgggtccaa   51   115   1               219660   Genomic   11   1759   agtccattggccagccagac   57   116   1               219661   Genomic   11   1779   ccaagcaggccaacactagt   52   117   1               219662   Genomic   11   1854   tgcgatgtctccagaagtgt   64   118   1               219663   Genomic   11   1966   gccagatccaggtttgaggt   57   119   1               219664   Genomic   11   2038   tggcctgccagccagcggcc   40   120   1               219665   Exon: Exon   11   2152   gtgtacttgcccatggcatg   43   121   1           Junction               219666   Coding   11   2250   agatctctgccagtgttgcc   65   122   1               219668   Genomic   11   2400   gacctacagggtggcagagc   62   123   1               219669   Genomic   11   2478   ctgggttcccagccacgctg   63   124   1               219670   3′UTR   11   2597   ggcatctgagaactccctgt   75   125   1               219671   3′UTR   11   2714   ccacttggccactgggtctg   52   126   1               219674   3′UTR   11   2864   agccttgaaggagtacagag   42   127   1               219676   3′UTR   11   2894   cacctttctgtggtccagca   78   128   1               219677   3′UTR   11   2920   atggccaggctggctgggct   30   129   1               219680   3′UTR   11   3013   tcacacaggagcagctgcat   55   130   1               219681   3′UTR   11   3024   caagaagtagatcacacagg   55   131   1               219682   3′UTR   11   3096   cattgctggtaccgtgagct   63   132   1               219683   3′UTR   11   3119   ctccagagcagaggcctggg   25   133   1               219684   3′UTR   11   3129   aaccacgcagctccagagca   64   134   1               219685   3′UTR   11   3139   tcatgttggaaaccacgcag   52   135   1               219686   3′UTR   11   3149   gctgctcaggtcatgttgga   29   136   1               219687   3′UTR   11   3214   gctgtggcctcatgtaggaa   56   137   1               219688   3′UTR   11   3224   catcagccgagctgtggcct   52   138   1               219689   3′UTR   11   3245   ccgggcaggacttgctcctg   36   139   1               219690   3′UTR   11   3299   tttgccactggaacctgccc   56   140   1               219691   3′UTR   11   3349   gtgtgctcccgccatgtggg   60   141   1               219692   3′UTR   11   3497   caggagcatctgctggcagt   24   142   1               219693   Stop Codon11   3537   gggtctagctggaagtgacg   28   143   1               219694   3′UTR   11   3632   tctgccactagaggtcggca   71   144   1               219695   3′UTR   11   3686   gcctacagagcaagagggtg   66   145   1               219697   3′UTR   11   3825   aaaatttctcaacctatgaa   57   146   1               219698   Genomic   98   32   tgagaacacttgtttcaagg   62   147   1               219699   Genomic   98   101   gccaatagctgcctttagat   59   148   1               219700   Genomic   98   227   gtgttcccaccgctggttcg   34   149   1               219701   Genomic   98   334   ttactggcggtcactgtcgt   50   150   1               219702   Intron   100   4956   ttggccgtacctttgcttca   41   151   1               219703   Intron   100   5918   ccacactattctcagctagc   74   152   1               219704   Intron   100   6071   agaaggcagcatcagggtgg   53   153   1               219705   Intron:   100   6211   ttcagtgattctgtaggcag   29   154   1           Exon           Junction               219706   Exon:   100   6426   agagtccaacctggctatcc   43   155   1           Intron           Junction               219707   Exon:   100   7009   cttacccgggccaaatccag   44   156   1           Intron           Junction               219708   Intron   100   7100   gggatgagacagactggaga   21   157   1               219709   Intron:   100   7547   gtgtacttgcctgcaaagtg   46   158   1           Exon           Junction                  
 
     [0301] As shown in Table 2, SEQ ID NOs 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 131, 132, 134, 135, 137, 138, 139, 140, 141, 144, 145, 146, 147, 148, 150, 151, 152, 153, 155, 156 and 158 demonstrated at least 35% inhibition of mouse sterol regulatory element-binding protein-1 expression in this experiment 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 3. 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 3 is the species in which each of the preferred target regions was found.  
                   TABLE 3                          Sequence and position of preferred target regions identified           in sterol regulatory element-binding protein-1.                                                 TARGET           REV                       SEQ ID   TARGET       COMP OF       SEQ ID       SITEID   NO   SITE   SEQUENCE   SEQ ID   ACTIVE IN   NO                                                     81314   4   981   ctggcaccactgtgcagaca   21     H. sapiens     159                   81320   4   1521   gcagtggcagtgactcggag   22     H. sapiens     160               136286   20   273   tcaacaaccaagacagtgac   24     H. sapiens     161               136287   20   278   aaccaagacagtgacttccc   25     H. sapiens     162               136288   20   283   agacagtgacttccctggcc   26     H. sapiens     163               136293   20   893   cacttcatcaaggcagactc   27     H. sapiens     164               136295   20   1088   ctcgcagctggcagcaaggc   28     H. sapiens     165               136298   20   1229   gcaaagctgaataaatctgc   29     H. sapiens     166               136299   20   1234   gctgaataaatctgctgtct   30     H. sapiens     167               136300   20   1239   ataaatctgctgtattgcgc   31     H. sapiens     168               136301   20   1244   tctgctgtcttgcgcaaggc   32     H. sapiens     169               136302   20   1249   tgtcttgcgcaaggccatcg   33     H. sapiens     170               136303   20   1254   tgcgcaaggccatcgactac   34     H. sapiens     171               136308   20   1610   atgctggaccgctcccgcct   35     H. sapiens     172               136318   20   2440   tgccatgcagtggctctgcc   36     H. sapiens     173               136323   20   2780   gtggccaagtggtgggcctc   37     H. sapiens     174               136324   20   2806   agctgtggtgatccactggc   38     H. sapiens     175               136326   20   2928   actccttcaaggctgcccgg   39     H. sapiens     176               136329   20   2994   ccatctgtgagaaggccagt   40     H. sapiens     177               136330   20   3004   gaaggccagtgggtacctgc   41     H. sapiens     178               136347   20   4091   tgcaaactttattttcatag   42     H. sapiens     179               123906   18   14369   ccttgacaggtgaagtcggc   43     H. sapiens     180               136698   18   18250   atttctgcaggaagccctcc   44     H. sapiens     181               136699   18   19601   caccgtgcagctgaataaat   45     H. sapiens     182               136701   18   22459   ctcctggccaaggctggtgc   47     H. sapiens     183               136704   18   24407   catgcggagggtgagtgccc   50     H. sapiens     184               136705   19   31   gtagggccaacggcctggac   51     H. sapiens     185               136706   19   73   gcggagccatggattgcact   52     H. sapiens     186               136707   19   97   aagacatgcttcagcttatc   53     H. sapiens     187               136708   20   33   ctctgaggccagggcaggac   54     H. sapiens     188               136709   20   159   gctgcgccatggacgagcca   55     H. sapiens     189               136710   20   174   agccacccttcagcgaggcg   56     H. sapiens     190               136711   20   249   acatcgaagacatgcttcag   57     H. sapiens     191               136712   20   386   ccacctcctgccacattgag   58     H. sapiens     192               136713   20   597   tcctgccacagagcttccca   59     H. sapiens     193               136714   20   718   gctgcctggcctgccactgg   60     H. sapiens     194               136715   20   998   acagggcctttgccgaccct   61     H. sapiens     195               136716   20   1074   tgcctatcaaccggctcgca   62     H. sapiens     196               136717   20   1127   cgtggagagaagcgcacagc   63     H. sapiens     197               136718   20   1204   caaggatctggtggtgggca   64     H. sapiens     198               136719   20   1310   caggagaacctaagtctgcg   65     H. sapiens     199               136720   20   1330   cactgctgtccacaaaagca   66     H. sapiens     200               136721   20   1364   ctggtgtcggcctgtggcag   67     H. sapiens     201               136722   20   1915   gaggcatcgcaagcaggctg   68     H. sapiens     202               136723   20   1927   gcaggctgacctggacctgg   69     H. sapiens     203               136724   20   2173   agccctggtctaccataagc   70     H. sapiens     204               136726   20   2310   tggccgagatctatgtggcg   72     H. sapiens     205               136727   20   2317   gatctatgtggcggctgcat   73     H. sapiens     206               136728   20   2584   ggaacatctcttagagcgag   74     H. sapiens     207               136729   20   2631   ccagccctgggtcagctgat   75     H. sapiens     208               136732   20   2966   aaggcagagtctggtccagc   78     H. sapiens     209               136733   20   3037   taccacaccagccagcagct   79     H. sapiens     210               136734   20   3182   gcttccgcccttgagctgcg   80     H. sapiens     211               136736   20   3560   cagcagatgctcatgcgcct   82     H. sapiens     212               136737   20   3585   gtgggaccactgtcacttcc   83     H. sapiens     213               136738   20   3595   tgtcacttccagctagaccc   84     H. sapiens     214               136739   20   3643   ctagccactttggtcccgtg   85     H. sapiens     215               136740   20   3657   cccgtgcagcttctgtcctg   86     H. sapiens     216               136741   20   3732   acctgcggctgctgtgtgcc   87     H. sapiens     217               136742   20   3745   gtgtgccttcgcggtggaag   88     H. sapiens     218               136744   20   3795   cggcggccatgatggtgctg   90     H. sapiens     219               136745   20   3874   ttgctctgcaggcaccttag   91     H. sapiens     220               136746   20   3923   agaggggtacatttccctgt   92     H. sapiens     221               136747   20   3937   ccctgtgctgacggaagcca   93     H. sapiens     222               136748   20   3953   gccaacttggctttcccgga   94     H. sapiens     223               136749   20   4043   cagcgtgcttagcctcctga   95     H. sapiens     224               136750   20   4079   tactttgccttttgcaaact   96     H. sapiens     225               136751   20   4118   agttttgtacagagaattaa   97     H. sapiens     226               136285   11   61   acggagccatggattgcaca   102     M. musculus     227               136290   11   396   cttcctgggaggacccaagg   104     M. musculus     228               136291   11   416   tgacacctgcacccttgtcc   105     M. musculus     229               136292   11   515   agccagtgccactcaccatc   106     M. musculus     230               136294   11   1026   cacagacaaactgcccatcc   107     M. musculus     231               136296   11   1090   cgtggtgagaagcgcacagc   108     M. musculus     232               136297   11   1110   ccacaatgccattgagaagc   109     M. musculus     233               136304   11   1326   ggtgtcagcttgtggcagtg   110     M. musculus     234               136305   11   1350   aggcacagatgtgtctatgg   111     M. musculus     235               136306   11   1476   tggtggcagtgactctgagc   112     M. musculus     236               136307   11   1515   ggatagccaggtcaaagccc   113     M. musculus     237               136310   11   1731   ttggacccagtggttgctgc   115     M. musculus     238               136311   11   1759   gtctggctggccaatggact   116     M. musculus     239               136312   11   1779   actagtgttggcctgcttgg   117     M. musculus     240               136313   11   1854   acacttctggagacatcgca   118     M. musculus     241               136314   11   1966   acctcaaacctggatctggc   119     M. musculus     242               136315   11   2038   ggccgctggctggcaggcca   120     M. musculus     243               136316   11   2152   catgccatgggcaagtacac   121     M. musculus     244               136317   11   2250   ggcaacactggcagagatct   122     M. musculus     245               136319   11   2400   gctctgccaccctgtaggtc   123     M. musculus     246               136320   11   2478   cagcgtggctgggaacccag   124     M. musculus     247               136321   11   2597   acagggagttctcagatgcc   125     M. musculus     248               136322   11   2714   cagacccagtggccaagtgg   126     M. musculus     249               136325   11   2864   ctctgtactccttcaaggct   127     M. musculus     250               136327   11   2894   tgctggaccacagaaaggtg   128     M. musculus     251               136331   11   3013   atgcagctgctcctgtgtga   130     M. musculus     252               136332   11   3024   cctgtgtgatctacttcttg   131     M. musculus     253               136333   11   3096   agctcacggtaccagcaatg   132     M. musculus     254               136335   11   3129   tgctctggagctgcgtggtt   134     M. musculus     255               136336   11   3139   ctgcgtggtttccaacatga   135     M. musculus     256               136338   11   3214   ttcctacatgaggccacagc   137     M. musculus     257               136339   11   3224   aggccacagctcggctgatg   138     M. musculus     258               136340   11   3245   caggagcaagtcctgcccgg   139     M. musculus     259               136341   11   3299   gggcaggttccagtggcaaa   140     M. musculus     260               136342   11   3349   cccacatggcgggagcacac   141     M. musculus     261               136345   11   3632   tgccgacctctagtggcaga   144     M. musculus     262               136346   11   3686   caccctcttgctctgtaggc   145     M. musculus     263               136348   11   3825   ttcataggttgagaaatttt   146     M. musculus     264               136349   98   32   ccttgaaacaagtgttctca   147     M. musculus     265               136350   98   101   atctaaaggcagctattggc   148     M. musculus     266               136352   98   334   acgacagtgaccgccagtaa   150     M. musculus     267               136353   100   4956   tgaagcaaaggtacggccaa   151     M. musculus     268               136354   100   5918   gctagctgagaatagtgtgg   152     M. musculus     269               136355   100   6071   ccaccctgatgctgccttct   153     M. musculus     270               136357   100   6426   ggatagccaggttqgactct   155     M. musculus     271               136358   100   7009   ctggatttggcccgggtaag   156     M. musculus     272               136360   100   7547   cactttgcaggcaagtacac   158     M. musculus     273                  
 
     [0302] 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 sterol regulatory element-binding protein-1.  
     Example 17  
     [0303] Western Blot Analysis of Sterol Regulatory Element-Binding Protein-1 Protein Levels  
     [0304] 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 sterol regulatory element-binding protein-1 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).  
    
     
       
         1 
         
           
             273  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

gtgcgcgcga gcccgaaatc                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            3 

atgcattctg cccccaagga                                                 20 

 
           
             4  
             4154  
             DNA  
             H. sapiens  
             
               CDS  
               (167)...(3610)  
             
           
            4 

taacgaggaa cttttcgccg gcgccgggcc gcctctgagg ccagggcagg acacgaacgc     60 

gcggagcggc ggcggcgact gagagccggg gccgcggcgg cgctccctag gaagggccgt    120 

acgaggcggc gggcccggcg ggcctcccgg aggaggcggc tgcgcc atg gac gag       175 
                                                   Met Asp Glu 
                                                     1 

cca ccc ttc agc gag gcg gct ttg gag cag gcg ctg ggc gag ccg tgc      223 
Pro Pro Phe Ser Glu Ala Ala Leu Glu Gln Ala Leu Gly Glu Pro Cys 
      5                  10                  15 

gat ctg gac gcg gcg ctg ctg acc gac atc gaa gac atg ctt cag ctt      271 
Asp Leu Asp Ala Ala Leu Leu Thr Asp Ile Glu Asp Met Leu Gln Leu 
 20                  25                  30                  35 

atc aac aac caa gac agt gac ttc cct ggc cta ttt gac cca ccc tat      319 
Ile Asn Asn Gln Asp Ser Asp Phe Pro Gly Leu Phe Asp Pro Pro Tyr 
                 40                  45                  50 

gct ggg agt ggg gca ggg ggc aca gac cct gcc agc ccc gat acc agc      367 
Ala Gly Ser Gly Ala Gly Gly Thr Asp Pro Ala Ser Pro Asp Thr Ser 
             55                  60                  65 

tcc cca ggc agc ttg tct cca cct cct gcc aca ttg agc tcc tct ctt      415 
Ser Pro Gly Ser Leu Ser Pro Pro Pro Ala Thr Leu Ser Ser Ser Leu 
         70                  75                  80 

gaa gcc ttc ctg agc ggg ccg cag gca gcg ccc tca ccc ctg tcc cct      463 
Glu Ala Phe Leu Ser Gly Pro Gln Ala Ala Pro Ser Pro Leu Ser Pro 
     85                  90                  95 

ccc cag cct gca ccc act cca ttg aag atg tac ccg tcc atg ccc gct      511 
Pro Gln Pro Ala Pro Thr Pro Leu Lys Met Tyr Pro Ser Met Pro Ala 
100                 105                 110                 115 

ttc tcc cct ggg cct ggt atc aag gaa gag tca gtg cca ctg agc atc      559 
Phe Ser Pro Gly Pro Gly Ile Lys Glu Glu Ser Val Pro Leu Ser Ile 
                120                 125                 130 

ctg cag acc ccc acc cca cag ccc ctg cca ggg gcc ctc ctg cca cag      607 
Leu Gln Thr Pro Thr Pro Gln Pro Leu Pro Gly Ala Leu Leu Pro Gln 
            135                 140                 145 

agc ttc cca gcc cca gcc cca ccg cag ttc agc tcc acc cct gtg tta      655 
Ser Phe Pro Ala Pro Ala Pro Pro Gln Phe Ser Ser Thr Pro Val Leu 
        150                 155                 160 

ggc tac ccc agc cct ccg gga ggc ttc tct aca gga agc cct ccc ggg      703 
Gly Tyr Pro Ser Pro Pro Gly Gly Phe Ser Thr Gly Ser Pro Pro Gly 
    165                 170                 175 

aac acc cag cag ccg ctg cct ggc ctg cca ctg gct tcc ccg cca ggg      751 
Asn Thr Gln Gln Pro Leu Pro Gly Leu Pro Leu Ala Ser Pro Pro Gly 
180                 185                 190                 195 

gtc ccg ccc gtc tcc ttg cac acc cag gtc cag agt gtg gtc ccc cag      799 
Val Pro Pro Val Ser Leu His Thr Gln Val Gln Ser Val Val Pro Gln 
                200                 205                 210 

cag cta ctg aca gtc aca gct gcc ccc acg gca gcc cct gta acg acc      847 
Gln Leu Leu Thr Val Thr Ala Ala Pro Thr Ala Ala Pro Val Thr Thr 
            215                 220                 225 

act gtg acc tcg cag atc cag cag gtc ccg gtc ctg ctg cag ccc cac      895 
Thr Val Thr Ser Gln Ile Gln Gln Val Pro Val Leu Leu Gln Pro His 
        230                 235                 240 

ttc atc aag gca gac tcg ctg ctt ctg aca gcc atg aag aca gac gga      943 
Phe Ile Lys Ala Asp Ser Leu Leu Leu Thr Ala Met Lys Thr Asp Gly 
    245                 250                 255 

gcc act gtg aag gcg gca ggt ctc agt ccc ctg gtc tct ggc acc act      991 
Ala Thr Val Lys Ala Ala Gly Leu Ser Pro Leu Val Ser Gly Thr Thr 
260                 265                 270                 275 

gtg cag aca ggg cct ttg ccg acc ctg gtg agt ggc gga acc atc ttg     1039 
Val Gln Thr Gly Pro Leu Pro Thr Leu Val Ser Gly Gly Thr Ile Leu 
                280                 285                 290 

gca aca gtc cca ctg gtc gta gat gcg gag aag ctg cct atc aac cgg     1087 
Ala Thr Val Pro Leu Val Val Asp Ala Glu Lys Leu Pro Ile Asn Arg 
            295                 300                 305 

ctc gca gct ggc agc aag gcc ccg gcc tct gcc cag agc cgt gga gag     1135 
Leu Ala Ala Gly Ser Lys Ala Pro Ala Ser Ala Gln Ser Arg Gly Glu 
        310                 315                 320 

aag cgc aca gcc cac aac gcc att gag aag cgc tac cgc tcc tcc atc     1183 
Lys Arg Thr Ala His Asn Ala Ile Glu Lys Arg Tyr Arg Ser Ser Ile 
    325                 330                 335 

aat gac aaa atc att gag ctc aag gat ctg gtg gtg ggc act gag gca     1231 
Asn Asp Lys Ile Ile Glu Leu Lys Asp Leu Val Val Gly Thr Glu Ala 
340                 345                 350                 355 

aag ctg aat aaa tct gct gtc ttg cgc aag gcc atc gac tac att cgc     1279 
Lys Leu Asn Lys Ser Ala Val Leu Arg Lys Ala Ile Asp Tyr Ile Arg 
                360                 365                 370 

ttt ctg caa cac agc aac cag aaa ctc aag cag gag aac cta agt ctg     1327 
Phe Leu Gln His Ser Asn Gln Lys Leu Lys Gln Glu Asn Leu Ser Leu 
            375                 380                 385 

cgc act gct gtc cac aaa agc aaa tct ctg aag gat ctg gtg tcg gcc     1375 
Arg Thr Ala Val His Lys Ser Lys Ser Leu Lys Asp Leu Val Ser Ala 
        390                 395                 400 

tgt ggc agt gga ggg aac aca gac gtg ctc atg gag ggc gtg aag act     1423 
Cys Gly Ser Gly Gly Asn Thr Asp Val Leu Met Glu Gly Val Lys Thr 
    405                 410                 415 

gag gtg gag gac aca ctg acc cca ccc ccc tcg gat gct ggc tca cct     1471 
Glu Val Glu Asp Thr Leu Thr Pro Pro Pro Ser Asp Ala Gly Ser Pro 
420                 425                 430                 435 

ttc cag agc agc ccc ttg tcc ctt ggc agc agg ggc agt ggc agc ggt     1519 
Phe Gln Ser Ser Pro Leu Ser Leu Gly Ser Arg Gly Ser Gly Ser Gly 
                440                 445                 450 

ggc agt ggc agt gac tcg gag cct gac agc cca gtc ttt gag gac agc     1567 
Gly Ser Gly Ser Asp Ser Glu Pro Asp Ser Pro Val Phe Glu Asp Ser 
            455                 460                 465 

aag gca aag cca gag cag cgg ccg tct ctg cac agc cgg ggc atg ctg     1615 
Lys Ala Lys Pro Glu Gln Arg Pro Ser Leu His Ser Arg Gly Met Leu 
        470                 475                 480 

gac cgc tcc cgc ctg gcc ctg tgc acg ctc gtc ttc ctc tgc ctg tcc     1663 
Asp Arg Ser Arg Leu Ala Leu Cys Thr Leu Val Phe Leu Cys Leu Ser 
    485                 490                 495 

tgc aac ccc ttg gcc tcc ttg ctg ggg gcc cgg ggg ctt ccc agc ccc     1711 
Cys Asn Pro Leu Ala Ser Leu Leu Gly Ala Arg Gly Leu Pro Ser Pro 
500                 505                 510                 515 

tca gat acc acc agc gtc tac cat agc cct ggg cgc aac gtg ctg ggc     1759 
Ser Asp Thr Thr Ser Val Tyr His Ser Pro Gly Arg Asn Val Leu Gly 
                520                 525                 530 

acc gag agc aga gat ggc cct ggc tgg gcc cag tgg ctg ctg ccc cca     1807 
Thr Glu Ser Arg Asp Gly Pro Gly Trp Ala Gln Trp Leu Leu Pro Pro 
            535                 540                 545 

gtg gtc tgg ctg ctc aat ggg ctg ttg gtg ctc gtc tcc ttg gtg ctt     1855 
Val Val Trp Leu Leu Asn Gly Leu Leu Val Leu Val Ser Leu Val Leu 
        550                 555                 560 

ctc ttt gtc tac ggt gag cca gtc aca cgg ccc cac tca ggc ccc gcc     1903 
Leu Phe Val Tyr Gly Glu Pro Val Thr Arg Pro His Ser Gly Pro Ala 
    565                 570                 575 

gtg tac ttc tgg agg cat cgc aag cag gct gac ctg gac ctg gcc cgg     1951 
Val Tyr Phe Trp Arg His Arg Lys Gln Ala Asp Leu Asp Leu Ala Arg 
580                 585                 590                 595 

gga gac ttt gcc cag gct gcc cag cag ctg tgg ctg gcc ctg cgg gca     1999 
Gly Asp Phe Ala Gln Ala Ala Gln Gln Leu Trp Leu Ala Leu Arg Ala 
                600                 605                 610 

ctg ggc cgg ccc ctg ccc acc tcc cac ctg gac ctg gct tgt agc ctc     2047 
Leu Gly Arg Pro Leu Pro Thr Ser His Leu Asp Leu Ala Cys Ser Leu 
            615                 620                 625 

ctc tgg aac ctc atc cgt cac ctg ctg cag cgt ctc tgg gtg ggc cgc     2095 
Leu Trp Asn Leu Ile Arg His Leu Leu Gln Arg Leu Trp Val Gly Arg 
        630                 635                 640 

tgg ctg gca ggc cgg gca ggg ggc ctg cag cag gac tgt gct ctg cga     2143 
Trp Leu Ala Gly Arg Ala Gly Gly Leu Gln Gln Asp Cys Ala Leu Arg 
    645                 650                 655 

gtg gat gct agc gcc agc gcc cga gac gca gcc ctg gtc tac cat aag     2191 
Val Asp Ala Ser Ala Ser Ala Arg Asp Ala Ala Leu Val Tyr His Lys 
660                 665                 670                 675 

ctg cac cag ctg cac acc atg ggg aag cac aca ggc ggg cac ctc act     2239 
Leu His Gln Leu His Thr Met Gly Lys His Thr Gly Gly His Leu Thr 
                680                 685                 690 

gcc acc aac ctg gcg ctg agt gcc ctg aac ctg gca gag tgt gca ggg     2287 
Ala Thr Asn Leu Ala Leu Ser Ala Leu Asn Leu Ala Glu Cys Ala Gly 
            695                 700                 705 

gat gcc gtg tct gtg gcg acg ctg gcc gag atc tat gtg gcg gct gca     2335 
Asp Ala Val Ser Val Ala Thr Leu Ala Glu Ile Tyr Val Ala Ala Ala 
        710                 715                 720 

ttg aga gtg aag acc agt ctc cca cgg gcc ttg cat ttt ctg aca cgc     2383 
Leu Arg Val Lys Thr Ser Leu Pro Arg Ala Leu His Phe Leu Thr Arg 
    725                 730                 735 

ttc ttc ctg agc agt gcc cgc cag gcc tgc ctg gca cag agt ggc tca     2431 
Phe Phe Leu Ser Ser Ala Arg Gln Ala Cys Leu Ala Gln Ser Gly Ser 
740                 745                 750                 755 

gtg cct cct gcc atg cag tgg ctc tgc cac ccc gtg ggc cac cgt ttc     2479 
Val Pro Pro Ala Met Gln Trp Leu Cys His Pro Val Gly His Arg Phe 
                760                 765                 770 

ttc gtg gat ggg gac tgg tcc gtg ctc agt acc cca tgg gag agc ctg     2527 
Phe Val Asp Gly Asp Trp Ser Val Leu Ser Thr Pro Trp Glu Ser Leu 
            775                 780                 785 

tac agc ttg gcc ggg aac cca gtg gac ccc ctg gcc cag gtg act cag     2575 
Tyr Ser Leu Ala Gly Asn Pro Val Asp Pro Leu Ala Gln Val Thr Gln 
        790                 795                 800 

cta ttc cgg gaa cat ctc tta gag cga gca ctg aac tgt gtg acc cag     2623 
Leu Phe Arg Glu His Leu Leu Glu Arg Ala Leu Asn Cys Val Thr Gln 
    805                 810                 815 

ccc aac ccc agc cct ggg tca gct gat ggg gac aag gaa ttc tcg gat     2671 
Pro Asn Pro Ser Pro Gly Ser Ala Asp Gly Asp Lys Glu Phe Ser Asp 
820                 825                 830                 835 

gcc ctc ggg tac ctg cag ctg ctg aac agc tgt tct gat gct gcg ggg     2719 
Ala Leu Gly Tyr Leu Gln Leu Leu Asn Ser Cys Ser Asp Ala Ala Gly 
                840                 845                 850 

gct cct gcc tac agc ttc tcc atc agt tcc agc atg gcc acc acc acc     2767 
Ala Pro Ala Tyr Ser Phe Ser Ile Ser Ser Ser Met Ala Thr Thr Thr 
            855                 860                 865 

ggc gta gac ccg gtg gcc aag tgg tgg gcc tct ctg aca gct gtg gtg     2815 
Gly Val Asp Pro Val Ala Lys Trp Trp Ala Ser Leu Thr Ala Val Val 
        870                 875                 880 

atc cac tgg ctg cgg cgg gat gag gag gcg gct gag cgg ctg tgc ccg     2863 
Ile His Trp Leu Arg Arg Asp Glu Glu Ala Ala Glu Arg Leu Cys Pro 
    885                 890                 895 

ctg gtg gag cac ctg ccc cgg gtg ctg cag gag tct gag aga ccc ctg     2911 
Leu Val Glu His Leu Pro Arg Val Leu Gln Glu Ser Glu Arg Pro Leu 
900                 905                 910                 915 

ccc agg gca gct ctg cac tcc ttc aag gct gcc cgg gcc ctg ctg ggc     2959 
Pro Arg Ala Ala Leu His Ser Phe Lys Ala Ala Arg Ala Leu Leu Gly 
                920                 925                 930 

tgt gcc aag gca gag tct ggt cca gcc agc ctg acc atc tgt gag aag     3007 
Cys Ala Lys Ala Glu Ser Gly Pro Ala Ser Leu Thr Ile Cys Glu Lys 
            935                 940                 945 

gcc agt ggg tac ctg cag gac agc ctg gct acc aca cca gcc agc agc     3055 
Ala Ser Gly Tyr Leu Gln Asp Ser Leu Ala Thr Thr Pro Ala Ser Ser 
        950                 955                 960 

tcc att gac aag gcc gtg cag ctg ttc ctg tgt gac ctg ctt ctt gtg     3103 
Ser Ile Asp Lys Ala Val Gln Leu Phe Leu Cys Asp Leu Leu Leu Val 
    965                 970                 975 

gtg cgc acc agc ctg tgg cgg cag cag cag ccc ccg gcc ccg gcc cca     3151 
Val Arg Thr Ser Leu Trp Arg Gln Gln Gln Pro Pro Ala Pro Ala Pro 
980                 985                 990                 995 

gca gcc cag ggc gcc agc agc agg ccc cag gct tcc gcc ctt gag ctg     3199 
Ala Ala Gln Gly Ala Ser Ser Arg Pro Gln Ala Ser Ala Leu Glu Leu 
                1000                1005                1010 

cgt ggc ttc caa cgg gac ctg agc agc ctg agg cgg ctg gca cag agc     3247 
Arg Gly Phe Gln Arg Asp Leu Ser Ser Leu Arg Arg Leu Ala Gln Ser 
            1015                1020                1025 

ttc cgg ccc gcc atg cgg agg gtg ttc cta cat gag gcc acg gcc cgg     3295 
Phe Arg Pro Ala Met Arg Arg Val Phe Leu His Glu Ala Thr Ala Arg 
        1030                1035                1040 

ctg atg gcg ggg gcc agc ccc aca cgg aca cac cag ctc ctc gac cgc     3343 
Leu Met Ala Gly Ala Ser Pro Thr Arg Thr His Gln Leu Leu Asp Arg 
    1045                1050                1055 

agt ctg agg cgg cgg gca ggc ccc ggt ggc aaa gga ggc gcg gtg gcg     3391 
Ser Leu Arg Arg Arg Ala Gly Pro Gly Gly Lys Gly Gly Ala Val Ala 
1060                1065                1070                1075 

gag ctg gag ccg cgg ccc acg cgg cgg gag cac gcg gag gcc ttg ctg     3439 
Glu Leu Glu Pro Arg Pro Thr Arg Arg Glu His Ala Glu Ala Leu Leu 
                1080                1085                1090 

ctg gcc tcc tgc tac ctg ccc ccc ggc ttc ctg tcg gcg ccc ggg cag     3487 
Leu Ala Ser Cys Tyr Leu Pro Pro Gly Phe Leu Ser Ala Pro Gly Gln 
            1095                1100                1105 

cgc gtg ggc atg ctg gct gag gcg gcg cgc aca ctc gag aag ctt ggc     3535 
Arg Val Gly Met Leu Ala Glu Ala Ala Arg Thr Leu Glu Lys Leu Gly 
        1110                1115                1120 

gat cgc cgg ctg ctg cac gac tgt cag cag atg ctc atg cgc ctg ggc     3583 
Asp Arg Arg Leu Leu His Asp Cys Gln Gln Met Leu Met Arg Leu Gly 
    1125                1130                1135 

ggt ggg acc act gtc act tcc agc tag accccgtgtc cccggcctca           3630 
Gly Gly Thr Thr Val Thr Ser Ser 
1140                1145 

gcacccctgt ctctagccac tttggtcccg tgcagcttct gtcctgcgtc gaagctttga   3690 

aggccgaagg cagtgcaaga gactctggcc tccacagttc gacctgcggc tgctgtgtgc   3750 

cttcgcggtg gaaggcccga ggggcgcgat cttgacccta agaccggcgg ccatgatggt   3810 

gctgacctct ggtggccgat cggggcactg caggggccga gccattttgg ggggcccccc   3870 

tccttgctct gcaggcacct tagtggcttt tttcctcctg tgtacaggga agagaggggt   3930 

acatttccct gtgctgacgg aagccaactt ggctttcccg gactgcaagc agggctctgc   3990 

cccagaggcc tctctctccg tcgtgggaga gagacgtgta catagtgtag gtcagcgtgc   4050 

ttagcctcct gacctgaggc tcctgtgcta ctttgccttt tgcaaacttt attttcatag   4110 

attgagaagt tttgtacaga gaattaaaaa tgaaattatt tata                    4154 

 
           
             5  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

gtcctgcgtc gaagctttg                                                  19 

 
           
             6  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

aggtcgaact gtggaggcc                                                  19 

 
           
             7  
             25  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

aggccgaagg cagtgcaaga gactc                                           25 

 
           
             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  
             3891  
             DNA  
             M. musculus  
             
 
           
            11 

aaaatcggcg cggaagctgt cggggtagcg tctgcacgcc ctaggggcgg ggcgcggacc     60 

acggagccat ggattgcaca tttcccagtt tccggggaac ttttccttaa cgtgggccta    120 

gtccgaagcc gggtgggcgc cggcgccatg gacgagctgg ccttcggtga ggcggctctg    180 

gaacagacac tggccgagat gtgcgaactg gacacagcgg ttttgaacga catcgaagac    240 

atgctccagc tcatcaacaa ccaagacagt gacttccctg gcctgtttga cgccccctat    300 

gctgggggtg agacagggga cacaggcccc agcagcccag gtgccaactc tcctgagagc    360 

ttctcttctg cttctctggc ctcctctctg gaagccttcc tgggaggacc caaggtgaca    420 

cctgcaccct tgtcccctcc accatcggca cccgctgctt taaagatgta cccgtccgtg    480 

tccccctttt cccctgggcc tgggatcaaa gaggagccag tgccactcac catcctacag    540 

cctgcagcgc cacagccgtc accggggacc ctcctgcctc cgagcttccc cgcaccaccc    600 

gtacagctca gccctgcgcc cgtgctgggt tactcgagcc tgccttcagg cttctcaggg    660 

acccttccag gaaacactca gcagccacca tctagcctgc cgctggcccc tgcaccagga    720 

gtcttgccca cccctgccct gcacacccag gtccaaagct tggcctccca gcagccgctg    780 

ccagcctcag cagcccctag aacaaacact gtgacctcac aggtccagca ggtcccagtt    840 

gtactgcagc cacacttcat caaggcagac tcactgctgc tgacagctgt gaagacagat    900 

gcaggagcca ccgtgaagac tgcaggcatc agcaccctgg ctcctggcac agccgtgcag    960 

gcaggtcccc tgcagaccct ggtgagtgga gggaccatct tggccacagt acctttggtt   1020 

gtggacacag acaaactgcc catccaccga ctcgcagctg gcagcaaggc cctaggctca   1080 

gctcagagcc gtggtgagaa gcgcacagcc cacaatgcca ttgagaagcg ctaccggtct   1140 

tctatcaatg acaagattgt ggagctcaaa gacctggtgg tgggcactga agcaaagctg   1200 

aataaatctg ctgtcttgcg caaggccatc gactacatcc gcttcttgca gcacagcaac   1260 

cagaagctca agcaggagaa cctgacccta ctttgtgcac acaaaagcaa atcactgaag   1320 

gacctggtgt cagcttgtgg cagtggagga ggcacagatg tgtctatgga gggcatgaaa   1380 

cccgaagtgg tggagacgct tacccctcca ccctcagacg ccggctcacc ctcccagagt   1440 

agccccttgt cttttggcag cagagctagc agcagtggtg gcagtgactc tgagcccgac   1500 

agtccagcct ttgaggatag ccaggtcaaa gcccagcggc tgccttcaca cagccgaggc   1560 

atgctggacc gctcccgcct ggccctgtgt gtactggcct ttctgtgtct gacctgcaat   1620 

cctttggcct cgctgttcgg ctggggcatt ctcactccct ctgatgctac gggtacacac   1680 

cgtagttctg ggcgcagcat gctggaggca gagagcagag atggctctaa ttggacccag   1740 

tggttgctgc cacccctagt ctggctggcc aatggactac tagtgttggc ctgcttggct   1800 

cttctctatg tctatgggga acctgtgact aggccacact ctggcccagc tgtacacttc   1860 

tggagacatc gcaaacaagc tgacctgaat ttggcccggg gagatgttcg cccagctgct   1920 

caacagctgt ggctagccct gcaagcgctt ggccggcccc tgcccacctc aaacctggat   1980 

ctggcctgca gtctgctttg gaacctcatc cgccacctgc tccagcgtct ctgggtgggc   2040 

cgctggctgg caggccaggc cgggggcctg ctgagggacc gtgggctgag aaaggatgcc   2100 

cgtgccagtg cccgggatgc ggctgttgtc taccataagc tgcaccagct gcatgccatg   2160 

ggcaagtaca caggaggaca tcttgctgct tctaacctgg cactaagtgc cctcaacctg   2220 

gctgagtgcg caggagatgc tatctccatg gcaacactgg cagagatcta tgtggcagcg   2280 

tgcctgaggg tcaaaaccag cctcccaaga gccctgcact tcttgacacg tttcttcctg   2340 

agcagcgccc gccaggcctg cctagcacag agcggctcgg tgcctcttgc catgcagtgg   2400 

ctctgccacc ctgtaggtca ccgtttcttt gtggacgggg actgggccgt gcacggtgcc   2460 

cccccggaga gcctgtacag cgtggctggg aacccagtgg atccgctggc ccaggtgacc   2520 

cggctattcc gtgaacatct cctagagcga gcgttgaact gtattgctca gcccagccca   2580 

ggggcagctg acggagacag ggagttctca gatgcccttg gatatctgca gttgctaaat   2640 

agctgttctg atgctgccgg ggctcctgcg tgcagtttct ctgtcagctc cagcatggct   2700 

gccaccactg gcccagaccc agtggccaag tggtgggcct cactgacagc tgtggtgatc   2760 

cactggctga ggcgggatga agaggcagct gagcgcttgt acccactggt agagcatatc   2820 

ccccaggtgc tgcaggacac tgagagaccc ctgcccaggg cagctctgta ctccttcaag   2880 

gctgcccggg ctctgctgga ccacagaaag gtggaatcta gcccagccag cctggccatc   2940 

tgtgagaagg ccagtgggta cctgcgggac agcttagcct ctacaccaac tggcagttcc   3000 

attgacaagg ccatgcagct gctcctgtgt gatctacttc ttgtggcccg taccagtctg   3060 

tggcagcggc agcagtcacc agcttcagtc caggtagctc acggtaccag caatggaccc   3120 

caggcctctg ctctggagct gcgtggtttc caacatgacc tgagcagcct gcggcggttg   3180 

gcacagagct tccggcctgc tatgaggagg gtattcctac atgaggccac agctcggctg   3240 

atggcaggag caagtcctgc ccggacacac cagctcctgg atcgcagtct gaggaggagg   3300 

gcaggttcca gtggcaaagg aggcactaca gctgagctgg agccacggcc cacatggcgg   3360 

gagcacaccg aggccctgct gttggcatcc tgctatctgc cccctgcctt cctgtcggct   3420 

cctgggcagc gaatgagcat gctggccgag gcggcacgca ccgtagagaa gcttggcgat   3480 

caccggctac tgctggactg ccagcagatg ctcctgcgcc tgggcggcgg aaccaccgtc   3540 

acttccagct agaccccaaa gctttccctt gaggaccttt gtcattggct gtggtcttcc   3600 

agagggtgag cctgacaagc aatcaggacc atgccgacct ctagtggcag atctggaaat   3660 

tgcagaggct gcactggccc gatggcaccc tcttgctctg taggcacctt agtggctttt   3720 

ccctagctga ggctcaccct gggagacctg tacatagtgt agatccggct gggcctggct   3780 

ccagggcagg cccatgtact actttgactt ttgcaaactt tattttcata ggttgagaaa   3840 

ttttgtacag aatattaaaa aatgaaatta tttataaaaa aaaaaaaaaa a            3891 

 
           
             12  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            12 

ttggccacag tacctttggt t                                               21 

 
           
             13  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            13 

ctgagcctag ggccttgct                                                  19 

 
           
             14  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            14 

catccaccga ctcgcagctg g                                              21 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            15 

ggcaaattca acggcacagt                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            16 

gggtctcgct cctggaagat                                                 20 

 
           
             17  
             27  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            17 

aaggccgaga atgggaagct tgtcatc                                         27 

 
           
             18  
             27001  
             DNA  
             H. sapiens  
             
 
           
            18 

ttaattctgg tttatttcaa cccacctcat tgggacccct tccctccttc ctgccccacc     60 

tggctctgtc cctaggccac agaaccaggt tcggtttcca gccctcttct caacagggct    120 

gcctgctctg atctagtccc agcttgtgat gatccagggc agcctggctc tgatctaaag    180 

cacagctacc tcttccttgc ggcccctatc ctggctgctc ctgggaataa gtgccaaatc    240 

tggggtcaga cagccctggg gccagtcttc cttgggtact ggcttcctcc ttcaggagct    300 

gcactgggcc cactggtatc ctatccctac agctggatct gggaggaaac cagatgacga    360 

aattccagcc tctttctttg gccactcctg tcctcaagag gccaatcttc tggtttcttt    420 

gcagagaggg ggcaggctga tctcacaggt catgctcccc tccacattgt cactagcctc    480 

ccagcctgcc cgtgagaaag catcattagg cccatgttac aaatgaggaa aattgaggca    540 

gagtgatgta actggcccag cagttacatc aggcctgctc acaacacagc aggcctggga    600 

cccctataac ttggatcctg gtctgtcttg ttctaaagag tcaaatctag gaaatgagga    660 

aatgaagttt gggatgggcc caggcctggg gcttccactc ggcttccttg cttggtgctg    720 

gagaaacaga ggcccagaga gggggctcgg cttgcccgcg ttcccgcagc agccggccag    780 

aggccgctgc cattgtgcgc gaggctggat aaaatgaatg actggagggc gctctggagg    840 

aggggccggc tgaggggaga tttgtggcgc agaccgggga tcaggggtcc cccgctctct    900 

caaggtgggg cggggccgtc tatctgggag ggcgggtcct ccccgaaagg ccccgcctcc    960 

gcctcgaccg cccagcagag ctgcggccgg gggaacccag tttccgagga acttttcgcc   1020 

ggcgccgggc cgcctctgag gccagggcag gacaggaacg cgcggagcgg cggcggcgac   1080 

tgagagccgg ggccgcggcg gcgctcccta ggaagggccg tacgaggcgg cgggcccggc   1140 

gggcctcccg gaggaggcgg ctgcgccatg gacgagccac ccttcagcga ggcggctttg   1200 

gagcaggcgc tgggcgagcc gtgcgatctg gacgcggcgc tgctgaccga catcgaaggt   1260 

gcgtcagggc gggcagggct tgaagctgcg ccgggtggcg cgagtagggg gcgcgcaggt   1320 

gtctccctgg cctttgtctc ccccacgggc gccagctccg tgctgtgctc gcgcgggact   1380 

tcccggtgtc tctgagctcg gtgtcccgag cctcaccgag cctccctggt tcccgcgcta   1440 

gcgtctcggg ccgcgcgctt gtgggtgagg gctcctgggc cgggccgggg tcccttggcg   1500 

gctccgggcc gggacacgtg cgcctctacg cgtcccaggc cgggtgccgc ccgaccggtg   1560 

actctccagc cctgtgatgg ccacggctga agctggggac ccaggcgtcg ccgaagctcc   1620 

gccccagccc cagccgtgac gtaattgcga ggttactcac ggtcattccc tccggcccga   1680 

gagttcagct cggcgtcgga gctcttgcgc atgcgcatgg gcgctgcctc gcgcccttcc   1740 

cccgcctcgt gtcgggttct cccggtctgc gacgggcaca gcctccgcac tcattcactg   1800 

acatccaccg aatgccaggc cccgtcttag gcaccgaggg tttacagaca gacctgggta   1860 

ccccctcttt tagggaacac aaaaatctcc cgggaaacca aacgggtatt tagttgtacc   1920 

ttgggtggag cgaggctggg ggagggcagg gatgtggcta ctttgggtag agcggtcagg   1980 

gacttctaag ctgagacctg agggtcaccc ccaggaccag caaggaaaga tgttttccag   2040 

gccacggcaa gggaagggca aaggcctcga ggcagggcct aagtgtgagg agttagaggc   2100 

ttgcaaagga gtgaggtcag ggaggaggag gacgcaaacc gacttggtcg gccagggaaa   2160 

gggcggagca gaacagtggc accggcttcc atctttggag catcaccctg gctgtgatga   2220 

gaaggggttt ggggccaatg gtggcaccaa gtgccaatta ggaggcccgt tgcttccatt   2280 

ttgtagatag agcaaacgga agcccctagc aaattgcctg catggtttct gtgcaggagt   2340 

tttagcagca ctagctaagt tgcacttggt tgatgaggaa actgaggcca aggtcgcagg   2400 

aacaagatgc ctagactcac agcctgaatg gacatgtcca tggaacccgt ggccaccctg   2460 

gggttggcaa aacagatata tctatgccac caccactcct gccctactgc agccttgcag   2520 

atgagcccag ctggttgcca gccccagaag cttcccagcc ctccctcctt ccccctgggg   2580 

ctgggctagg ggaggacccc agaggagagg ccctgattgt gaggcttttc caaaacagcc   2640 

tcccctatcc ctggcacgag gggttgtcct tcactgccct ctggagtgat gaaccctgaa   2700 

atcccaagcc ctagggagat ctgggcctga ctcaactacc agttccacat cactgggccc   2760 

agtgagtgta gtcccaagag gcaacgtgac caagccagga ggacatgcgc tttggggtca   2820 

gaacttgaac ctggacactc ctcacttcct ttgtcatcct gctcaagccc tctcaccctc   2880 

taaaccttag tttccacctc cagaaaaatg atgcaaaccc tcccttcatg ggcaagttgg   2940 

acaacagaac ccgttctggg ccacaggtct gatacagacc tttgtttgtt tgtttgtttg   3000 

ttttctgcag tggcgcaatt ttggctcact gcaagctcct cctcctgggt tcacgccatt   3060 

ctcctgcctt agtctcccaa gtagctggga ctacaggcgc cagccaccac gcctggctaa   3120 

ttttttgtat ttttagtaga gacggggttt cactgtgcta gccaggatgg tctcgatctc   3180 

ctgaccttgt gatccgcccg cctcagcctc ccaaagtgct gggattacag gtgtgagcca   3240 

ccgctcccag cccagacctt tcttactgac agaatctggt ctgggccaga ggtctgatac   3300 

agacctttct tactgactca tggataaaaa cattgtctct ccagaaccaa aggccaggca   3360 

tgggcagcca tgtggcccaa ggtctagtct atgagagagt gggggcagtc ccagcccctt   3420 

gaagactggg ggcagcccct tctcactagg cagggctcag ctttacccac ttcagtagag   3480 

gatttttcag tttttattca aacttcctgt ttttcttccc aattacacac atcttttttc   3540 

attgtagaaa acttagaaaa tgcaagtgag caaaaagaag aaaataaaat ctttagacct   3600 

ggggtggtgg ctcacaccta taatcccagc acttgggagg tcgaagcaag aggatgactt   3660 

gtgtccagga gtttgagacc agcctgggca acatgacaaa atcctgtctc tacaaaaata   3720 

aaaaattagc tgggtgtggg tgacatgtgc ctgtagtctc agctactctg gaggctgaag   3780 

tgggaggatt gcttgagcct ggacttagag gctgcagtga gctataacca tgccgttgca   3840 

ctcagcctgg atgacagagt gagattctgt ttcaaaaaaa actttaaacc taccacccag   3900 

agataagccc tgctaattat gtgaaagagc ttttcttctc tctctctctc tctctctgtg   3960 

tgtttatatg tgtttgggga tgggtgcaca ctcttcataa actttttttt ttttgagaca   4020 

gggtctcgct cttttgccca tgctgtagtc cagtggcatg atctcagctc actgcaaact   4080 

ctgcctctca ggttcaagag attctccagc tcccaagtag ctgggattac agtcatgcac   4140 

cgccacgcct ggttaaattt tgtattttta gtagagatgg ccatgttggc caggctggtc   4200 

tcgaactcct gagctcaggt gatctgccca cctcagcctc tcaaaagtgc tgggattaca   4260 

gggcatgaac caccatgccc ggccttcatc aattttttaa aaacgacttt attgaggtat   4320 

actttatgta tcacaaaatt tacccatttt tagtatatca ttcaatgatt tttagttaac   4380 

tttttgagtt gtgtgacaat tactagctgt cgaacatttt tatcacacag tgagatccct   4440 

tatacttctt tagtcagttc ctgttcctgc tcccagcccc gggcagctgt ggatctgtat   4500 

gtgtgtgtgt atatatatat atatatatat atattttttt tttttttttt tttttttttt   4560 

ttttgagacg gagtcttgct ctgtcgccca ggctggagtg cagtggtgcg atcttggctc   4620 

actgcaagct ccgcctccca ggttcaaaca gttctgcctc agcctcccga gtagctggga   4680 

ttacaggcac ctgccaccac gcccggctaa tttttttgta tttttagtag agatggggtt   4740 

tcaccatgtt agccaggatg gtctcgatct cctgaccttg tgatctgccc acctcggcct   4800 

cccaacgttc tggaattaca ggcgtgagcc accgcgcccg gctggatctg tatttttata   4860 

aattaaaata gggtccattg gttcacagct gattggaatc tgcttggttc catgtcaaca   4920 

gccagacgac agtaaggttt cctcttatta cccacctgat tccctgtcga tggacaccta   4980 

ggttgtttta tctttataaa ctgctgcagt ggacactgag gccggttttt ttctttgttt   5040 

tttttttttt gtttgtttgt ttttgagaca gagtcttgct ctgtcaccca ggctggagtg   5100 

cagtggcgcg atctcggctc actgcaaact ccgcctcccg ggttcacacc attctcctgc   5160 

ctcagcctcc cgagtagctt gggactatag gtgcgtgcca ccatgcctgg ctaatttttt   5220 

gtatttttag tagagacggg gtttcaccgt gttagccagg atggtctcga tctcctgacc   5280 

tcatgatctg cccgcctcgg cctcccaaag tgctgggatt acaggcgtga gccactgtgc   5340 

ctggccactg aggccagtct ttgcccggat cctcactgtg ttcctaggat gaggttctgg   5400 

gaggggaatt gctggtcaga ggtcgagcct gcttttgaag cttcttctac caggagtgga   5460 

gctgagcagg tttgataagg tctgaagatt tgggggtgga aatgccaggt cccttgagag   5520 

acatgaggga taagaggggg ccaggctggc cttgagtgcc agagtgcaga gctgggctag   5580 

atgtgaggac agtcgggggt cagagcaggg gcacaccgag cttcagttcc ctctggctgc   5640 

ttggatggag gatcgtaatg tgaacagaaa acactaattg agtacttact gtgtttcaga   5700 

cagtgtgttg ataatcccac ttaatcccct gacaacccca agtaggtaga catatgatga   5760 

agatgacggc cttgaggacc agagaggtta agtgatttgc ctgagatcac acagccagat   5820 

gatggcaaag ccagaattca aacccaggct gtgggctcca gagcctagct cttaagctct   5880 

taagcactgg gctcctaaga atggggatga ggggttgagg gaggctcctc cacaggggct   5940 

actctggggg cctggaagtg ggtcacagag gggtcagagg ctatgtggct acctccccat   6000 

cccagtccag agcagtgttt gagtcattag actgggaacc agccctggtg agccagccaa   6060 

gggccttggg ccccatccgg tcctgctgcc tgccacagcc aaactcttgt catgtgaatg   6120 

gatttgggga tggagctgcc tccatgagtc cttgcatctg tgggtgaagg cactgccctg   6180 

gctatagtgt ccctgggttt gagtcctgca tctgcaccaa gacctcaggt gagcctgtct   6240 

ccttctgggc ctcagagtac cttgcagctg tcgggggagg atggatcagg agatggccct   6300 

gtacctgtgt tggggattat tgttaagccc gtggcagtct tcacctccct gctgaggatt   6360 

aatttatcca attttgcaca agcttatgag tgcagaagag gcagacggaa acagagttct   6420 

ggccaagagc ctggaacagg gcctcggggt ctctttccta tgcctggacc ccgtcatgtc   6480 

tgctctttgt ctgtcggacc ccagatgtct gccaagcccc gtcagaggct gcttcccaga   6540 

aagcccttct gggtgtcacc ttgccccgag cagtgcgttc tcagagttct cccgccctga   6600 

tgtccctccc agcatgccca gcccagccac aacagggcct tgcttctagt catgtgtctg   6660 

gctgtttgct gggtccaggc cagccctggt agggcacaat gggggcccgc tctgccaccc   6720 

catacctctc cccaggatat ctcatgcccc agttctctcc ctagttccac caagcactgg   6780 

cactccttag aaaacacagc tctagactag ttactgccct agcttacagc acagaactcc   6840 

cctggtctcc aaccattcat ggctccctag tgctccaaga taaagttccc ttgtctcagc   6900 

cgggttggga gttaccttct gcccaacatt cacctagctg gacacaaaca tcctgagtga   6960 

cccggtcagc tccaggcagg agtcactgcc agcagaggcc tgggatctgg actttgcctg   7020 

ctgacaggtg gagcccaggc cggccagagg aagtgcctct gaccttgtct cctagcagcc   7080 

acgggccatg tggacatgcc ttttgaccct gggcactgac agtgtgtgac agcctgcacc   7140 

atgtgctcca caggggcggc tgtgtgtgtc gggggtgagg tggggaaagc cttaactggc   7200 

tcaggggtga gaggtcaggg agccattgag actggctcca ggtgtgggtc ccctgctggg   7260 

ttggggcttg tgggaggtgg gacggggctg ggggtccatc cccctagggg gaatttgtgg   7320 

cctaccccga accctgtttg agctcctttc ctaactgact ccccgtccct gcacctgtct   7380 

cccagcaggc cttgcctctg catgctgccc ctgccaggct ctggggtccc tgtgctccct   7440 

gcagctagaa ggctgggatc aggggtctta acaagcagcc ctactgtatg accttggaca   7500 

agtccaagaa ccttcaggtt cttaacaatg taaagggagc agtactaaaa gcagcttctt   7560 

ggaattgtgg ggatccgatg agtgaaggct taagcagtgc atggcacata gtaggccctg   7620 

aaccaatgcc agttagtgtt attattatca ccatttagcc agatgcagtg gctcacgcct   7680 

ataatcttat tgactttaga ggctgaggtt ggaggattgc ttgagaccag gagttcaaga   7740 

ccagcctggg caacatagca aggccctgtt tgttttagag aaaacaaaca aatcaccatt   7800 

tagagcacct aaccagtacc tggcacgcga taggtttagc tcaacaaatg ttagcagcaa   7860 

ttacccaagg agcctgtgct ggaagtttct aggatgtacc aggctatggt tccaagttct   7920 

gagcatctac catgtggtgg tctggagttg gtgagagaca ggatggggct gactaggcca   7980 

gtggggagca ccccccgcca tggggaacaa gcaccctatc cttggcttcc atggaagata   8040 

attgatgctg ggcacagtgg ctcacgcctg taatcccagc actttgggag gctgaggcag   8100 

ggggatcgct tgagtctggg agttcaagac cagcctgggc aacattgtga gacccaaact   8160 

aaaaaaatta gcttggcatg gtggagtgtg cctgtcgtcc cagctactca ggaggctgag   8220 

gctaaagctg gaggattgct tgagcccagg aggttgaggc tgcagtgagc catgatcata   8280 

ccactacact ccagcctggg caacatagtg aggccctgtc tcaaaacaaa caaacaaaaa   8340 

gaacctgctg aggaagcagt gtttctggct gggggaggac gggcagagtg gccatctggc   8400 

cacagatggc ggtttctgtg caaaacacat caaggcagcc ttggaaatgt gagtgaaagc   8460 

accttcaaag ttctggtcac agccttggga ctaagcaaag ccaccaaaag tacataaaag   8520 

acaatgacca tcacccagtg ccggtgatgc tagaaggaaa gggaatacgt tgtagggaag   8580 

gttgtaaagg gctttatctt ttccagactg gagcctggca gctcgaaaac atcttgctgc   8640 

cttcatatga gctttaaaac aagctgcaga gaaacaactc aagagggaga aatatatata   8700 

tatatgtgtg tgtgtgtgta tgtgtgagtg tgtgtgtgtg tgtgtataca tatatatata   8760 

tatatatata tatatatatt tttttttttt tttaagatgg agtctcgttc tgtcaccagg   8820 

ctggagtgca gtggtacaat ctcggctcac tgcaacctcc gcctcctggg ttcaaatgat   8880 

tctcctgcgt cagcctccca agcagctggg actataggca cataccacca cgcccagcta   8940 

atttttgtat ttttagtaga ggctggattt caccatgttg gccaggatgg ttttgatctc   9000 

ctgacctcgt gatctgcctg ccttagcctc ccaaagtgtt gggattacag gcgtgagcca   9060 

gtttgttttt agagacgggg tcttgctctg tcacccaggc tggaatacca tggcacaatc   9120 

acagctcgct gcaatgttga actcccgggt tcaagggatc ctcccacctc agcctccaga   9180 

gtaatggaga ctacaggctc atgccaccat gcccagctat ttttaaaact ttgtagagat   9240 

ggggccttgc tacattgccc aggctggtct tgaactcctg ggctcaagtg atctgcctgc   9300 

ctttgcctcc caaagtgctg ttattacagg tgtgagcccc tgcgcctaac cttagcactg   9360 

ccattttgac tgaaaacagg tgcccagcag caggggctac tcccagaatt gccactgcat   9420 

caggcccgtg ggttgttttc agctgccagt gataagtatg tgccctgggc cacctctcgg   9480 

acaaggtgtc tgaattggtg ccgaccagca tcacatgtaa ttgccatctc gcaggtgctg   9540 

ctgagggtaa ttccgcacac ctgtagctcc gggaagagcc tagtggggag gaggaaacgt   9600 

ggctctgagg tttatagggt cagacggtca gtatgttggg agctggcatg tggaggggca   9660 

cagacaaggg aagaatggga ggtggcatca gagcaagttt tgatggagga ataggaattc   9720 

accaggtgga aagggcattc ctggtggagg gaacagcctg gccttcaata gcttgtggtg   9780 

ttcagaaagc aggcagggaa agggaggccc agggagacac cagttagggg atgggggtgg   9840 

aggcagacga gggtggagga agccatggct ggagtctgca cggcctctga ctggggtccc   9900 

tgctgtggtc agccctgtgc tgggtgaggc tggggtcaca gctggttcag gccctgacag   9960 

gaggggcccc cagctgaggc ccagcctcta atttggcagg gcaggtggat aggtctgggg  10020 

gggtggtggt taggaagcct ccaggaggag gcagtgccgg agctgagcct taaagagctt  10080 

cgtgttgtcc tctctgtctt tgcactctgc acacactcac tgaactgcga caaatgagga  10140 

tagctggtca gggcagaggc aggccggagt tggggctcac tgctgtcccc cacaggctgg  10200 

ggctgaaggg caggctctgg ggccgcagaa tggggtttgt gtaccagatt cttcatatgg  10260 

cagctgtggg actttgggca cgaggcctcc gtctgagcct tagtttcctc aagaggacct  10320 

gcgcccaggt gcacctgggg ctccagccat gggtgcgtcc cattccggga agagctggca  10380 

cacacttgtg cccccggggc agccatgagt gcacaaaggg cagcctgtgc cactgctgga  10440 

tacacgacca gctgagaaca cgaggaccgc cgactccagt taggaggatc aaggaagtgc  10500 

ctggtgggag cagaacagca ggtggggtgc agcccagctc cctggaggga tggtgggcac  10560 

ccatcctcac cctgctgcct ccattagcag gccgagaggg tgtgctctgg aatcccatga  10620 

gcacctgtgc cacatcctcc cctgtggctg acccttcttc acagttggtg cagctttgtg  10680 

gtctgtagtg cagggatcaa ttggcaaatc cctttcccac ccattccctg gagaattggg  10740 

gtccttggct cagatgacag accaacctga gttggaatcc cagctccttg gtggccgtcc  10800 

tggcctccac cccctcactg cctccgctcc tcctatcctg cccacgccca ctgcagggcc  10860 

tttgcacaca ctgtttcttc tgccctccct tccggcccac tccctcatat cattcagtcc  10920 

tcctttcaga tgtcacctcc taagatgggc tgccctgacc acctcatcta taatggcccc  10980 

agtgcctggc acaggattgg cacacagtag atattgtcag agatggatct gggttctgtg  11040 

gacaaggctg tgggggcagg tgaagagctc cctcttccag gaggttgttt ggggttcaag  11100 

gccttgtttg ggttgtaggc ttctgtgctg gtcagcgttg ggccctacaa gcgcatgcca  11160 

tgaggcctgc ccaggatttc cctcatggcc tcacagaata catcggccag agtcattaaa  11220 

gggcgcctgc atctgccttc agagagaggt ttgaaggtag aactggggag ggatgccagg  11280 

tgggggttca ggtttcctgt tgggtcctga tagaatcagg gcaggagagg aagaagaaga  11340 

gggaagagga ggaacccagg cttggggagg ggtggcaggg cttcacaagc ctggggaagg  11400 

tgaactaggg agcagttggg gccaccatgg cccagagtct atgcctcctc ttccttcctg  11460 

tgttcagagt gtgtgtggga accacaaggg ccttctcagt gttcataggg aagcccggtt  11520 

cacccatggg tgggccgcaa tttgggtgcc acagtgagcc cctagagacc agctctccca  11580 

gcttccagga cagggactag gggaggcaag agaggctctt ccttaaattg tgcacccaag  11640 

gtgcctcagc tgccttactc tagactggcc ccgttaactc cccttaaaaa aaaaaaaaaa  11700 

aagactcagt cgaatggtaa tggagctcca acgtgaatac tgcaagtatc aggcaactca  11760 

ctacctgact ttccagttct aaaccattct aattgctgta gagagaacta acctttgttg  11820 

agactgttga gtgatggatg ttttacacac ttgctttccc agaattccca cctctggaga  11880 

tcgtaggtgt gggagctcag agggtgggga gtggactgtc cccatcacac agcaagggag  11940 

gggctaaagg aagagcaggg cctggcatgc agccccagat agcccacttg ggtgtgtctc  12000 

tgagggaggc tgcagggctg gctctagagt ttcctttttc agtcttaacc tggtgaccag  12060 

cttccacaga aattggcacg gtgactcatg cctgtaatcg caacacattg ggaggccgag  12120 

gtgggaggat cacctgaggt caggagttcg agaccagcct ggccaacgtg gtgaaaccct  12180 

gtctctacta aaaatacaaa aattacattt cattacaggt gtggtggcgc acacctgtaa  12240 

tcccagctac tcaggaggct gaggcaggag agtcacttga acccgggagg tagaggttgc  12300 

agtgagctga gatcgtgtca ctgcactcca gcctgggtga cagagccaga ctctgtctca  12360 

aaaaaaaaaa aaaaaaaaaa agaaattggc cagtagatca gccccagggg agagtgagcc  12420 

agggtttggc caggccttga gtttcagagg ctggccatgg ccagtggcac ccaggccctt  12480 

cccccttcct cggggcatct tagcttagtc tgtgccctct gcccaagggc cagccctctg  12540 

ttcccaggtc acaccccctc ctcttggaag gccccccccg ccccaccccc atcagagtct  12600 

ttaatgactc tgctgcccct ggggctcaga gagcaaccgc cctctcccat cgcgcttcct  12660 

cagtgggatg ggagggggtt agagcaggaa gatgagacaa ataaagacac aataagaggc  12720 

aggaatatgt ggtaaagcca agatgggtaa ggggagggga caggcttgac tgttcacagt  12780 

ggccctggcc ctgctgtctc aggctagtat ctgcttgttg gtctcaccac attctaggct  12840 

cagaaactgg ggagcaaagt aatgaaagaa ccaggctggg aggccatggg gaactcatgc  12900 

ctggagttca gctctcagtg tgcttttggg tcaaggacgc ttccctgtct taagtcactc  12960 

atgtcagagc ctttgccaag agcaatgctg tgttttgttt tgggggtgag ggaacacccg  13020 

cgggctgagg ggagggttgg gccatgctag agaggccgtc tgttgtcctt gaacctccca  13080 

aagctgggaa ataagggcct gggctggacg gcggtggcga ggacaggttg cgagagagac  13140 

atggctgggt tttcttgctt agggtcctga atagagagca aggttgaggc cgcagggacc  13200 

ccagccccca atggactgct gagtcgctgg gtctgcccag ggttcaggca ccctctcagg  13260 

ttgcagccaa ctggggtgtg gaccaggcag aggcgctggc ctgcagtttg gggcagaggc  13320 

aggctttgct ggtggtctac ttggctgcaa aatcaactgg ccaggctctg atcactttgt  13380 

gtgtgtgtgt gtgtgtaact tttacctttg acaaaagagg gaagacaggc ccaggcacct  13440 

cctcaaaaga accctagagc ctgtcacccc ttccttaccc atcttctgtc ctagggactg  13500 

cagcccttcc tggcttccca gggccctaca atgaatagtg ggtcgggact cacttggtga  13560 

ctgctgggtt gtgaggcctt gagggggagg ggcagacttc acccatctgg cagagggaca  13620 

tcggtgctgg cagtcaggaa acccttattt ccaggcctca gtttcccgga agtgacctgt  13680 

tttcaggagt ggcctcatcc cagaccatca gccccgctgt ggtgaggggt ggccccttcc  13740 

tggggctgcc ctagaagggg gaggtccctg cacccaccgc agctgccact cggcagccct  13800 

tggccttaat taaacgcttc ttgcgtacta agtgctgcac ccatattatc tcccttctac  13860 

cattcgacgc cagggagata atgactgtcc tgttttctgg aggagtaaac ggagggttgg  13920 

agcggttaag gctcgctcag ggtgccagcg aaccagtgat ttcgaacaca gagttctggt  13980 

gtgttgggcc aggacttctc tgctttgacc ctttaacgaa gggggcggga gctgagggcc  14040 

agtgaccgcc agtaaccccg gcagacgctg gcaccgagcg ggttaaaggc ggacgtccgc  14100 

tagtaacccc aaccccattc agcgccgcgg ggtgaaactc gagcccccgc cgccgtgggg  14160 

aggtggggcg ggggccgggg ccgggcccta gcgaggcggc agcgcggccg ctgattggcc  14220 

gcgcgcgctc accccatgcc cggcccgcag ccccgaaggg cggggcgggg cgggacctgc  14280 

aggcggggcg gggctggggc ggggctgggg gcggggcggg gcggggcggg cgcgccgcag  14340 

cgctcaacgg cttcaaaaat ccgccgcgcc ttgacaggtg aagtcggcgc ggggaggggt  14400 

agggccaacg gcctggacgc cccaagggcg ggcgcagatc gcggagccat ggattgcact  14460 

ttcgaaggta tttttggagg cctccccacc agccctttat acaatgcctc cgtctcctgc  14520 

aggttctcct ggggtgggcg ggcatgcggg ctacgcaact tgagcaggaa agagcccctt  14580 

cccgagggag aaggtgtgac agttaccagc tcgctgggga agtggagggc tacctccaac  14640 

caaattagtg tcccctgcaa ctcaaggggg aagggtttgc ttagagaccc aaaagcagca  14700 

tcccgaccta agagggtttg gagggagagg gtggtcttct ctacattctc tgcacccgct  14760 

ttgggacagg accaggagga agcagggagg agggcccgtt gtccctctgc cacagcgtct  14820 

gccctattca gcacccctgc ctattgtggg catcttagac ttttcaggaa gacagtggga  14880 

gccctagatt gtcaaaattg tcagtttttc tttcaggcct cagtttcccc catctatcga  14940 

agaggctcac acggactggg gtaaagggat gggaaaccct gcagttgaaa gtccattatg  15000 

acttgatgac ttgtgacctg gggggtccac aaaccaggag agtttctact tgagaagcca  15060 

ggaagactgg ggctgccacc ccatcctgtt ctgccaactg ctctaggaaa ttcccctcct  15120 

gcagtagctt ccctgcctgg gtacctgtca gtaggcaatg ttgggtctcc actcggtgcc  15180 

agctgcctgc caagcaaagc ctcgggcagc cgtaccaaaa ggggtttagt cttttctgtt  15240 

gtacagatga ggaaactggg gccagtgaga ggaggctgtt ggtccaggct ccacttcaag  15300 

ctggtggtgg gcagggctgg gagctcaggc tggggatcct gagagcactg gaggccccca  15360 

tgggtcctgt agagcattct gacccagtgg gtgccaccac gagtgggtta gagggccctg  15420 

ggctgagcca gataggctgc tagtcaccag ctgggggaga gggcccttgg ccaggtgggg  15480 

ctgaggtggg agtgtgtccc agtctgtatg aggaggaagg agtcaggaca gacagcactt  15540 

gcttttacag agatgaaatc aaagccctga gtggccaggc ctgggtcttg aggctacttg  15600 

gctgcaggca aagcctggac ttgagcccag aactctacac agagacacac tggttggcca  15660 

tgtggccagc agctggcttg gccctaagcc ttggtctgtt ccactgagta atgggttggt  15720 

gatggcagcc tggctcttgg cttcttagtg gggcaagaaa aggcagagag acaatagatt  15780 

tgggattttg tagacctggg tttgaacccc actgcatgct cttgggctgc ttgtggtcct  15840 

ccctgagcct cagtgtcttt tcttgtctcc aagatgaggt gagctaatct tttgaggtag  15900 

tctagggtag tggccagtgg ttggggcatt ggagtcaaaa tagggtctgg actcagttga  15960 

gtctctgact ctataagaac ttaggccagt aagtcacctc tctacagctc agtttcttca  16020 

cgtgtagaat ggggccaatg atcacatcac cctctcagct gtgggtgagg attaggggtc  16080 

tagcctggcc ccatcaatgt gggtagcccc acagcgggcc tggcttttgg accagaccca  16140 

cccttctgac atgggccccc acccttagag tccttctagt gtggatgagg accctgctct  16200 

gatctggggt cctcttgggg gacttccctg tctgccattc tctttgggga tcctgcgctg  16260 

ccctaggaag agtgggccca ggctgcacag ttggtccttg gtcacagagg atcccaccac  16320 

ttcttcaggg cctcaaggca atcctgcctc tctctgcacc cctcttcccc ctgtaaactg  16380 

aggggagggg aaaatcaccc actcctcagc agtttctaag ttgctttgtc aaattcagtg  16440 

cccagaggat cctgctgggg gtgcgtttta ggatgagacc aggagtggcc aatggtgggg  16500 

tgtggggccc atcgctccta tatgaagacc ccctctgccc tagactgctc ctccctcccc  16560 

atccccatct ccatcccaaa gactggagct gctggatctg tggatggagg cgtgcccccc  16620 

gtttcacaca ttgagaaaca ggccccaagt ggagccaggg aaggctgcac ctgggcctct  16680 

ggattccttt tgttctgtgt ggggttgggg gtgatggact gtggagaggg caggagagct  16740 

gtctggaagg gttggtcacc tcatgggcaa atgcttggaa gctggtctga gtccacggtg  16800 

cagtgtgtat gtgtgtgtgt gtgtgtgtgt gtatgtgtgt ggactcagag gtggatgtct  16860 

tgtagaatgc atgccccatg aagacaggag taaaagttta ccaccatcca catcaagcta  16920 

caggacactc ccagctcccc agaaagttgc ttagttctag gcagggattt cccttattca  16980 

cagccgggag cagtgcctgg catagtgtgg gcactcagca ctcagcacat gctcactgga  17040 

tgagtgaatg aatgtgagcc tgctgtttgc tgtggactaa ggatgtttct agatgtttgg  17100 

gcaaataccg gatggtggga agagctcagg ctctgaagtc tgcagtcttg ggcccgaccc  17160 

tgggctcagc cccagcctag ctgtggggca agattgtgag ccttgtggtg cccaccttgt  17220 

ccaggtattg tgatgcactc gcagcagcag gcattgcttt agacagcaca ggtgctcgca  17280 

aaatggctgt atgtccggga acaccagctc ctgtgggtgg ctttctgtcc tggtggcatt  17340 

gcccacacat acagctgtgt gccaacaagg gttgtgcaaa taaggttgtg tttggatgtg  17400 

tgtgatgccc tgtttggggg tcagtctctg cctcactcac gcaccctctt ctccttttca  17460 

cagacatgct tcagcttatc aacaaccaag acagtgactt ccctggccta tttgacccac  17520 

cctatgctgg gagtggggca gggggcacag accctgccag ccccgatacc agctccccag  17580 

gcagcttgtc tccacctcct gccacattga gctcctctct tgaagccttc ctgagcgggc  17640 

cgcaggcagc gccctcaccc ctgtcccctc cccagcctgc acccactcca ttgaagatgt  17700 

acccgtccat gcccgctttc tcccctgggc ctggtatcaa ggaagagtca gtgccactga  17760 

gcatcctgca gacccccacc ccacagcccc tgccaggggc cctcctgcca cagagcttcc  17820 

cagccccagc cccaccgcag ttcagctcca cccctgtgtt aggctacccc agccctccgg  17880 

gaggcttctc tacaggtaag ggggatgtgt ggcgggaggg gacacccggg gtggggcttc  17940 

caggagcaca ggaagaagct tctgctgtga tgtgagtaga ggtctgtgca ggctttagaa  18000 

actggggctc cactcggctg cttgagatgc cctgttacta gcagtcctgg tgtgcttgtt  18060 

gccggggtag gcgcaacctc gcactggagg cctggcttga agccagtgca tttgcatcag  18120 

agcccaggca gggactgtcc ataggaagcc acatggggca atgactcatc caaggccagt  18180 

cggtgataga gacctgaaga gcaggttgaa agtgggagag ggaggtctgt gtctgcagcc  18240 

ccatgcttta tttctgcagg aagccctccc gggaacaccc agcagccgct gcctggcctg  18300 

ccactggctt ccccgccagg ggtcccgccc gtctccttgc acacccaggt ccagagtgtg  18360 

gtcccccagc agctactgac agtcacagct gcccccacgg cagcccctgt aacgaccact  18420 

gtgacctcgc agatccagca ggtcccggtg agggggtctg gccaggggtt ggggaggggg  18480 

cagccccagc ccagacacac agcttacagc caagcctctc ccaccctcag gtcctgctgc  18540 

agccccactt catcaaggca gactcgctgc ttctgacagc catgaagaca gacggagcca  18600 

ctgtgaaggc ggcaggtctc agtcccctgg tctctggcac cactgtgcag acagggcctt  18660 

tgccggtggg tgacgtgggc agggcataag ggagtggggt ctacacacac acacacatgc  18720 

ccacctggta acatgtgcct ggccctgcag accctggtga gtggcggaac catcttggca  18780 

acagtcccac tggtcgtaga tgcggagaag ctgcctatca accggctcgc agctggcagc  18840 

aaggccccgg cctctgccca gagccgtgga gagaagcgca cagcccacaa cgccattgag  18900 

aagcgctacc gctcctccat caatgacaaa atcattgagc tcaaggatct ggtggtgggc  18960 

actgaggcaa aggtgtggag aggcctgcag gggcacagac cggggtgtcc ctaggaagga  19020 

acagatcagg ggcaactgga aggaagagag ggagtgagac tgagcctgga caagcaggga  19080 

attggaattc agcctcccca ggcctggcca gcctcgttta tttagttaaa ctggtttgca  19140 

ggcctcttca ataaaggtgg ggctgtgcta ggcattgggg atgcagcaat gaacaagaca  19200 

gacaaaaatt gtccctcaaa gaagagccga ccttctggtg ggggagatgg acagtaggca  19260 

ggatgaataa gtgctcgaga ccaccacgtt tggctcgttg cagagaaagc aggaagagga  19320 

tggtgagggt cccctggtgg tagccaggga aggcctccct gagatggcgg caggcacagc  19380 

agcagctagc cagaccctgc tgtctgcatc ttacattcta accctatgcc cggcctggga  19440 

ggtgggtgct actaggcgag gaacggttca ggtagaagga acaagtgcaa aggtcctgag  19500 

gcagtaatgt tgcaaagcag ctccgcaccc ccttgctagg gctctccaac cccacaaccc  19560 

ccgacctgac aggccacctg tgcgctcccc ctccctccca caccgtgcag ctgaataaat  19620 

ctgctgtctt gcgcaaggcc atcgactaca ttcgctttct gcaacacagc aaccagaaac  19680 

tcaagcagga gaacctaagt ctgcgcactg ctgtccacaa aagcagtgag tcctggcttt  19740 

attgagctcc agtctggcct cttctctagc cttgctccac ctcccggccc caccccatcc  19800 

ctagccccac cccacccttg gttctggccc accctctgcc ctgcccacct cacccttggc  19860 

tgtagccctg cattcagctc tagtcccttg gttacctctg gtcctgaaag agacctggtg  19920 

cctccctttg gccctaaccc agccccatca aagcgtcctg ggctagcttt aggagctaca  19980 

gtagtcccta ggcctccaag ggcctaggct ctgatttggg gtcacatatc cagcctttac  20040 

tcctggctct gttcctttcg gcccacagaa tctctgaagg atctggtgtc ggcctgtggc  20100 

agtggaggga acacagacgt gctcatggag ggcgtgaaga ctgaggtgga ggacacactg  20160 

accccacccc cctcggatgc tggctcacct ttccagagca gccccttgtc ccttggcagc  20220 

aggggcagtg gcagcggtgg cagtggcagt gactcggagc ctgacagccc agtctttgag  20280 

gacagcaagg ttgggccctg ccacggtgcc cccttcccca ctcccagcca tatcctctga  20340 

gcctcatgac agggccggga agaccctaac agatcctacc tcccatttca tagacagaat  20400 

aactgaggcc tggagccacg tggggtccca cagtaaggtg ggcagaatcc tgaccccccc  20460 

cttcccagcc ccatgctctc tggggtccct ccgattctgc cctcaccacc ctgcccaacc  20520 

ccaccaggca aagccagagc agcggccgtc tctgcacagc cggggcatgc tggaccgctc  20580 

ccgcctggcc ctgtgcacgc tcgtcttcct ctgcctgtcc tgcaacccct tggcctcctt  20640 

gctgggggcc cgggggcttc ccagcccctc agataccacc agcgtctacc atagccctgg  20700 

gcgcaacgtg ctgggcaccg agagcagagg tgggaccggc cagcctgggc atctttggga  20760 

gggacactcg gggtgagccc ccaggcttgt gaacttgggg ctctggattt cctgggagct  20820 

gtgtccccag ctttccctct gtccatagat ggccctggct gggcccagtg gctgctgccc  20880 

ccagtggtct ggctgctcaa tgggctgttg gtgctcgtct ccttggtgct tctctttgtc  20940 

tacggtgagc cagtcacacg gccccactca ggccccgccg tgtacttctg gaggcatcgc  21000 

aagcaggctg acctggacct ggcccgggta aggggctggc cccggcagag tgggcagggc  21060 

agggacccca ggctgtgaag gtgctgggtg tcaacccttg ttcctgctcc ctgtgcacac  21120 

catgaatctg tcccgtcctc cctgtgccta gccacgcatc cgcagacccc caccacccct  21180 

ccagagcctg ctgtggacgg ctcttctgag ctttggggca gctgctctga cctcactttt  21240 

ctcacctgga aaaccctcat ccacagggag actttgccca ggctgcccag cagctgtggc  21300 

tggccctgcg ggcactgggc cggcccctgc ccacctccca cctggacctg gcttgtagcc  21360 

tcctctggaa cctcatccgt cacctgctgc agcgtctctg ggtgggccgc tggctggcag  21420 

gccgggcagg gggcctgcag caggactgtg ctctgcgagt ggatgctagc gccagcgccc  21480 

gagacgcagc cctggtctac cataagctgc accagctgca caccatgggt aggactgagc  21540 

gtggggcggg ctccgaggtg ctccctgctg cctgtgctcc acccacagcc tcatgcctgc  21600 

ttgccttcca gggaagcaca caggcgggca cctcactgcc accaacctgg cgctgagtgc  21660 

cctgaacctg gcagagtgtg caggggatgc cgtgtctgtg gcgacgctgg ccgagatcta  21720 

tgtggcggct gcattgagag tgaagaccag tctcccacgg gccttgcatt ttctgacagt  21780 

gagtgggttg gggggatggc gggagtgggg agggtggggc gcctgaggct ccctgggtaa  21840 

gagctacacg ggatgtggca gtggttacca gggggactcc aggccaagct gggactcggc  21900 

ccggggtctg gccccaggct gtgtccactg tgacagccca gtacccaccc ctacagcgct  21960 

tcttcctgag cagtgcccgc caggcctgcc tggcacagag tggctcagtg cctcctgcca  22020 

tgcagtggct ctgccacccc gtgggccacc gtttcttcgt ggatggggac tggtccgtgc  22080 

tcagtacccc atgggagagc ctgtacagct tggccgggaa cccaggtgct ctcttacccc  22140 

ttccctgtcc cctctcctgt ccctcatcct cattcctgtc ctgtcccttg tcgcctgaat  22200 

ctctggctgt ctctggccac cccagtcctt ctccctgcca tgggttgttg ctgtgggggt  22260 

tgcaggaagg gaaaggcctg ggtgcctctc gttcccattg gggctttcag aagcacatgc  22320 

agggattgat gggcagatgg ctaattggag aagtgacccc aggcagtgcc gctgtggagt  22380 

aaggaagcgg agccaacaat ggcatcttct caagtcggtt ttcctttgga agcagtgtag  22440 

ggcaggcctc agtgttgtct cctggccaag gctggtgctg gtgatagtta tgtccacccg  22500 

ctttcccctg tccttggcag gggctgcacc caggggcatg ccggcacttc ccagtggccc  22560 

taggtgtggc cccagcccac ccaggaaaaa gcccttagct tggagaggag ggtggggccc  22620 

tgctccccac cccactcacc tcctcctctc cacagtggac cccctggccc aggtgactca  22680 

gctattccgg gaacatctct tagagcgagc actgaactgt gtgacccagc ccaaccccag  22740 

ccctgggtca gctgatgggg acaagtaagt gtcgttgtgc cctcctccag gcaaggcccc  22800 

tccggcggga ttctgagaat agctctggcc tcaaccctgt ggagagagcc cagagctggg  22860 

ctaccgtgcg tgccatgcac gcttcattcc tctctgagtt tcctctcccc accagcctgt  22920 

gggaggagac agtggcactt tgcagagcca ggggccaggc tgtactctgg agggcaggtg  22980 

gggagcaccc tcctaggacc cctgccatct gttccgacag ccagctctct ccttccacag  23040 

ggaattctcg gatgccctcg ggtacctgca gctgctgaac agctgttctg atgctgcggg  23100 

ggctcctgcc tacagcttct ccatcagttc cagcatggcc accaccaccg gtgagtcccc  23160 

ggcccctgtc ctggctccct tctcagctcc cccgtgcagc gtgactgagg gttcagggga  23220 

ccctccctct tctgcaggcg tagacccggt ggccaagtgg tgggcctctc tgacagctgt  23280 

ggtgatccac tggctgcggc gggatgagga ggcggctgag cggctgtgcc cgctggtgga  23340 

gcacctgccc cgggtgctgc aggagtctga gtgagtgcac ggcaggttcc tcctgcctgg  23400 

tcccgggctc agccttcctc atcccctggg cactgtgcct cactcagcct ttgttctgtg  23460 

caggaggagt caccaccttt tttcctcagg gaactcgagc cagggaagtg gggggcactc  23520 

agccagggct tgtggactgg tctgactggc actcttctgc cctggtccca acaggagacc  23580 

cctgcccagg gcagctctgc actccttcaa ggctgcccgg gccctgctgg gctgtgccaa  23640 

ggcagagtct ggtccagcca gcctgaccat ctgtgagaag gccagtgggt acctgcagga  23700 

cagcctggct accacaccag ccagcagctc cattgacaag gtgaggggtg gggtcagggg  23760 

cctggcaggg ctgggggatt cagctttcca ttccctggtt cctctcccca gcccccaggg  23820 

gctgcagaag accatggggt tagcccaagc agcacaggat agggggtcca gcagaccctg  23880 

ctttttggct aaggcttctg tccagaggag aggggttgcc cctatctggc ctcagtttcc  23940 

ccatccctgg gaggaggggg gtggatggtg tggtaggatc cctttggagg ccctgcatca  24000 

ggagggctgg acagctgctc ccgggccggt ggcgggtgtg ggggccgaga gaggcgggcg  24060 

gccccgcggt gcattgctgt tgcattgcac gtgtgtgagg cgggtgcagt gcctcggcag  24120 

tgcagcccgg agccggcccc tggcaccacg ggcccccatc ctgcccctcc cagagctgga  24180 

gccctggtga cccctgccct gcctgccacc cccaggccgt gcagctgttc ctgtgtgacc  24240 

tgcttcttgt ggtgcgcacc agcctgtggc ggcagcagca gcccccggcc ccggccccag  24300 

cagcccaggg caccagcagc aggccccagg cttccgccct tgagctgcgt ggcttccaac  24360 

gggacctgag cagcctgagg cggctggcac agagcttccg gcccgccatg cggagggtga  24420 

gtgcccgatg gccctgtcct caagacgggg agtcaggcag tggtggagat ggagagccct  24480 

gagcctccac tctcctggcc cccaggtgtt cctacatgag gccacggccc ggctgatggc  24540 

gggggccagc cccacacgga cacaccagct cctcgaccgc agtctgaggc ggcgggcagg  24600 

ccccggtggc aaaggaggtg agggggcagc tgctgaccag ggatgtgctg tctgctcagc  24660 

agggaagggc gcacatggga tgtgatacca agggaggctg tgtgtgtgtc agacgggaca  24720 

gacaggcctg gcgcagtggc tcacacctag cactttggga ggctcagttg ggaggacagc  24780 

ttgagcccag gagttggagg ccgcagtgag cctgagtgac agggagagtc cctgtctcaa  24840 

aaaaaaaaaa agaccaagca tcttcttgat ggttacctga tgacaattcc tttcacaagg  24900 

aatcagtggg gtgactgtca tttgtgggat acatgactgc acgtgcgtga ctcagtctgt  24960 

ggactttgtg tgtgggctga gactagggtg gggagagggg aacccgccag gcccccgcca  25020 

ggtacctgtg tgccaggtac aggcggctgg tgccgtggct tgtgtgtggg cagggctccc  25080 

gcgggggcgt ggccagcttg agacccatcc ctgacacatc ctcgtgtgcg caggcgcggt  25140 

ggcggagctg gagccgcggc ccacgcggcg ggagcacgcg gaggccttgc tgctggcctc  25200 

ctgctacctg ccccccggct tcctgtcggc gcccgggcag cgcgtgggca tgctggctga  25260 

ggcggcgcgc acactcgaga agcttggcga tcgccggctg ctgcacgact gtcagcagat  25320 

gctcatgcgc ctgggcggtg ggaccactgt cacttccagc tagaccccgt gtccccggcc  25380 

tcagcacccc tgtctctagc cactttggtc ccgtgcagct tctgtcctgc gtcgaagctt  25440 

tgaaggccga aggcagtgca agagactctg gcctccacag ttcgacctgc ggctgctgtg  25500 

tgccttcgcg gtggaaggcc cgaggggcgc gatcttgacc ctaagaccgg cggccatgat  25560 

ggtgctgacc tctggtggcc gatcggggca ctgcaggggc cgagccattt tggggggccc  25620 

ccctccttgc tctgcaggca ccttagtggc ttttttcctc ctgtgtacag ggaagagagg  25680 

ggtacatttc cctgtgctga cggaagccaa cttggctttc ccggactgca agcagggctc  25740 

tgccccagag gcctctctct ccgtcgtggg agagagacgt gtacatagtg taggtcagcg  25800 

tgcttagcct cctgacctga ggctcctgtg ctactttgcc ttttgcaaac tttattttca  25860 

tagattgaga agttttgtac agagaattaa aaatgaaatt atttataatc tgggttttgt  25920 

gtcttcagct gatggatgtg ctgactagtg agagtgcttg ggccctcccc cagcacctag  25980 

ggaaaggctt cccctccccc tccggccaca aggtacacaa cttttaactt agctcttccc  26040 

gatgtttgtt tgttagtggg aggagtgggg agggctggct gtatggcctc cagcctacct  26100 

gttccccctg ctcccagggc acatggttgg gctgtgtcaa cccttagggc ctccatgggg  26160 

tcagttgtcc cttctcacct cccagctctg tccccatcag gtccctgggt ggcacgggag  26220 

gatggactga cttccaggac ctgttgtgtg acaggagcta cagcttgggt ctccctgcaa  26280 

gaagtctggc acgtctcacc tcccccatcc cggcccctgg tcatctcaca gcaaagaagc  26340 

ctcctccctc ccgacctgcc gccacactgg agagggggca caggggcggg ggaggtttcc  26400 

tgttctgtga aaggccgact ccctgactcc attcatgccc ccccccccag cccctccctt  26460 

cattcccatt ccccaaccta aagcctggcc cggctcccag ctgaatctgg tcggaatcca  26520 

cgggctgcag attttccaaa acaatcgttg tatctttatt gacttttttt tttttttttt  26580 

tctgaatgca atgactgttt tttactctta aggaaaataa acatctttta gaaacagctc  26640 

gatacacaca atcttcagtg tgaagcaata tactaataag aacactagtc gtcttaacat  26700 

ttacagtctt catatatatt atatatatgt atatgtatac atatatatac actatataac  26760 

gaggccagat ataatacaca cgtttaccat tttacagtca tatgtacagg aagttgctag  26820 

ggcggccctg ggctgggggc tgcgtcaggc ctatcgaagc gtggacagag ctgaggacac  26880 

ggacggacag gcggacggac tggcagggac tggcccgggc cggtggtggc tgcgtggaca  26940 

agtggcgtcg cggtagcccc ttacccggca aaggcccggt tggggctctg ttgcgggcgc  27000 

a                                                                  27001 

 
           
             19  
             698  
             DNA  
             H. sapiens  
             
 
           
            19 

ccttgacagg tgaagtcggc gcggggaggg gtagggccaa cggcctggac gccccaaggg     60 

cgggcgcaga tcgcggagcc atggattgca ctttcgaaga catgcttcag cttatcaaca    120 

accaagacag tgacttccct ggcctatttg acccacccta tgctgggagt ggggcagggg    180 

gcacagaccc tgccagcccc gataccagct ccccaggcag ctagtctcca cctcctgcca    240 

cattgagctc ctctcttgaa gccttcctga gcgggccgca ggcagcgccc tcacccctgt    300 

cccctcccca gcctgcaccc actccattga agatgtaccc gtccatgccc gctttctccc    360 

ctgggcctgg tatcaaggaa gagtcagtgc cactgagcat cctgcagacc cccaccccac    420 

agcccctgcc aggggccctc ctgccacaga gcttcccagc cccagcccca cctgagttca    480 

gctccacccc tgtgttaggc taccccagcc ctcctggagg ctactctaca ggaagccctc    540 

ccgggaacac ccagcagccg ctgcctggcc tgccactggc ttccccgaca ggggtcccgc    600 

ccgtctcctt gcacacccgg gtccagagtg tggtccccca gtagctactg acagtcacag    660 

ctggccccac tgcagcccct tgaacgacca ctgtgact                            698 

 
           
             20  
             4154  
             DNA  
             H. sapiens  
             
               CDS  
               (167)...(3610)  
             
           
            20 

taacgaggaa cttttcgccg gcgccgggcc gcctctgagg ccagggcagg acacgaacgc     60 

gcggagcggc ggcggcgact gagagccggg gccgcggcgg cgctccctag gaagggccgt    120 

acgaggcggc gggcccggcg ggcctcccgg aggaggcggc tgcgcc atg gac gag       175 
                                                   Met Asp Glu 
                                                     1 

cca ccc ttc agc gag gcg gct ttg gag cag gcg ctg ggc gag ccg tgc      223 
Pro Pro Phe Ser Glu Ala Ala Leu Glu Gln Ala Leu Gly Glu Pro Cys 
      5                  10                  15 

gat ctg gac gcg gcg ctg ctg acc gac atc gaa gac atg ctt cag ctt      271 
Asp Leu Asp Ala Ala Leu Leu Thr Asp Ile Glu Asp Met Leu Gln Leu 
 20                  25                  30                  35 

atc aac aac caa gac agt gac ttc cct ggc cta ttt gac cca ccc tat      319 
Ile Asn Asn Gln Asp Ser Asp Phe Pro Gly Leu Phe Asp Pro Pro Tyr 
                 40                  45                  50 

gct ggg agt ggg gca ggg ggc aca gac cct gcc agc ccc gat acc agc      367 
Ala Gly Ser Gly Ala Gly Gly Thr Asp Pro Ala Ser Pro Asp Thr Ser 
             55                  60                  65 

tcc cca ggc agc ttg tct cca cct cct gcc aca ttg agc tcc tct ctt      415 
Ser Pro Gly Ser Leu Ser Pro Pro Pro Ala Thr Leu Ser Ser Ser Leu 
         70                  75                  80 

gaa gcc ttc ctg agc ggg ccg cag gca gcg ccc tca ccc ctg tcc cct      463 
Glu Ala Phe Leu Ser Gly Pro Gln Ala Ala Pro Ser Pro Leu Ser Pro 
     85                  90                  95 

ccc cag cct gca ccc act cca ttg aag atg tac ccg tcc atg ccc gct      511 
Pro Gln Pro Ala Pro Thr Pro Leu Lys Met Tyr Pro Ser Met Pro Ala 
100                 105                 110                 115 

ttc tcc cct ggg cct ggt atc aag gaa gag tca gtg cca ctg agc atc      559 
Phe Ser Pro Gly Pro Gly Ile Lys Glu Glu Ser Val Pro Leu Ser Ile 
                120                 125                 130 

ctg cag acc ccc acc cca cag ccc ctg cca ggg gcc ctc ctg cca cag      607 
Leu Gln Thr Pro Thr Pro Gln Pro Leu Pro Gly Ala Leu Leu Pro Gln 
            135                 140                 145 

agc ttc cca gcc cca gcc cca ccg cag ttc agc tcc acc cct gtg tta      655 
Ser Phe Pro Ala Pro Ala Pro Pro Gln Phe Ser Ser Thr Pro Val Leu 
        150                 155                 160 

ggc tac ccc agc cct ccg gga ggc ttc tct aca gga agc cct ccc ggg      703 
Gly Tyr Pro Ser Pro Pro Gly Gly Phe Ser Thr Gly Ser Pro Pro Gly 
    165                 170                 175 

aac acc cag cag ccg ctg cct ggc ctg cca ctg gct tcc ccg cca ggg      751 
Asn Thr Gln Gln Pro Leu Pro Gly Leu Pro Leu Ala Ser Pro Pro Gly 
180                 185                 190                 195 

gtc ccg ccc gtc tcc ttg cac acc cag gtc cag agt gtg gtc ccc cag      799 
Val Pro Pro Val Ser Leu His Thr Gln Val Gln Ser Val Val Pro Gln 
                200                 205                 210 

cag cta ctg aca gtc aca gct gcc ccc acg gca gcc cct gta acg acc      847 
Gln Leu Leu Thr Val Thr Ala Ala Pro Thr Ala Ala Pro Val Thr Thr 
            215                 220                 225 

act gtg acc tcg cag atc cag cag gtc ccg gtc ctg ctg cag ccc cac      895 
Thr Val Thr Ser Gln Ile Gln Gln Val Pro Val Leu Leu Gln Pro His 
        230                 235                 240 

ttc atc aag gca gac tcg ctg ctt ctg aca gcc atg aag aca gac gga      943 
Phe Ile Lys Ala Asp Ser Leu Leu Leu Thr Ala Met Lys Thr Asp Gly 
    245                 250                 255 

gcc act gtg aag gcg gca ggt ctc agt ccc ctg gtc tct ggc acc act      991 
Ala Thr Val Lys Ala Ala Gly Leu Ser Pro Leu Val Ser Gly Thr Thr 
260                 265                 270                 275 

gtg cag aca ggg cct ttg ccg acc ctg gtg agt ggc gga acc atc ttg     1039 
Val Gln Thr Gly Pro Leu Pro Thr Leu Val Ser Gly Gly Thr Ile Leu 
                280                 285                 290 

gca aca gtc cca ctg gtc gta gat gcg gag aag ctg cct atc aac cgg     1087 
Ala Thr Val Pro Leu Val Val Asp Ala Glu Lys Leu Pro Ile Asn Arg 
            295                 300                 305 

ctc gca gct ggc agc aag gcc ccg gcc tct gcc cag agc cgt gga gag     1135 
Leu Ala Ala Gly Ser Lys Ala Pro Ala Ser Ala Gln Ser Arg Gly Glu 
        310                 315                 320 

aag cgc aca gcc cac aac gcc att gag aag cgc tac cgc tcc tcc atc     1183 
Lys Arg Thr Ala His Asn Ala Ile Glu Lys Arg Tyr Arg Ser Ser Ile 
    325                 330                 335 

aat gac aaa atc att gag ctc aag gat ctg gtg gtg ggc act gag gca     1231 
Asn Asp Lys Ile Ile Glu Leu Lys Asp Leu Val Val Gly Thr Glu Ala 
340                 345                 350                 355 

aag ctg aat aaa tct gct gtc ttg cgc aag gcc atc gac tac att cgc     1279 
Lys Leu Asn Lys Ser Ala Val Leu Arg Lys Ala Ile Asp Tyr Ile Arg 
                360                 365                 370 

ttt ctg caa cac agc aac cag aaa ctc aag cag gag aac cta agt ctg     1327 
Phe Leu Gln His Ser Asn Gln Lys Leu Lys Gln Glu Asn Leu Ser Leu 
            375                 380                 385 

cgc act gct gtc cac aaa agc aaa tct ctg aag gat ctg gtg tcg gcc     1375 
Arg Thr Ala Val His Lys Ser Lys Ser Leu Lys Asp Leu Val Ser Ala 
        390                 395                 400 

tgt ggc agt gga ggg aac aca gac gtg ctc atg gag ggc gtg aag act     1423 
Cys Gly Ser Gly Gly Asn Thr Asp Val Leu Met Glu Gly Val Lys Thr 
    405                 410                 415 

gag gtg gag gac aca ctg acc cca ccc ccc tcg gat gct ggc tca cct     1471 
Glu Val Glu Asp Thr Leu Thr Pro Pro Pro Ser Asp Ala Gly Ser Pro 
420                 425                 430                 435 

ttc cag agc agc ccc ttg tcc ctt ggc agc agg ggc agt ggc agc ggt     1519 
Phe Gln Ser Ser Pro Leu Ser Leu Gly Ser Arg Gly Ser Gly Ser Gly 
                440                 445                 450 

ggc agt ggc agt gac tcg gag cct gac agc cca gtc ttt gag gac agc     1567 
Gly Ser Gly Ser Asp Ser Glu Pro Asp Ser Pro Val Phe Glu Asp Ser 
            455                 460                 465 

aag gca aag cca gag cag cgg ccg tct ctg cac agc cgg ggc atg ctg     1615 
Lys Ala Lys Pro Glu Gln Arg Pro Ser Leu His Ser Arg Gly Met Leu 
        470                 475                 480 

gac cgc tcc cgc ctg gcc ctg tgc acg ctc gtc ttc ctc tgc ctg tcc     1663 
Asp Arg Ser Arg Leu Ala Leu Cys Thr Leu Val Phe Leu Cys Leu Ser 
    485                 490                 495 

tgc aac ccc ttg gcc tcc ttg ctg ggg gcc cgg ggg ctt ccc agc ccc     1711 
Cys Asn Pro Leu Ala Ser Leu Leu Gly Ala Arg Gly Leu Pro Ser Pro 
500                 505                 510                 515 

tca gat acc acc agc gtc tac cat agc cct ggg cgc aac gtg ctg ggc     1759 
Ser Asp Thr Thr Ser Val Tyr His Ser Pro Gly Arg Asn Val Leu Gly 
                520                 525                 530 

acc gag agc aga gat ggc cct ggc tgg gcc cag tgg ctg ctg ccc cca     1807 
Thr Glu Ser Arg Asp Gly Pro Gly Trp Ala Gln Trp Leu Leu Pro Pro 
            535                 540                 545 

gtg gtc tgg ctg ctc aat ggg ctg ttg gtg ctc gtc tcc ttg gtg ctt     1855 
Val Val Trp Leu Leu Asn Gly Leu Leu Val Leu Val Ser Leu Val Leu 
        550                 555                 560 

ctc ttt gtc tac ggt gag cca gtc aca cgg ccc cac tca ggc ccc gcc     1903 
Leu Phe Val Tyr Gly Glu Pro Val Thr Arg Pro His Ser Gly Pro Ala 
    565                 570                 575 

gtg tac ttc tgg agg cat cgc aag cag gct gac ctg gac ctg gcc cgg     1951 
Val Tyr Phe Trp Arg His Arg Lys Gln Ala Asp Leu Asp Leu Ala Arg 
580                 585                 590                 595 

gga gac ttt gcc cag gct gcc cag cag ctg tgg ctg gcc ctg cgg gca     1999 
Gly Asp Phe Ala Gln Ala Ala Gln Gln Leu Trp Leu Ala Leu Arg Ala 
                600                 605                 610 

ctg ggc cgg ccc ctg ccc acc tcc cac ctg gac ctg gct tgt agc ctc     2047 
Leu Gly Arg Pro Leu Pro Thr Ser His Leu Asp Leu Ala Cys Ser Leu 
            615                 620                 625 

ctc tgg aac ctc atc cgt cac ctg ctg cag cgt ctc tgg gtg ggc cgc     2095 
Leu Trp Asn Leu Ile Arg His Leu Leu Gln Arg Leu Trp Val Gly Arg 
        630                 635                 640 

tgg ctg gca ggc cgg gca ggg ggc ctg cag cag gac tgt gct ctg cga     2143 
Trp Leu Ala Gly Arg Ala Gly Gly Leu Gln Gln Asp Cys Ala Leu Arg 
    645                 650                 655 

gtg gat gct agc gcc agc gcc cga gac gca gcc ctg gtc tac cat aag     2191 
Val Asp Ala Ser Ala Ser Ala Arg Asp Ala Ala Leu Val Tyr His Lys 
660                 665                 670                 675 

ctg cac cag ctg cac acc atg ggg aag cac aca ggc ggg cac ctc act     2239 
Leu His Gln Leu His Thr Met Gly Lys His Thr Gly Gly His Leu Thr 
                680                 685                 690 

gcc acc aac ctg gcg ctg agt gcc ctg aac ctg gca gag tgt gca ggg     2287 
Ala Thr Asn Leu Ala Leu Ser Ala Leu Asn Leu Ala Glu Cys Ala Gly 
            695                 700                 705 

gat gcc gtg tct gtg gcg acg ctg gcc gag atc tat gtg gcg gct gca     2335 
Asp Ala Val Ser Val Ala Thr Leu Ala Glu Ile Tyr Val Ala Ala Ala 
        710                 715                 720 

ttg aga gtg aag acc agt ctc cca cgg gcc ttg cat ttt ctg aca cgc     2383 
Leu Arg Val Lys Thr Ser Leu Pro Arg Ala Leu His Phe Leu Thr Arg 
    725                 730                 735 

ttc ttc ctg agc agt gcc cgc cag gcc tgc ctg gca cag agt ggc tca     2431 
Phe Phe Leu Ser Ser Ala Arg Gln Ala Cys Leu Ala Gln Ser Gly Ser 
740                 745                 750                 755 

gtg cct cct gcc atg cag tgg ctc tgc cac ccc gtg ggc cac cgt ttc     2479 
Val Pro Pro Ala Met Gln Trp Leu Cys His Pro Val Gly His Arg Phe 
                760                 765                 770 

ttc gtg gat ggg gac tgg tcc gtg ctc agt acc cca tgg gag agc ctg     2527 
Phe Val Asp Gly Asp Trp Ser Val Leu Ser Thr Pro Trp Glu Ser Leu 
            775                 780                 785 

tac agc ttg gcc ggg aac cca gtg gac ccc ctg gcc cag gtg act cag     2575 
Tyr Ser Leu Ala Gly Asn Pro Val Asp Pro Leu Ala Gln Val Thr Gln 
        790                 795                 800 

cta ttc cgg gaa cat ctc tta gag cga gca ctg aac tgt gtg acc cag     2623 
Leu Phe Arg Glu His Leu Leu Glu Arg Ala Leu Asn Cys Val Thr Gln 
    805                 810                 815 

ccc aac ccc agc cct ggg tca gct gat ggg gac aag gaa ttc tcg gat     2671 
Pro Asn Pro Ser Pro Gly Ser Ala Asp Gly Asp Lys Glu Phe Ser Asp 
820                 825                 830                 835 

gcc ctc ggg tac ctg cag ctg ctg aac agc tgt tct gat gct gcg ggg     2719 
Ala Leu Gly Tyr Leu Gln Leu Leu Asn Ser Cys Ser Asp Ala Ala Gly 
                840                 845                 850 

gct cct gcc tac agc ttc tcc atc agt tcc agc atg gcc acc acc acc     2767 
Ala Pro Ala Tyr Ser Phe Ser Ile Ser Ser Ser Met Ala Thr Thr Thr 
            855                 860                 865 

ggc gta gac ccg gtg gcc aag tgg tgg gcc tct ctg aca gct gtg gtg     2815 
Gly Val Asp Pro Val Ala Lys Trp Trp Ala Ser Leu Thr Ala Val Val 
        870                 875                 880 

atc cac tgg ctg cgg cgg gat gag gag gcg gct gag cgg ctg tgc ccg     2863 
Ile His Trp Leu Arg Arg Asp Glu Glu Ala Ala Glu Arg Leu Cys Pro 
    885                 890                 895 

ctg gtg gag cac ctg ccc cgg gtg ctg cag gag tct gag aga ccc ctg     2911 
Leu Val Glu His Leu Pro Arg Val Leu Gln Glu Ser Glu Arg Pro Leu 
900                 905                 910                 915 

ccc agg gca gct ctg cac tcc ttc aag gct gcc cgg gcc ctg ctg ggc     2959 
Pro Arg Ala Ala Leu His Ser Phe Lys Ala Ala Arg Ala Leu Leu Gly 
                920                 925                 930 

tgt gcc aag gca gag tct ggt cca gcc agc ctg acc atc tgt gag aag     3007 
Cys Ala Lys Ala Glu Ser Gly Pro Ala Ser Leu Thr Ile Cys Glu Lys 
            935                 940                 945 

gcc agt ggg tac ctg cag gac agc ctg gct acc aca cca gcc agc agc     3055 
Ala Ser Gly Tyr Leu Gln Asp Ser Leu Ala Thr Thr Pro Ala Ser Ser 
        950                 955                 960 

tcc att gac aag gcc gtg cag ctg ttc ctg tgt gac ctg ctt ctt gtg     3103 
Ser Ile Asp Lys Ala Val Gln Leu Phe Leu Cys Asp Leu Leu Leu Val 
    965                 970                 975 

gtg cgc acc agc ctg tgg cgg cag cag cag ccc ccg gcc ccg gcc cca     3151 
Val Arg Thr Ser Leu Trp Arg Gln Gln Gln Pro Pro Ala Pro Ala Pro 
980                 985                 990                 995 

gca gcc cag ggc gcc agc agc agg ccc cag gct tcc gcc ctt gag ctg     3199 
Ala Ala Gln Gly Ala Ser Ser Arg Pro Gln Ala Ser Ala Leu Glu Leu 
                1000                1005                1010 

cgt ggc ttc caa cgg gac ctg agc agc ctg agg cgg ctg gca cag agc     3247 
Arg Gly Phe Gln Arg Asp Leu Ser Ser Leu Arg Arg Leu Ala Gln Ser 
            1015                1020                1025 

ttc cgg ccc gcc atg cgg agg gtg ttc cta cat gag gcc acg gcc cgg     3295 
Phe Arg Pro Ala Met Arg Arg Val Phe Leu His Glu Ala Thr Ala Arg 
        1030                1035                1040 

ctg atg gcg ggg gcc agc ccc aca cgg aca cac cag ctc ctc gac cgc     3343 
Leu Met Ala Gly Ala Ser Pro Thr Arg Thr His Gln Leu Leu Asp Arg 
    1045                1050                1055 

agt ctg agg cgg cgg gca ggc ccc ggt ggc aaa gga ggc gcg gtg gcg     3391 
Ser Leu Arg Arg Arg Ala Gly Pro Gly Gly Lys Gly Gly Ala Val Ala 
1060                1065                1070                1075 

gag ctg gag ccg cgg ccc acg cgg cgg gag cac gcg gag gcc ttg ctg     3439 
Glu Leu Glu Pro Arg Pro Thr Arg Arg Glu His Ala Glu Ala Leu Leu 
                1080                1085                1090 

ctg gcc tcc tgc tac ctg ccc ccc ggc ttc ctg tcg gcg ccc ggg cag     3487 
Leu Ala Ser Cys Tyr Leu Pro Pro Gly Phe Leu Ser Ala Pro Gly Gln 
            1095                1100                1105 

cgc gtg ggc atg ctg gct gag gcg gcg cgc aca ctc gag aag ctt ggc     3535 
Arg Val Gly Met Leu Ala Glu Ala Ala Arg Thr Leu Glu Lys Leu Gly 
        1110                1115                1120 

gat cgc cgg ctg ctg cac gac tgt cag cag atg ctc atg cgc ctg ggc     3583 
Asp Arg Arg Leu Leu His Asp Cys Gln Gln Met Leu Met Arg Leu Gly 
    1125                1130                1135 

ggt ggg acc act gtc act tcc agc tag accccgtgtc cccggcctca           3630 
Gly Gly Thr Thr Val Thr Ser Ser 
1140                1145 

gcacccctgt ctctagccac tttggtcccg tgcagcttct gtcctgcgtc gaagctttga   3690 

aggccgaagg cagtgcaaga gactctggcc tccacagttc gacctgcggc tgctgtgtgc   3750 

cttcgcggtg gaaggcccga ggggcgcgat cttgacccta agaccggcgg ccatgatggt   3810 

gctgacctct ggtggccgat cggggcactg caggggccga gccattttgg ggggcccccc   3870 

tccttgctct gcaggcacct tagtggcttt tttcctcctg tgtacaggga agagaggggt   3930 

acatttccct gtgctgacgg aagccaactt ggctttcccg gactgcaagc agggctctgc   3990 

cccagaggcc tctctctccg tcgtgggaga gagacgtgta catagtgtag gtcagcgtgc   4050 

ttagcctcct gacctgaggc tcctgtgcta ctttgccttt tgcaaacttt attttcatag   4110 

attgagaagt tttgtacaga gaattaaaaa tgaaattatt tata                    4154 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

tgtctgcaca gtggtgccag                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

ctccgagtca ctgccactgc                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

tgaagcatgt cttcgaaagt                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

gtcactgtct tggttgttga                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

gggaagtcac tgtcttggtt                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

ggccagggaa gtcactgtct                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

gagtctgcct tgatgaagtg                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

gccttgctgc cagctgcgag                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

gcagatttat tcagctttgc                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

agacagcaga tttattcagc                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

gcgcaagaca gcagatttat                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

gccttgcgca agacagcaga                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

cgatggcctt gcgcaagaca                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

gtagtcgatg gccttgcgca                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

aggcgggagc ggtccagcat                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

ggcagagcca ctgcatggca                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

gaggcccacc acttggccac                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

gccagtggat caccacagct                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

ccgggcagcc ttgaaggagt                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

actggccttc tcacagatgg                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

gcaggtaccc actggccttc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

ctatgaaaat aaagtttgca                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

gccgacttca cctgtcaagg                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

ggagggcttc ctgcagaaat                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

atttattcag ctgcacggtg                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

gtgcttccct ggaaggcaag                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

gcaccagcct tggccaggag                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

ccctgtggaa ggagagagct                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

gggtctacgc ctgcagaaga                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

gggcactcac cctccgcatg                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

gtccaggccg ttggccctac                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

agtgcaatcc atggctccgc                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

gataagctga agcatgtctt                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

gtcctgccct ggcctcagag                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

tggctcgtcc atggcgcagc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

cgcctcgctg aagggtggct                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

ctgaagcatg tcttcgatgt                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

ctcaatgtgg caggaggtgg                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

tgggaagctc tgtggcagga                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

ccagtggcag gccaggcagc                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

agggtcggca aaggccctgt                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

tgcgagccgg ttgataggca                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

gctgtgcgct tctctccacg                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

tgcccaccac cagatccttg                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

cgcagactta ggttctcctg                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

tgcttttgtg gacagcagtg                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

ctgccacagg ccgacaccag                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

cagcctgctt gcgatgcctc                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

ccaggtccag gtcagcctgc                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

gcttatggta gaccagggct                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

gtgtgcttcc ccatggtgtg                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

cgccacatag atctcggcca                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

atgcagccgc cacatagatc                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

ctcgctctaa gagatgttcc                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

atcagctgac ccagggctgg                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

ggcatccgag aattccttgt                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

tacgccggtg gtggtggcca                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

gctggaccag actctgcctt                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

agctgctggc tggtgtggta                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

cgcagctcaa gggcggaagc                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

tctgctgaca gtcgtgcagc                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

aggcgcatga gcatctgctg                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

ggaagtgaca gtggtcccac                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

gggtctagct ggaagtgaca                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

cacgggacca aagtggctag                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

caggacagaa gctgcacggg                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

ggcacacagc agccgcaggt                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

cttccaccgc gaaggcacac                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

atggccgccg gtcttagggt                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            90 

cagcaccatc atggccgccg                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

ctaaggtgcc tgcagagcaa                                                 20 

 
           
             92  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            92 

acagggaaat gtacccctct                                                 20 

 
           
             93  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            93 

tggcttccgt cagcacaggg                                                 20 

 
           
             94  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            94 

tccgggaaag ccaagttggc                                                 20 

 
           
             95  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            95 

tcaggaggct aagcacgctg                                                 20 

 
           
             96  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            96 

agtttgcaaa aggcaaagta                                                 20 

 
           
             97  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            97 

ttaattctct gtacaaaact                                                 20 

 
           
             98  
             616  
             DNA  
             M. musculus  
             
 
           
            98 

ggatccagaa ctggatcatc agcccccccc tccttgaaac aagtgttctc atcctggggc     60 

gctctgctag ctagatgacc ctgcaccacc aactgccact atctaaaggc agctattggc    120 

cttcctcaga ctgtaggcaa atcttgctgc tgccattcga tgcgaagggc caggagtggg    180 

taaactgagg ctaaaatggt ccaggcaagt tctgggtgtg tgcgaacgaa ccagcggtgg    240 

gaacacagag cttccgggat caaagccaga cgccgtccgg attccggacc caggctcttt    300 

tcggggatgg ttgcctgtgc ggcaggggtt gggacgacag tgaccgccag taaccccagc    360 

gcgcgctggc gcagacgcgg ttaaaggcgg acgcccgcta gtaaccccgg ccccattcag    420 

agcaccggga gaaacccgag ctgccgccgt cgggggtggg cggggcccta atggggcgcg    480 

gcgcggctgc tgattggcca tgtgcgctca cccgaggggc ggggcacgga ggcgatcggc    540 

gggctttaaa gcctcgcggg gcctgacagg tgaaatcggc gcggaagctg tcggggtagc    600 

gtctgcacgc cctagg                                                    616 

 
           
             99  
             491  
             DNA  
             M. musculus  
             
               unsure  
               352  
               unknown  
             
           
            99 

aaaatcggcg cggaagctgt cggggtagcg tctgcacgcc ctaggggcgg ggcgcggacc     60 

acggagccat ggattgcaca tttgaagaca tgctccagct catcaacaac caagacagtg    120 

acttcccggg cctgtttgac gccccctatg ctgggggtga gacaggggac acaggcccca    180 

gcagcccagg tgccaactct cctgagagct tctcttctgc ttctctggcc tcctctctgg    240 

aagccttcct gggaggaccc aaggtgacac ctgcaccctt gtcccctcca ccatcggcac    300 

ccgctgcttt aaagatgtac ccgtccgtgt cccccttttc ccctgggcct gngatcaaag    360 

aggagccagt gccactcacc atcctacagc ctgcagcgcc acagccgtca ccggngaccc    420 

tcctgcctcc gagcttcccc gcaccacccg tacagctcag ccctgcgccc gtgctgggtt    480 

actcgagcct g                                                         491 

 
           
             100  
             8128  
             DNA  
             M. musculus  
             
               unsure  
               3861  
               unknown  
             
           
            100 

cagctcacaa attgactaca aaggcagttt ggccatcaaa caaggaatgt ccttgtgcag     60 

cccctcagac ctgagattat aagcatcagc tgtcataccc ggttccccca ccccacctcc    120 

ccctgctttt taaatttatt ttttgcttct ttatttttct atacctggct ttttgtgggg    180 

gttaaactcg ggtccctccc tttgcctgca cagcaagcac ccactaatgg agctgtcttc    240 

ccagcccctc tgcataagtg gggcttgctg tgtaagtggt tgaggcccag atgactgtgg    300 

gccttttcgg aggcctgcca cagcaccctg tgctgtctct ctgcatatac gaaggcgata    360 

aaggctgctt ggcccagggc tcacctcagg ccgtgactga ctatatagga gcagactgta    420 

taggcaccgt ggatcagcag aactgagcca gggtctcaag tgcttcccga ggccactgag    480 

ggctcttgat ccttctctgg accttggtgt cctcactggg aagaggtcct gagcacaagc    540 

gtgactgttt catcagcctg cgtgtagcct atccccttcc aggaagaacc acattctttt    600 

aatgccctgg agcagggcct ttgagtgcac aaaaggcagt ctatacccct gtgccctggc    660 

acccatacga cagccaagga ccagagtgcc tgccagggac ttctgaggag taagggcctg    720 

gggagcagca gggcaggctg catgcctgaa aaaacagtga gccatagccc agtcctctaa    780 

cctgcaagtc cccaagcagg gggcactgtc ctgtgtcctc ggtgggaggt ggtgccactt    840 

ctctatgcag cctgctcccc ttctctctcc tgcgctcctt caggggatgg gataggttgg    900 

aaatcctgta ggctcactgg gatcccagca taacctgtcc ttacccgagc cactgtttct    960 

gcctctgccc tcacacctag cttgtacggt ttccgtcttt ggctttgcct tttcttctgg   1020 

ccagagagtt ttccttccct tgtagcccta tttattcaga ctacactcaa gtgtcacgtc   1080 

cccaggcagc cttgataccc acctgtcttt gcttgcccag cctctcacct ctgccactcg   1140 

tctcacatcc ctcccccaac cccaccccga gcatgtgcgc agctggttcc ttggtggagt   1200 

ggaagtatcc accaggggct ggatctctcg tgttgtcccc agcaagtggc tttcacctag   1260 

gatggtcctt tgattctgtt ggggaggggc agccgaggct tcaggtttcc ggttgaagcc   1320 

agataggatc agggcttgag aagggagtat aggaggcttg tgcccgggtc cccttttgtc   1380 

cttttgcttc aaatcacata tgtgacctgg aagtctgtgc acggttgtga gaagtcagta   1440 

ttcagcatgc cctgatggct cgtagcttgg ttactgtggt gcccctttcc agactgcagg   1500 

acctactgag ccctagtcct tcctagggtg aggcaaggaa cactctcacg ttaggtgtgt   1560 

agcgtgttag gtgtgtagcg tgctggctga tgtctcccct cagttcttgg gtggccctac   1620 

tcattccctt taaaatgtta aaaacctacc aggtgcccag gactgactca gtcctgcagc   1680 

tcagggtcta gtttgcaggt ctagccaatt ccagcggctg ttgagaggaa acacctttgc   1740 

tgaaaccttt ttgagtgggt agattcttta ttaacttgtt ctggaatcgc caccccaggg   1800 

aggggtagag tctggacctg ggggctctta gaggcatccg gctcccgatg catagctggt   1860 

ggggaaaaga aaagaaaggc cgcagcacac agctgcagat ccttggcaag gcttattctc   1920 

aaggagcttg caaagctggc tttaaggtcc cgtttcctct caagacttcc ccctggccac   1980 

cagcatctac agacatgagc tagcgacccg gctcagaagg tggtgagggg ggaggccagg   2040 

cagcatggac acacattctg ctagttgtca ggcctgcccc cggtccagtg cttgactaag   2100 

gcttttgtac tcacaagcgt gcccacatgc ttgggtcaca cttgtccagt gtccagatac   2160 

ggacaggggt ggggagacgt gaccccacct gtacggagtt tcgatgagcc tccccgcctc   2220 

tgcaagtctt tctgtattcg ggactcagat gtcagaagga gcagagtagg gtcaacactg   2280 

ggaagcctca tgcctggact ccagcccccc cccccccccc cgtgttgggg tcagggctct   2340 

tccctgcctt cagttgggtg aggtcagagg ttttcccagg agctgtgcat ggtttgggga   2400 

ctctcgagca cttgcaggct ggacagaacg gtgtcataaa aagatgtttt ctttggaatg   2460 

aacctcctat gaggatgtga aaagacctag aaaggggatc aggggaatgt cagacacacg   2520 

tgtctgtttc ccagacaaga ctctgaaaag agagatgggc cacaagtccc tgacacacat   2580 

aaggtgacta cttggtcgct ggacccctca cagactgtgt gagtccctgg tctgccaact   2640 

aggctgccag accttgctgg gccactgcca cagaagctag gttgctggcc atcactgtgt   2700 

ggtgatggta atggcgggag tatgtgtgtg cacatgcttg tgtgtgcaca ggtatgaaag   2760 

ctttcaattt gccagcaagg gacagggaca gatttggcat acccttaata tccactgcct   2820 

ttcccttctg tcccagagac tggttcctgt gcaggccttt gcagagtgct ataagagaat   2880 

cgagtaaggc ttcacttgtt gactgctggg ggctgtgata cctggaggga agacactgac   2940 

ccagcctagg ggcatcagag ctgagagcag gatatcctgg acgcgtgatt tgaggaagga   3000 

tttccctagc tcactcctga aggcagtttc atgagggatc cagaactgga tcatcagccc   3060 

ccccctcctt gaaacaagtg ttctcatcct ggggcgctct gctagctaga tgaccctgca   3120 

ccaccaactg ccactatcta aaggcaacta ttggccttcc tcagactgta ggcaaatctt   3180 

gctgctgcca ttcgatgcga agggccagga gtgggtaaac tgaggctaaa atggtccagg   3240 

caagttctgg gtgtgtgcga acgaaccagc ggtgggaaca cagagcttcc gggatcaaag   3300 

ccagacgccg tccggattcc ggacccaggc tcttttcggg gatggttgcc tgtgcggcag   3360 

gggttgggac gacagtgacc gccagtaacc ccagcgcgcg ctggcgcaga cgcggttaaa   3420 

ggcggacgcc cgctagtaac cccggcccca ttcagagcac cgggagaaac ccgagctgcc   3480 

gccgtcgggg gtgggcgggg ccctaatggg gcgcggcgcg gctgctgatt ggccatgtgc   3540 

gctcacccga ggggcggggc acggaggcga tcggcgggct ttaaagcctc gcggggcctg   3600 

acaggtgaaa tcggcgcgga agctgtcggg gtagcgtctg cacgccctag gggcggggcg   3660 

cggaccacgg agccatggat tgcacatttg aaggtacttt ggggaggacc ctgcactcta   3720 

ttactttgcc agggtctctg cagcggactg cagtacggtg ttctaacaga gaatgcagga   3780 

cggcccttcc ccaccttggg ctggaaattg gtgggcctct ttatcctgct taaggaccga   3840 

caccttgcaa tttgcaactt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn   3900 

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn   3960 

gagcctgcct tcaggcttct caggtgagcg agtgatggaa gaagagtggc cgctgtgctc   4020 

ttacagagga attcccaggc ttcagaagtt aggtggtcat cctgcgacct gagatgccct   4080 

ttggttctgg gcccagtgca tccccccaac ccccagttgt gcagctggaa ggtgacatgt   4140 

gcagggtctg tcctgctatg aagtaatggg gatagttatg tgaggccagt cggggtaaag   4200 

gtcggcaagg cagcctgtgc cagcaacctt aaactctgtc tctgcaggga cccttccagg   4260 

aaacactcag cagccaccat ctagcctgcc gctggcccct gcaccaggag tcttgcccac   4320 

ccctgccctg cacacccagg tccaaagctt ggcctcccag cagccgctgc cagcctcagc   4380 

agcccctaga acaaacactg tgacctcaca ggtccagcag gtcccagtga gtgggtctga   4440 

ccaggaaggt ggggggtggg gacgcctggc ttggatgctg ctcgcttaca gcttggcccc   4500 

tcccatccag gttgtactgc agccacactt catcaaggca gactcactgc tgctgacagc   4560 

tgtgaagaca gatgcaggag ccaccgtgaa gactgcaggc atcagcaccc tggctcctgg   4620 

cacagccgtg caggcaggtc ccctgcaggt agatggctca ggcacaaggg agactatggg   4680 

ggggggggga gggttggctg cgcatgtgtc tgtccacctg gtgagatgca tctgacccca   4740 

cagaccctgg tgagtggagg gaccatcttg gccacagtac ctttggttgt ggacacagac   4800 

aaactgccca tccaccgact cgcagctggc agcaaggccc taggctcagc tcagagccgt   4860 

ggtgagaagc gcacagccca caatgccatt gagaagcgct accggtcttc tatcaatgac   4920 

aagattgtgg agctcaaaga cctggtggtg ggcactgaag caaaggtacg gccaaaggcc   4980 

tgcgagactc aggtcagggt gaccagggaa gaaatggggc acatcagcca gccggggatg   5040 

ggattaggtc agtcctcgtc acttagtcat atgcatcaac ttgtctgggt ctaggcagtc   5100 

ccgtttgcgg agttaggtct tatcaagggc agcctggata aagaaagctg gtctatgcat   5160 

tgagggggcg tggtgatgaa gcacagaaat cctgtcctgg aggaactgac tccctagggg   5220 

agtagtggga attgcagcgg ctggctccca tgttcgggga agaaaccagg accagtgaaa   5280 

gttgtggttg tgaactgggt ggtcaaggaa ggtctcaccg tagagagctg agggtgtagg   5340 

gaatgtgagg tggagacagc aggggccgca gctgggagac accgttgtga gtattcacag   5400 

ggtgactttt atctctgccc tgtggagtgg gtactgtcag gagacagcag cataggagag   5460 

ttgtagtcag aaggaaccgt cccgtccaga ggccccgagg cagctgtgac gcagagcggc   5520 

tcttacctgc tctcgtacct gtggtcaggt ccacttggct ggctgagccc tctccctctc   5580 

ctcacagctg aataaatctg ctgtcttgcg caaggccatc gactacatcc gcttcttgca   5640 

gcacagcaac cagaagctca agcaggagaa cctgacccta cgaagtgcac acaaaagcag   5700 

tgagtcccag cccctccccc ccgccccccc ccccctgctg tcctggccac tatgccgttg   5760 

ctgtgaagac actatgacca tggtcaggtt tattaaaggc ttacagtttc aggggtgaac   5820 

ccatgaccac agtggtggcg gcaggcagac aggcttggcg cttggagcag tagccgagag   5880 

ctcaaatatt gagacagcca caaggccaag agaaagagct agctgagaat agtgtggggt   5940 

tttgaaattt caaagcctac cacagtgaca cccctcctcc agcaaggcca cacctcccaa   6000 

tccttcccaa acaggaatgg gaaccaagcg gtcaaacggg accctctgaa agccattctc   6060 

attcagattg ccaccctgat gctgccttct ctatccctgc ccaaccttgt ctctggctct   6120 

caccctacct tggcccctgt tttgagcata acagaaccat ccaagtcctg gcgcttggcg   6180 

gccaggcctc tctcaccagc cctgttcttt ctgcctacag aatcactgaa ggacctggtg   6240 

tcagcttgtg gcagtggagg aggcacagat gtgtctatgg agggcatgaa acccgaagtg   6300 

gtggagacgc ttacccctcc accctcagac gccggctcac cctcccagag tagccccttg   6360 

tcttttggca gcagagctag cagcagtggt ggtagtgact ctgagcccga cagtccagcc   6420 

tttgaggata gccaggttgg actctgcaat atggcccctt ccctctccca gcagccctgc   6480 

agtctcctcc accttttagc ctcgcctttg gggctagctg agctctatgc ccttacctcc   6540 

cttgctccct gccaggtcaa agcccagcgg ctgccttcac acagccgagg catgctggac   6600 

cgctcccgcc tggccctgtg tgtactggcc tttctgtgtc tgacctgcaa tcctttggcc   6660 

tcgcttttcg gctggggcat tctcactccc tctgatgcta cgggtacaca ccgtagttct   6720 

gggcgcagca tgctggaggc agagagcaga ggtgagtcag gtcagcccag gtgttgtcgg   6780 

cagagacctt tgggactttg gatttccgga gaactgagtt ctcagacctt ttctttgcct   6840 

gtagatggct ctaattggac ccagtggttg ctgccacccc tagtctggct ggccaatgga   6900 

ctactagtgt tggcctgctt ggctcttctc tttgtctatg gggaacctgt gactaggcca   6960 

cactctggcc cggctgtaca cttctggaga catcgcaaac aagctgacct ggatttggcc   7020 

cgggtaaggg gctgaccctg aggaggcggg gtggggcccc gggcctggaa ggtgctgggt   7080 

gcctctgctc acttcatttt ctccagtctg tctcatcccc cgccttcaga gctcctgact   7140 

ctaggggccc agacaagggg gtaccctgct gccatccctg ctgccatttt tcttactgag   7200 

aatcttttct ctagggagat ttcccccagg ctgctcaaca gctgtggctg gccctgcaag   7260 

cgctgggccg gcccctgccc acctcaaacc tggatctggc ctgcagtctg ctttggaacc   7320 

tcatccgcca cctgctccag cgtctctggg tgggccgctg gctggcaggc caggccgggg   7380 

gcctgctgag ggaccgtggg ctgaggaagg atgcccgtgc cagtgcccgg gatgcggctg   7440 

ttgtctacca taagctgcac cagctgcatg ccatgggtat ggctggctgg gagctgggct   7500 

ccgagggtcc ccaccacacc gtcacctcct gtcctcatgc ctcacccact ttgcaggcaa   7560 

gtacacagga ggacatcttg ctgcttctaa cctggcacta agtgccctca acctggctga   7620 

gtgcgcagga gatgctatct ccatggcaac actggcagag atctatgtgg cagcggccct   7680 

gagggtcaaa accagcctcc caagagccct gcacttcttg acagtgagta ggctgatggg   7740 

gacagggctg ggggctcctc tttacaactc tcaacctgtc acttccaggg caaggggcta   7800 

aacaggatgt ggcagtggtt agcaggtggg ctgtaggccc tcctgggatc caactgggag   7860 

ccagtgtgac agttctgttc cttccctaca gcgtttcttc ctgagcagcg cccgccaggc   7920 

ctgcctagca cagagcggct cggtgcctct tgccatgcag tggctctgcc accctgtagg   7980 

tcaccgtttc tttgtggacg gggactgggc cgtgcacggt gcccccccgg agagcctgta   8040 

cagcgtggct gggaacccag gtgctttctc gttctgttct tacccctgcc tcatccctgt   8100 

ccctatgtca cattgcactg tcccctct                                      8128 

 
           
             101  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            101 

tggagcatgt cttcaaatgt                                                 20 

 
           
             102  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            102 

tgtgcaatcc atggctccgt                                                 20 

 
           
             103  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            103 

aagagaagct ctcaggagag                                                 20 

 
           
             104  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            104 

ccttgggtcc tcccaggaag                                                 20 

 
           
             105  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            105 

ggacaagggt gcaggtgtca                                                 20 

 
           
             106  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            106 

gatggtgagt ggcactggct                                                 20 

 
           
             107  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            107 

ggatgggcag tttgtctgtg                                                 20 

 
           
             108  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            108 

gctgtgcgct tctcaccacg                                                 20 

 
           
             109  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            109 

gcttctcaat ggcattgtgg                                                 20 

 
           
             110  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            110 

cactgccaca agctgacacc                                                 20 

 
           
             111  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            111 

ccatagacac atctgtgcct                                                 20 

 
           
             112  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            112 

gctcagagtc actgccacca                                                 20 

 
           
             113  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            113 

gggctttgac ctggctatcc                                                 20 

 
           
             114  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            114 

ttagagccat ctctgctctc                                                 20 

 
           
             115  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            115 

gcagcaacca ctgggtccaa                                                 20 

 
           
             116  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            116 

agtccattgg ccagccagac                                                 20 

 
           
             117  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            117 

ccaagcaggc caacactagt                                                 20 

 
           
             118  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            118 

tgcgatgtct ccagaagtgt                                                 20 

 
           
             119  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            119 

gccagatcca ggtttgaggt                                                 20 

 
           
             120  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            120 

tggcctgcca gccagcggcc                                                 20 

 
           
             121  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            121 

gtgtacttgc ccatggcatg                                                 20 

 
           
             122  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            122 

agatctctgc cagtgttgcc                                                 20 

 
           
             123  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            123 

gacctacagg gtggcagagc                                                 20 

 
           
             124  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            124 

ctgggttccc agccacgctg                                                 20 

 
           
             125  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            125 

ggcatctgag aactccctgt                                                 20 

 
           
             126  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            126 

ccacttggcc actgggtctg                                                 20 

 
           
             127  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            127 

agccttgaag gagtacagag                                                 20 

 
           
             128  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            128 

cacctttctg tggtccagca                                                 20 

 
           
             129  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            129 

atggccaggc tggctgggct                                                 20 

 
           
             130  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            130 

tcacacagga gcagctgcat                                                 20 

 
           
             131  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            131 

caagaagtag atcacacagg                                                 20 

 
           
             132  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            132 

cattgctggt accgtgagct                                                 20 

 
           
             133  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            133 

ctccagagca gaggcctggg                                                 20 

 
           
             134  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            134 

aaccacgcag ctccagagca                                                 20 

 
           
             135  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            135 

tcatgttgga aaccacgcag                                                 20 

 
           
             136  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            136 

gctgctcagg tcatgttgga                                                 20 

 
           
             137  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            137 

gctgtggcct catgtaggaa                                                 20 

 
           
             138  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            138 

catcagccga gctgtggcct                                                 20 

 
           
             139  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            139 

ccgggcagga cttgctcctg                                                 20 

 
           
             140  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            140 

tttgccactg gaacctgccc                                                 20 

 
           
             141  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            141 

gtgtgctccc gccatgtggg                                                 20 

 
           
             142  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            142 

caggagcatc tgctggcagt                                                 20 

 
           
             143  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            143 

gggtctagct ggaagtgacg                                                 20 

 
           
             144  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            144 

tctgccacta gaggtcggca                                                 20 

 
           
             145  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            145 

gcctacagag caagagggtg                                                 20 

 
           
             146  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            146 

aaaatttctc aacctatgaa                                                 20 

 
           
             147  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            147 

tgagaacact tgtttcaagg                                                 20 

 
           
             148  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            148 

gccaatagct gcctttagat                                                 20 

 
           
             149  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            149 

gtgttcccac cgctggttcg                                                 20 

 
           
             150  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            150 

ttactggcgg tcactgtcgt                                                 20 

 
           
             151  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            151 

ttggccgtac ctttgcttca                                                 20 

 
           
             152  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            152 

ccacactatt ctcagctagc                                                 20 

 
           
             153  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            153 

agaaggcagc atcagggtgg                                                 20 

 
           
             154  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            154 

ttcagtgatt ctgtaggcag                                                 20 

 
           
             155  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            155 

agagtccaac ctggctatcc                                                 20 

 
           
             156  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            156 

cttacccggg ccaaatccag                                                 20 

 
           
             157  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            157 

gggatgagac agactggaga                                                 20 

 
           
             158  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            158 

gtgtacttgc ctgcaaagtg                                                 20 

 
           
             159  
             20  
             DNA  
             H. sapiens  
             
 
           
            159 

ctggcaccac tgtgcagaca                                                 20 

 
           
             160  
             20  
             DNA  
             H. sapiens  
             
 
           
            160 

gcagtggcag tgactcggag                                                 20 

 
           
             161  
             20  
             DNA  
             H. sapiens  
             
 
           
            161 

tcaacaacca agacagtgac                                                 20 

 
           
             162  
             20  
             DNA  
             H. sapiens  
             
 
           
            162 

aaccaagaca gtgacttccc                                                 20 

 
           
             163  
             20  
             DNA  
             H. sapiens  
             
 
           
            163 

agacagtgac ttccctggcc                                                 20 

 
           
             164  
             20  
             DNA  
             H. sapiens  
             
 
           
            164 

cacttcatca aggcagactc                                                 20 

 
           
             165  
             20  
             DNA  
             H. sapiens  
             
 
           
            165 

ctcgcagctg gcagcaaggc                                                 20 

 
           
             166  
             20  
             DNA  
             H. sapiens  
             
 
           
            166 

gcaaagctga ataaatctgc                                                 20 

 
           
             167  
             20  
             DNA  
             H. sapiens  
             
 
           
            167 

gctgaataaa tctgctgtct                                                 20 

 
           
             168  
             20  
             DNA  
             H. sapiens  
             
 
           
            168 

ataaatctgc tgtcttgcgc                                                 20 

 
           
             169  
             20  
             DNA  
             H. sapiens  
             
 
           
            169 

tctgctgtct tgcgcaaggc                                                 20 

 
           
             170  
             20  
             DNA  
             H. sapiens  
             
 
           
            170 

tgtcttgcgc aaggccatcg                                                 20 

 
           
             171  
             20  
             DNA  
             H. sapiens  
             
 
           
            171 

tgcgcaaggc catcgactac                                                 20 

 
           
             172  
             20  
             DNA  
             H. sapiens  
             
 
           
            172 

atgctggacc gctcccgcct                                                 20 

 
           
             173  
             20  
             DNA  
             H. sapiens  
             
 
           
            173 

tgccatgcag tggctctgcc                                                 20 

 
           
             174  
             20  
             DNA  
             H. sapiens  
             
 
           
            174 

gtggccaagt ggtgggcctc                                                 20 

 
           
             175  
             20  
             DNA  
             H. sapiens  
             
 
           
            175 

agctgtggtg atccactggc                                                 20 

 
           
             176  
             20  
             DNA  
             H. sapiens  
             
 
           
            176 

actccttcaa ggctgcccgg                                                 20 

 
           
             177  
             20  
             DNA  
             H. sapiens  
             
 
           
            177 

ccatctgtga gaaggccagt                                                 20 

 
           
             178  
             20  
             DNA  
             H. sapiens  
             
 
           
            178 

gaaggccagt gggtacctgc                                                 20 

 
           
             179  
             20  
             DNA  
             H. sapiens  
             
 
           
            179 

tgcaaacttt attttcatag                                                 20 

 
           
             180  
             20  
             DNA  
             H. sapiens  
             
 
           
            180 

ccttgacagg tgaagtcggc                                                 20 

 
           
             181  
             20  
             DNA  
             H. sapiens  
             
 
           
            181 

atttctgcag gaagccctcc                                                 20 

 
           
             182  
             20  
             DNA  
             H. sapiens  
             
 
           
            182 

caccgtgcag ctgaataaat                                                 20 

 
           
             183  
             20  
             DNA  
             H. sapiens  
             
 
           
            183 

ctcctggcca aggctggtgc                                                 20 

 
           
             184  
             20  
             DNA  
             H. sapiens  
             
 
           
            184 

catgcggagg gtgagtgccc                                                 20 

 
           
             185  
             20  
             DNA  
             H. sapiens  
             
 
           
            185 

gtagggccaa cggcctggac                                                 20 

 
           
             186  
             20  
             DNA  
             H. sapiens  
             
 
           
            186 

gcggagccat ggattgcact                                                 20 

 
           
             187  
             20  
             DNA  
             H. sapiens  
             
 
           
            187 

aagacatgct tcagcttatc                                                 20 

 
           
             188  
             20  
             DNA  
             H. sapiens  
             
 
           
            188 

ctctgaggcc agggcaggac                                                 20 

 
           
             189  
             20  
             DNA  
             H. sapiens  
             
 
           
            189 

gctgcgccat ggacgagcca                                                 20 

 
           
             190  
             20  
             DNA  
             H. sapiens  
             
 
           
            190 

agccaccctt cagcgaggcg                                                 20 

 
           
             191  
             20  
             DNA  
             H. sapiens  
             
 
           
            191 

acatcgaaga catgcttcag                                                 20 

 
           
             192  
             20  
             DNA  
             H. sapiens  
             
 
           
            192 

ccacctcctg ccacattgag                                                 20 

 
           
             193  
             20  
             DNA  
             H. sapiens  
             
 
           
            193 

tcctgccaca gagcttccca                                                 20 

 
           
             194  
             20  
             DNA  
             H. sapiens  
             
 
           
            194 

gctgcctggc ctgccactgg                                                 20 

 
           
             195  
             20  
             DNA  
             H. sapiens  
             
 
           
            195 

acagggcctt tgccgaccct                                                 20 

 
           
             196  
             20  
             DNA  
             H. sapiens  
             
 
           
            196 

tgcctatcaa ccggctcgca                                                 20 

 
           
             197  
             20  
             DNA  
             H. sapiens  
             
 
           
            197 

cgtggagaga agcgcacagc                                                 20 

 
           
             198  
             20  
             DNA  
             H. sapiens  
             
 
           
            198 

caaggatctg gtggtgggca                                                 20 

 
           
             199  
             20  
             DNA  
             H. sapiens  
             
 
           
            199 

caggagaacc taagtctgcg                                                 20 

 
           
             200  
             20  
             DNA  
             H. sapiens  
             
 
           
            200 

cactgctgtc cacaaaagca                                                 20 

 
           
             201  
             20  
             DNA  
             H. sapiens  
             
 
           
            201 

ctggtgtcgg cctgtggcag                                                 20 

 
           
             202  
             20  
             DNA  
             H. sapiens  
             
 
           
            202 

gaggcatcgc aagcaggctg                                                 20 

 
           
             203  
             20  
             DNA  
             H. sapiens  
             
 
           
            203 

gcaggctgac ctggacctgg                                                 20 

 
           
             204  
             20  
             DNA  
             H. sapiens  
             
 
           
            204 

agccctggtc taccataagc                                                 20 

 
           
             205  
             20  
             DNA  
             H. sapiens  
             
 
           
            205 

tggccgagat ctatgtggcg                                                 20 

 
           
             206  
             20  
             DNA  
             H. sapiens  
             
 
           
            206 

gatctatgtg gcggctgcat                                                 20 

 
           
             207  
             20  
             DNA  
             H. sapiens  
             
 
           
            207 

ggaacatctc ttagagcgag                                                 20 

 
           
             208  
             20  
             DNA  
             H. sapiens  
             
 
           
            208 

ccagccctgg gtcagctgat                                                 20 

 
           
             209  
             20  
             DNA  
             H. sapiens  
             
 
           
            209 

aaggcagagt ctggtccagc                                                 20 

 
           
             210  
             20  
             DNA  
             H. sapiens  
             
 
           
            210 

taccacacca gccagcagct                                                 20 

 
           
             211  
             20  
             DNA  
             H. sapiens  
             
 
           
            211 

gcttccgccc ttgagctgcg                                                 20 

 
           
             212  
             20  
             DNA  
             H. sapiens  
             
 
           
            212 

cagcagatgc tcatgcgcct                                                 20 

 
           
             213  
             20  
             DNA  
             H. sapiens  
             
 
           
            213 

gtgggaccac tgtcacttcc                                                 20 

 
           
             214  
             20  
             DNA  
             H. sapiens  
             
 
           
            214 

tgtcacttcc agctagaccc                                                 20 

 
           
             215  
             20  
             DNA  
             H. sapiens  
             
 
           
            215 

ctagccactt tggtcccgtg                                                 20 

 
           
             216  
             20  
             DNA  
             H. sapiens  
             
 
           
            216 

cccgtgcagc ttctgtcctg                                                 20 

 
           
             217  
             20  
             DNA  
             H. sapiens  
             
 
           
            217 

acctgcggct gctgtgtgcc                                                 20 

 
           
             218  
             20  
             DNA  
             H. sapiens  
             
 
           
            218 

gtgtgccttc gcggtggaag                                                 20 

 
           
             219  
             20  
             DNA  
             H. sapiens  
             
 
           
            219 

cggcggccat gatggtgctg                                                 20 

 
           
             220  
             20  
             DNA  
             H. sapiens  
             
 
           
            220 

ttgctctgca ggcaccttag                                                 20 

 
           
             221  
             20  
             DNA  
             H. sapiens  
             
 
           
            221 

agaggggtac atttccctgt                                                 20 

 
           
             222  
             20  
             DNA  
             H. sapiens  
             
 
           
            222 

ccctgtgctg acggaagcca                                                 20 

 
           
             223  
             20  
             DNA  
             H. sapiens  
             
 
           
            223 

gccaacttgg ctttcccgga                                                 20 

 
           
             224  
             20  
             DNA  
             H. sapiens  
             
 
           
            224 

cagcgtgctt agcctcctga                                                 20 

 
           
             225  
             20  
             DNA  
             H. sapiens  
             
 
           
            225 

tactttgcct tttgcaaact                                                 20 

 
           
             226  
             20  
             DNA  
             H. sapiens  
             
 
           
            226 

agttttgtac agagaattaa                                                 20 

 
           
             227  
             20  
             DNA  
             M. musculus  
             
 
           
            227 

acggagccat ggattgcaca                                                 20 

 
           
             228  
             20  
             DNA  
             M. musculus  
             
 
           
            228 

cttcctggga ggacccaagg                                                 20 

 
           
             229  
             20  
             DNA  
             M. musculus  
             
 
           
            229 

tgacacctgc acccttgtcc                                                 20 

 
           
             230  
             20  
             DNA  
             M. musculus  
             
 
           
            230 

agccagtgcc actcaccatc                                                 20 

 
           
             231  
             20  
             DNA  
             M. musculus  
             
 
           
            231 

cacagacaaa ctgcccatcc                                                 20 

 
           
             232  
             20  
             DNA  
             M. musculus  
             
 
           
            232 

cgtggtgaga agcgcacagc                                                 20 

 
           
             233  
             20  
             DNA  
             M. musculus  
             
 
           
            233 

ccacaatgcc attgagaagc                                                 20 

 
           
             234  
             20  
             DNA  
             M. musculus  
             
 
           
            234 

ggtgtcagct tgtggcagtg                                                 20 

 
           
             235  
             20  
             DNA  
             M. musculus  
             
 
           
            235 

aggcacagat gtgtctatgg                                                 20 

 
           
             236  
             20  
             DNA  
             M. musculus  
             
 
           
            236 

tggtggcagt gactctgagc                                                 20 

 
           
             237  
             20  
             DNA  
             M. musculus  
             
 
           
            237 

ggatagccag gtcaaagccc                                                 20 

 
           
             238  
             20  
             DNA  
             M. musculus  
             
 
           
            238 

ttggacccag tggttgctgc                                                 20 

 
           
             239  
             20  
             DNA  
             M. musculus  
             
 
           
            239 

gtctggctgg ccaatggact                                                 20 

 
           
             240  
             20  
             DNA  
             M. musculus  
             
 
           
            240 

actagtgttg gcctgcttgg                                                 20 

 
           
             241  
             20  
             DNA  
             M. musculus  
             
 
           
            241 

acacttctgg agacatcgca                                                 20 

 
           
             242  
             20  
             DNA  
             M. musculus  
             
 
           
            242 

acctcaaacc tggatctggc                                                 20 

 
           
             243  
             20  
             DNA  
             M. musculus  
             
 
           
            243 

ggccgctggc tggcaggcca                                                 20 

 
           
             244  
             20  
             DNA  
             M. musculus  
             
 
           
            244 

catgccatgg gcaagtacac                                                 20 

 
           
             245  
             20  
             DNA  
             M. musculus  
             
 
           
            245 

ggcaacactg gcagagatct                                                 20 

 
           
             246  
             20  
             DNA  
             M. musculus  
             
 
           
            246 

gctctgccac cctgtaggtc                                                 20 

 
           
             247  
             20  
             DNA  
             M. musculus  
             
 
           
            247 

cagcgtggct gggaacccag                                                 20 

 
           
             248  
             20  
             DNA  
             M. musculus  
             
 
           
            248 

acagggagtt ctcagatgcc                                                 20 

 
           
             249  
             20  
             DNA  
             M. musculus  
             
 
           
            249 

cagacccagt ggccaagtgg                                                 20 

 
           
             250  
             20  
             DNA  
             M. musculus  
             
 
           
            250 

ctctgtactc cttcaaggct                                                 20 

 
           
             251  
             20  
             DNA  
             M. musculus  
             
 
           
            251 

tgctggacca cagaaaggtg                                                 20 

 
           
             252  
             20  
             DNA  
             M. musculus  
             
 
           
            252 

atgcagctgc tcctgtgtga                                                 20 

 
           
             253  
             20  
             DNA  
             M. musculus  
               
           
            253 

cctgtgtgat ctacttcttg                                                 20 

 
           
             254  
             20  
             DNA  
             M. musculus  
             
 
           
            254 

agctcacggt accagcaatg                                                 20 

 
           
             255  
             20  
             DNA  
             M. musculus  
             
 
           
            255 

tgctctggag ctgcgtggtt                                                 20 

 
           
             256  
             20  
             DNA  
             M. musculus  
             
 
           
            256 

ctgcgtggtt tccaacatga                                                 20 

 
           
             257  
             20  
             DNA  
             M. musculus  
             
 
           
            257 

ttcctacatg aggccacagc                                                 20 

 
           
             258  
             20  
             DNA  
             M. musculus  
             
 
           
            258 

aggccacagc tcggctgatg                                                 20 

 
           
             259  
             20  
             DNA  
             M. musculus  
             
 
           
            259 

caggagcaag tcctgcccgg                                                 20 

 
           
             260  
             20  
             DNA  
             M. musculus  
             
 
           
            260 

gggcaggttc cagtggcaaa                                                 20 

 
           
             261  
             20  
             DNA  
             M. musculus  
             
 
           
            261 

cccacatggc gggagcacac                                                 20 

 
           
             262  
             20  
             DNA  
             M. musculus  
             
 
           
            262 

tgccgacctc tagtggcaga                                                 20 

 
           
             263  
             20  
             DNA  
             M. musculus  
             
 
           
            263 

caccctcttg ctctgtaggc                                                 20 

 
           
             264  
             20  
             DNA  
             M. musculus  
             
 
           
            264 

ttcataggtt gagaaatttt                                                 20 

 
           
             265  
             20  
             DNA  
             M. musculus  
             
 
           
            265 

ccttgaaaca agtgttctca                                                 20 

 
           
             266  
             20  
             DNA  
             M. musculus  
             
 
           
            266 

atctaaaggc agctattggc                                                  20 

 
           
             267  
             20  
             DNA  
             M. musculus  
             
 
           
            267 

acgacagtga ccgccagtaa                                                  20 

 
           
             268  
             20  
             DNA  
             M. musculus  
             
 
           
            268 

tgaagcaaag gtacggccaa                                                  20 

 
           
             269  
             20  
             DNA  
             M. musculus  
             
 
           
            269 

gctagctgag aatagtgtgg                                                  20 

 
           
             270  
             20  
             DNA  
             M. musculus  
             
 
           
            270 

ccaccctgat gctgccttct                                                  20 

 
           
             271  
             20  
             DNA  
             M. musculus  
             
 
           
            271 

ggatagccag gttggactct                                                  20 

 
           
             272  
             20  
             DNA  
             M. musculus  
             
 
           
            272 

ctggatttgg cccgggtaag                                                  20 

 
           
             273  
             20  
             DNA  
             M. musculus  
             
 
           
            273 

cactttgcag gcaagtacac                                                  20