Patent Publication Number: US-2003228690-A1

Title: Antisense modulation of IL-1 receptor-associated kinase-1 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of IL-1 receptor-associated kinase-1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding IL-1 receptor-associated kinase-1. Such compounds have been shown to modulate the expression of IL-1 receptor-associated kinase-1.  
       BACKGROUND OF THE INVENTION  
       [0002] The proinflammatory cytokine interleukin-1 (IL-1) is a central regulator in immune and inflammatory responses, involved in generating systemic and local response to infection, injury, and immunologic challenges. IL-1 is produced mainly by activated macrophages and monocytes, and participates in lymphocyte activation, induction of fever, leukocyte trafficking, the acute phase response, and cartilage remodeling. The expression of more than 90 genes is affected by IL-1, including genes that encode other cytokines, cytokine receptors, acute-phase reactants, growth factors, tissue-remodeling enzymes, extracellular matrix components, and cell adhesion molecules. IL-1 is a critical cytokine in the pathogenesis of viral infections and inflammatory diseases such as rheumatoid arthritis (O&#39;Neill and Greene,  J. Leukoc. Biol.,  1998, 63, 650-657).  
       [0003] Cellular responses are transduced through the type IIL-1 receptor (IL-1RI), located on the plasma membrane of a variety of IL-1-responsive cells. Binding of IL-1 to IL-1RI ultimately triggers activation of transcription factors in the NF-κB family, which are bound by inhibitory proteins (IκBS) and remain anchored in the cytoplasm until the inhibitory proteins are degraded. In response to IL-1, tumor necrosis factor (TNF), or other extracellular stimuli such as lipopolysaccharides, double-stranded RNA, or oxidative stress, and once unbound and activated, NF-κB is then transported to the nucleus, where it influences the activity of many genes (O&#39;Neill and Greene,  J. Leukoc. Biol.,  1998, 63, 650-657).  
       [0004] A family of proteins has been described that share significant homology with the type I IL-1 receptor in their signaling domains. This family includes IL-1 receptor accessory protein (IL1-RAcP), which does not bind IL-1, but is essential for IL-1 signaling; a Drosophila receptor protein, Toll; a number of human Toll-like receptors (hTLRs); the interferon-γ-inducing factor/IL-1γ/IL-18 receptor-related protein (IL-1Rrp), a number of plant proteins, and the IL-1 receptor-associated kinases (IRAK-1, IRAK-2, and IRAK-M). All members of this family appear to be involved in host responses to injury and infection (O&#39;Neill and Greene,  J. Leukoc. Biol.,  1998, 63, 650-657; Wesche et al.,  J. Biol. Chem.,  1999, 274, 19403-19410).  
       [0005] IL-1 receptor-associated kinase-1 (also known as interleukin 1 receptor-associated kinase 1, IRAKI, IRAK-1, Illrak, mouse Pelle-like kinase, and mPLK) was purified from human embryonic kidney epithelial cell line 293, and its amino acid sequence was determined by micropeptide sequencing. PCR was then used to amplify a probe, and the gene was cloned from a human teratocarcinoma cDNA library. The IL-1 receptor-associated kinase-1 gene encodes a 3.5-kb mRNA that was detected in all tissues examined (Cao et al.,  Science,  1996, 271, 1128-1131). The IL-1 receptor-associated kinase-1 gene has been mapped to the human X chromosome region q28, and a homologous region XA7-C on mouse chromosome X (Reichwald et al.,  Mamm. Genome,  2000, 11, 182-190).  
       [0006] The IL-1 signaling pathway in mammals is analogous to the Toll pathway in  Drosophila melanogaster . Homologues of IL-1 receptor-associated kinase-1 are found in  D. melanogaster  (Pelle) and in plants (Pto), and in these systems, the kinases have been shown to be components of a signal transduction system leading to the activation of NF-κB. In fact, because of their homology to several components of the NF-κB signaling pathway in  D. melanogaster , new mammalian proteins of the IL-1RI signaling system and their functions are being elucidated (Burns et al.,  Nat. Cell Biol.,  2000, 2, 346-351; O&#39;Neill and Greene,  J. Leukoc. Biol.,  1998, 63, 650-657; Vig et al.,  J. Biol. Chem.,  2001, 276, 7859-7866). A novel signaling molecule that associates with the mouse Pelle-like kinase, SIMPL, was recently identified in mice, and found to bind to IL-1 receptor associated kinase (Vig et al.,  J. Biol. Chem.,  2001, 276, 7859-7866). Additionally, a novel protein, Tollip, was found to associate with the activated IL-1RI/IL-1RAcP complex (Burns et al.,  Nat. Cell Biol.,  2000, 2, 346-351).  
       [0007] When cells receive the extracellular IL-1 signal, a complex between IL-1RI and IL-1RAcP is formed (Huang et al.,  Proc. Natl. Acad. Sci. U.S.A.,  1997, 94, 12829-12832), the cytosolic adapter protein MyD88 interacts with IL-1RAcP in the receptor complex (Burns et al.,  J. Biol. Chem.,  1998, 273, 12203-12209), and MyD88 rapidly recruits IL-1 receptor-associated kinase-1 into the complex. Tollip also interacts with IL-1RAcP and is believed to block autophosphorylation of the IL-1 receptor-associated kinase-1 or its association with another kinase; thus, the association of Tollip with IL-1 receptor-associated kinase-1 is inhibitory (Burns et al.,  Nat. Cell Biol.,  2000, 2, 346-351). At some point after its IL-1-dependent association with the receptor complex, IL-1 receptor-associated kinase-1 is extensively phosphorylated and its own serine/threonine kinase catalytic activity becomes activated (Cao et al.,  Science,  1996, 271, 1128-1131). IL-1 receptor-associated kinase-1 then interacts with an adapter protein, TRAF6, a protein critical for IL-1-dependent activation of NF-κB, which dissociates from the receptor complex. TRAF6 relays a signal via NF-κB-inducing kinase (NIK) to two I-κB kinases (IKK-1 and -2), culminating in activation of NF-κB (Bacher et al.,  FEBS Lett.,  2001, 497, 153-158; Jefferies et al.,  Mol. Cell. Biol.,  2001, 21, 4544-4552; O&#39;Neill and Greene,  J. Leukoc. Biol.,  1998, 63, 650-657).  
       [0008] Cellular trafficking and nuclear importation may play a role in the timing and/or activity of IL-1 receptor-associated kinase-1 mediated signaling. Association of IL-1 receptor-associated kinase-1 with the receptor complex is detectable 30 seconds after IL-1 stimulation of human umbilical cord vein ECV 304 cells, and significant levels of IL-1 receptor-associated kinase-1 accumulate in the nucleus within 30 minutes (Bol et al.,  FEBS Lett.,  2000, 477, 73-78).  
       [0009] There is evidence that hyperphosphorylation of IL-1 receptor-associated kinase-1 is regulatory and results in proteolytic degradation. A nonspecific kinase inhibitor blocks proteolysis of IL-1 receptor-associated kinase-1 as well as its phosphorylation, and the translocation of IL-1 receptor-associated kinase-1 to the IL-1RI/IL-1RAcP complex is independent of this treatment. Degradation of the IL-1 receptor-associated kinase-1 component of this signaling pathway may explain why some IL-1 responses are transient (Yamin and Miller,  J. Biol. Chem.,  1997, 272, 21540-21547).  
       [0010] IL-1 receptor-associated kinase-1 plays a role in regulation of multiple signaling pathways. In addition to transducing the IL-1 signal, IL-1 receptor-associated kinase-1 also transduces a signal initiated by binding of tumor necrosis factor (TNF)-α to its receptor, again leading to activation of NF-κB (Vig et al.,  J. Biol. Chem.,  1999, 274, 13077-13084; Vig et al.,  J. Biol. Chem.,  2001, 276, 7859-7866). In another signal transduction pathway separate from its activation of NF-κB, IL-1 receptor-associated kinase-1 has also been implicated in activation of the Jun amino-terminal kinase (JNK) and the transcription factor AP-1 (Bacher et al.,  FEBS Lett.,  2001, 497, 153-158).  
       [0011] The IL-1 receptor-associated kinase-1 gene has been disrupted in mice. Upon IL-1 treatment, IL-1 receptor-associated kinase-1-deficient embryonic fibroblasts derived from these mice are defective in activation of JNK, p38 MAP kinase, and NF-κB (Kanakaraj et al.,  J. Exp. Med.,  1998, 187, 2073-2079). Furthermore, in T helper cell type 1 (Th1) cells from the IL-1 receptor-associated kinase-1 null mice, IL-18-induced production of interferon (IFN)-γ was substantially reduced, proliferation of the Th1 cells was decreased, activation of natural killer (NK) cells was defective, and these defects resulted in an impaired immune response to murine cytomegalovirus (MCMV) infection (Kanakaraj et al.,  J. Exp. Med.,  1999, 189, 1129-1138).  
       [0012] In a separate study, IL-1 receptor-associated kinase-1-deficient mice and fibroblasts were generated, and it was further demonstrated that IL-1 receptor-associated kinase-1 null mice retain a normal response to  Listeria monocytogenes  infection (Thomas et al.,  J. Immunol.,  1999, 163, 978-984).  
       [0013] The pharmacological modulation of IL-1 receptor-associated kinase-1 activity and/or expression is therefore believed to be an appropriate point of therapeutic intervention in pathological conditions such as viral infections, rheumatoid arthritis, and other inflammatory disease and immune disorders.  
       [0014] To date, investigative strategies aimed at studying the localization and function of IL-1 receptor-associated kinase-1 have involved the use of antibodies, transgenic animals and IL-1 receptor-associated kinase-1-deficient cell lines, and an antisense oligonucleotide.  
       [0015] Disclosed and claimed in U.S. Pat. No. 5,654,397 are nucleic acids which encode IL-1 receptor-associated kinase-1 and methods for screening chemical libraries and identifying lead compounds to be used as pharmacological agents in the diagnosis and treatment of disease associated with interleukin-1 signal transduction (Cao et al., 1997).  
       [0016] IL-1 receptor-associated kinase-1 specific antibodies have been raised and used to study IL-1 receptor-associated kinase-1 (Cao et al.,  Science,  1996, 271, 1128-1131) and its interactions with the IL-1RI/IL-1RAcP complex (Volpe et al.,  FEBS Lett.,  1997, 419, 41-44) and to demonstrate that the phosphorylated form of IL-1 receptor-associated kinase-1 is degraded (Yamin and Miller,  J. Biol. Chem.,  1997, 272, 21540-21547).  
       [0017] A phosphorothioate antisense oligonucleotide, 18 nucleotides in length, targeted to a region from nucleotide −6 to nucleotide 12 of the mRNA encoding the human IL-1 receptor-associated kinase-1 and spanning the translation initiation site, was used to show that inhibition of expression of IL-1 receptor-associated kinase-1 blocks activation of NF-κB (Guo and Wu,  Immunopharmacology,  2000, 49, 241-246).  
       [0018] Disclosed and claimed in U.S. Pat. No. 6,166,289 is a transgenic mouse whose somatic and germ cells comprise a disruption in an endogenous IL-1 receptor-associated kinase-1 gene, wherein the disruption is generated by targeted replacement with a non-functional IL-1 receptor-associated kinase-1 gene, and wherein said disruption results in IL-1 receptor-associated kinase-1-deficient cells from said mouse having a decrease in activation of JNK, activation of p38, and induction of IL-6 in response to IL-1 as compared to wild-type mice, as well as a method for producing this transgenic mouse (Harris et al., 2000).  
       [0019] Disclosed and claimed in U.S. Pat. No. 6,127,176 is a mutant cell that lacks a functional IL-1 receptor-associated kinase-1 and comprise an HSV thymidine kinase gene operatively linked to an IL-1 promoter and zeomycin resistance gene operatively linked to an IL-1 promoter, as well as a method of making a mutant mammalian cell that lacks a functional component of the IL-1 signaling pathway and the TNF signaling pathways (Stark and Li, 2000).  
       [0020] Disclosed and claimed in U.S. Pat. No. 5,817,479 are polynucleotides which identify and encode novel protein kinases expressed in various human cells and tissues, wherein one of the kinases is IL-1 receptor-associated kinase-1. Further claimed are expression vectors, host cells, and methods for the production of purified kinase peptides, antibodies capable of binding the kinases, and inhibitors of the kinases as well as antisense sequences and oligonucleotides designed from the polynucleotides or their complements (Au-Young et al., 1998).  
       [0021] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of IL-1 receptor-associated kinase-1. Consequently, there remains a long felt need for agents capable of effectively inhibiting IL-1 receptor-associated kinase-1 function.  
       [0022] 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 IL-1 receptor-associated kinase-1 expression.  
       [0023] The present invention provides compositions and methods for modulating IL-1 receptor-associated kinase-1 expression.  
       SUMMARY OF THE INVENTION  
       [0024] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding IL-1 receptor-associated kinase-1, and which modulate the expression of IL-1 receptor-associated kinase-1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-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  
       [0025] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding IL-1 receptor-associated kinase-1, ultimately modulating the amount of IL-1 receptor-associated kinase-1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding IL-1 receptor-associated kinase-1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding IL-1 receptor-associated kinase-1” encompass DNA encoding IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-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.  
       [0026] 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 IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-1, regardless of the sequence(s) of such codons.  
       [0027] 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.  
       [0028] 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.  
       [0029] 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.  
       [0030] 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.  
       [0031] 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.  
       [0032] 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.  
       [0033] 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.  
       [0034] 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.  
       [0035] 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).  
       [0036] 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.  
       [0037] 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.  
       [0038] 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.  
       [0039] 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.  
       [0040] 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.  
       [0041] 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.  
       [0042] 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.  
       [0043] 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).  
       [0044] 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.  
       [0045] 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.  
       [0046] 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.  
       [0047] 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.  
       [0048] 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.  
       [0049] 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.  
       [0050] 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.  
       [0051] 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.  
       [0052] 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.  
       [0053] 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.  
       [0054] 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.  
       [0055] 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.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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 1 -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.  
       [0059] 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 31 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.  
       [0060] 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.  
       [0061] 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]l[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.  
       [0062] 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.  
       [0063] 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.  
       [0064] 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.  
       [0065] 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.  
       [0066] 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.  
       [0067] 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.  
       [0068] 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.  
       [0069] 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.  
       [0070] 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.  
       [0071] 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.  
       [0072] 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.  
       [0073] 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.  
       [0074] 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 IL-1 receptor-associated kinase-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.  
       [0075] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-1 in a sample may also be prepared.  
       [0076] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.  
       [0077] 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.  
       [0078] 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, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0079] 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.  
       [0080] 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.  
       [0081] 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.  
       [0082] 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.  
       [0083] 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.  
       [0084] Emulsions  
       [0085] 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.  
       [0086] 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).  
       [0087] 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).  
       [0088] 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.  
       [0089] 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).  
       [0090] 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.  
       [0091] 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.  
       [0092] 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.  
       [0093] 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).  
       [0094] 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.  
       [0095] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.  
       [0096] 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.  
       [0097] 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.  
       [0098] Liposomes  
       [0099] 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.  
       [0100] 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.  
       [0101] 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.  
       [0102] 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.  
       [0103] 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.  
       [0104] 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.  
       [0105] 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.  
       [0106] 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).  
       [0107] 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).  
       [0108] 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.  
       [0109] 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).  
       [0110] 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).  
       [0111] 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).  
       [0112] 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.).  
       [0113] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. ( Bull. Chem. Soc. Jpn.,  1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Illum et al. ( FEBS Lett.,  1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. ( FEBS Lett.,  1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et  Biophysica Acta,  1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0114] 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.  
       [0115] 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.  
       [0116] 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).  
       [0117] 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.  
       [0118] 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.  
       [0119] 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.  
       [0120] 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.  
       [0121] 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).  
       [0122] Penetration Enhancers  
       [0123] 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.  
       [0124] 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.  
       [0125] 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).  
       [0126] 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).  
       [0127] 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).  
       [0128] 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 IN-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).  
       [0129] 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 l-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).  
       [0130] 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.  
       [0131] 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.  
       [0132] Carriers  
       [0133] 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).  
       [0134] Excipients  
       [0135] 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.).  
       [0136] 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.  
       [0137] 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.  
       [0138] 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.  
       [0139] Other Components  
       [0140] 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.  
       [0141] 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.  
       [0142] 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.  
       [0143] 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.  
       [0144] 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.  
       [0145] 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  
     [0146] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0147] 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.  
     [0148] 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).  
     [0149] 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:  
     [0150] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0151] 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 (Rf 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.  
     [0152] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0153] 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).  
     [0154] 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.  
     [0155] 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.  
     [0156] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0157] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH 2 Cl 2 -EtOAc; CH 2 Cl 2 -EtOAc 4:1; R f  0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L) The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.  
     [0158] 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.  
     [0159] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0160] 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%).  
     [0161] 2′-Fluoro Amidites  
     [0162] 2′-Fluorodeoxyadenosine Amidites  
     [0163] 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.  
     [0164] 2′-Fluorodeoxyguanosine  
     [0165] 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 isobutyrylarabinofuranosylguanosine. Alternatively, isobutyrylarabinofuranosylguanosine 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.  
     [0166] 2′-Fluorouridine  
     [0167] 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.  
     [0168] 2′-Fluorodeoxycytidine  
     [0169] 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 1 -DMT and 5′-DMT-3′phosphoramidites.  
     [0170] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0171] 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).  
     [0172] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate  
     [0173] 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.  
     [0174] 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.).  
     [0175] 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.  
     [0176] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0177] 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).  
     [0178] 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.  
     [0179] 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.  
     [0180] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0181] 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%).  
     [0182] Preparation of 5′-O-Dimethoxytrityl-2-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0183] 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  
     [0184] 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.  
     [0185] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0186] 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%.  
     [0187] 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)  
     [0188] 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%).  
     [0189] Preparation of [51-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite)  
     [0190] 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%).  
     [0191] 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)  
     [0192] 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%).  
     [0193] 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0194] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0195] 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.  
     [0196] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0197] 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 (Rf 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.  
     [0198] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0199] 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.  
     [0200] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0201] 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.  
     [0202] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0203] 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.  
     [0204] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine  
     [0205] 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.  
     [0206] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0207] 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.  
     [0208] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0209] 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.  
     [0210] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0211] 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.  
     [0212] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0213] 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.  
     [0214] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0215] 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 aminor 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].  
     [0216] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0217] 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.  
     [0218] 2-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0219] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetrahydrofuran (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.  
     [0220] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0221] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylaminoethoxy)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.  
     [0222] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0223] 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  
     [0224] Oligonucleotide Synthesis  
     [0225] 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.  
     [0226] 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.  
     [0227] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0228] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0229] 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.  
     [0230] 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.  
     [0231] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0232] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0233] 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  
     [0234] Oligonucleoside Synthesis  
     [0235] 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.  
     [0236] 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.  
     [0237] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0238] PNA Synthesis  
     [0239] 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  
     [0240] Synthesis of Chimeric Oligonucleotides  
     [0241] 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”.  
     [0242] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0243] 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.  
     [0244] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0245] [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.  
     [0246] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0247] [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.  
     [0248] 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  
     [0249] Oligonucleotide Isolation  
     [0250] 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  
     [0251] Oligonucleotide Synthesis—96 Well Plate Format  
     [0252] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected betacyanoethyldiisopropyl phosphoramidites.  
     [0253] 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  
     [0254] Oligonucleotide Analysis—96-Well Plate Format  
     [0255] 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  
     [0256] Cell Culture and Oligonucleotide Treatment  
     [0257] 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.  
     [0258] T-24 Cells:  
     [0259] 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.  
     [0260] 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.  
     [0261] A549 Cells:  
     [0262] 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.  
     [0263] NHDF Cells:  
     [0264] 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.  
     [0265] HEK Cells:  
     [0266] 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.  
     [0267] Treatment with Antisense Compounds:  
     [0268] 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-MEMTM-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.  
     [0269] 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  
     [0270] Analysis of Oligonucleotide Inhibition of IL-1 Receptor-Associated Kinase-1 Expression  
     [0271] Antisense modulation of IL-1 receptor-associated kinase-1 expression can be assayed in a variety of ways known in the art. For example, IL-1 receptor-associated kinase-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.  
     [0272] Protein levels of IL-1 receptor-associated kinase-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 IL-1 receptor-associated kinase-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).  
     [0273] 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  
     [0274] Poly(A)+mRNA Isolation  
     [0275] Poly(A)+mRNA was isolated according to Miura et al., ( Clin. Chem.,  1996, 42, 1758-1764). Other methods for poly(A)+mRNA isolation are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 1, pp. 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.  
     [0276] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0277] Total RNA Isolation  
     [0278] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer&#39;s recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.  
     [0279] 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  
     [0280] Real-time Quantitative PCR Analysis of IL-1 Receptor-Associated Kinase-1 mRNA Levels  
     [0281] Quantitation of IL-1 receptor-associated kinase-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 manufacturers 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.  
     [0282] 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.  
     [0283] 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 MgC12, 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).  
     [0284] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0285] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0286] Probes and primers to human IL-1 receptor-associated kinase-1 were designed to hybridize to a human IL-1 receptor-associated kinase-1 sequence, using published sequence information (GenBank accession number L76191.1, incorporated herein as SEQ ID NO:4). For human IL-1 receptor-associated kinase-1 the PCR primers were: forward primer: ACTTCTCGGAGGAGCTCAAGATC (SEQ ID NO: 5) reverse primer: GCATACACCGTGTTCCTCATCA (SEQ ID NO: 6) and the PCR probe was: FAM-CGCCCGGTACACGCACCCAA-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC— TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     [0287] Northern Blot Analysis of IL-1 Receptor-Associated Kinase-1 mRNA Levels  
     [0288] 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, OH). 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.  
     [0289] To detect human IL-1 receptor-associated kinase-1, a human IL-1 receptor-associated kinase-1 specific probe was prepared by PCR using the forward primer ACTTCTCGGAGGAGCTCAAGATC (SEQ ID NO: 5) and the reverse primer GCATACACCGTGTTCCTCATCA (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.).  
     [0290] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGERTM and IMAGEQUANTTM Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.  
     Example 15  
     [0291] Antisense Inhibition of Human IL-1 Receptor-Associated Kinase-1 Expression by Chimeric Phosphorothioate Oligonucleotides having 2′-MOE Wings and a Deoxy Gap  
     [0292] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human IL-1 receptor-associated kinase-1 RNA, using published sequences (GenBank accession number L76191.1, incorporated herein as SEQ ID NO: 4, nucleotides 1 to 13000 of GenBank accession number AF031075.1, the complement of which is incorporated herein as SEQ ID NO: 11, GenBank accession number BG479917.1, incorporated herein as SEQ ID NO: 12, and GenBank accession number AL581159.1, the complement of which is incorporated herein as SEQ ID NO: 13). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human IL-1 receptor-associated kinase-1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which T-24 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.  
                   TABLE 1                          Inhibition of human IL-1 receptor-associated kinase-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                                                         151391   3′UTR   4   3139   cctggcttgcaggccaccac   91   14   2                   151392   3′UTR   4   2424   gatgccagccttccttgccc   94   15   2               151393   Coding   4   965   cagtggagacggtcctccag   90   16   2               151394   3′UTR   4   3196   cttgtggcctccgaagcctg   96   17   2               151395   Coding   4   2127   ggacgacagcagctgcaggc   84   18   2               151396   Coding   4   1448   tctgcagccagacctgcttg   60   19   2               151397   Coding   4   1443   agccagacctgcttgcagtg   92   20   2               151398   3′UTR   4   3450   gtgaagcctgtgctacagcc   93   21   2               151399   Coding   4   191   tggcaccagtcggcgggctc   92   22   2               151400   Coding   4   2070   gaccatcttctgtcgggcag   81   23   2               151401   Coding   4   1264   ccagccttcccgtcttgatg   92   24   2               151402   3′UTR   4   2465   agccagcagcctcccaacat   94   25   2               151403   Stop   4   2199   tcagctctgaaattcatcac   94   26   2           Codon               151404   Coding   4   1995   cagtccttccacggctgtgg   90   27   2               151405   Coding   4   1315   ccaaggtctctagcactacc   95   28   2               151406   3′UTR   4   3269   ctaggtcttgggcctagcta   85   29   2               151407   Coding   4   766   acaccgtgttcctcatcacc   99   30   2               151408   Coding   4   2195   ctctgaaattcatcactttc   32   31   2               151409   Coding   4   1598   tgggtcataggaggcctcct   64   32   2               151410   3′UTR   4   3171   gctcggagctcgtctgtggc   11   33   2               151411   Coding   4   1348   tggcaccgtgcgtcttcaca   91   34   2               151412   Coding   4   146   cggcacatgacccagggcgg   94   35   2               151413   Coding   4   1322   tgaccagccaaggtctctag   0   36   2               151414   Coding   4   1383   ctcctcttccaccaggtctt   89   37   2               151415   3′UTR   4   2252   tgaccatgagaactttgact   97   38   2               151416   Coding   4   2001   aagggccagtccttccacgg   1   39   2               151417   Coding   4   1709   ctggacacgtaggagttctc   96   40   2               151418   Coding   4   1711   tgctggacacgtaggagttc   90   41   2               151419   Coding   4   2069   accatcttctgtcgggcagg   92   42   2               151420   Coding   4   1432   cttgcagtgtgctctgggtg   78   43   2               151421   Coding   4   854   ctggacagctgctccacctc   93   44   2               151422   3′UTR   4   3436   acagccctgggctacttttg   89   45   2               151423   3′UTR   4   2390   ggaggcagggttccactctg   90   46   2               151424   Coding   4   1446   tgcagccagacctgcttgca   71   47   2               151425   3′UTR   4   3246   cttgggtagtggcccctctg   94   48   2               151426   Coding   4   1998   ggccagtccttccacggctg   92   49   2               151427   Coding   4   901   tctgagcacagtagccagca   97   50   2               194303   Coding   4   341   cggagcagctgcaggtgcgt   79   51   2               194304   Coding   4   556   aagcagggcttggaaccagg   84   52   2               194305   Coding   4   715   cgatcttgagctcctccgag   81   53   2               194306   Coding   4   840   cacctcggtcaggaagctct   87   54   2               194307   Coding   4   933   caggaagccgtacaccaggc   88   55   2               194308   Coding   4   1211   actgtctgtgtccgggccac   76   56   2               194309   Coding   4   1548   cagctggcccaggcccaggc   50   57   2               194310   Coding   4   1574   gcccggcggtgcaggcagca   87   58   2               194311   Coding   4   1621   gcttctctagcctctcgtac   81   59   2               194312   Coding   4   1748   ggctgccatggagcagcccc   23   60   2               194313   Coding   4   1795   gcagctgctctgctgcctgg   64   61   2               194314   Coding   4   1848   agagaggccgcctaggctct   76   62   2               194315   Coding   4   1890   cagagggcagcttggagtca   43   63   2               194316   Coding   4   2084   agggccagcttctggaccat   87   64   2               194317   Stop   4   2209   ggtgaacacatcagctctga   0   65   2           Codon               194318   3′UTR   4   2577   agctgctgccagaggcctgg   81   66   2               194319   3′UTR   4   2994   ttacagccatacttcacttt   75   67   2               194320   3′UTR   4   3029   aattctcgcttcttgctagg   89   68   2               194321   3′UTR   4   3115   ggccagctcgcaggtcccca   91   69   2               194322   3′UTR   4   3332   ccttccctgtctgccatgct   90   70   2               194323   3′UTR   4   3413   acgcaagaggacactcggtt   92   71   2               194324   3′UTR   4   3469   ggctgaacacaaaatcactg   88   72   2               194325   3′UTR   4   3477   tgactcacggctgaacacaa   85   73   2               194326   3′UTR   4   3544   atacgtttttattactcaag   89   74   2               194327   3′UTR   4   3551   agggaacatacgtttttatt   30   75   2               194328   intron   11   2441   cctggaaaagcttcataaag   32   76   2               194329   exon   11   3229   ctggcctcacctggacagct   42   77   2               194330   intron   11   4066   agaccctccagctacgctgc   36   78   2               194331   intron   11   5276   ggagagcccacttgaagaca   61   79   2               194332   exon   11   7478   agctggctacctgggtcata   37   80   2               194333   intron   11   7534   tacagagcaaggcctggaat   62   81   2               194334   intron   11   7694   tccttctctctatgtgaagg   62   82   2               194335   intron   11   9602   aggcccaagcctacagaagg   34   83   2               194336   genomic   12   184   aggccagctggcccaggcgc   19   84   2               194337   3′UTR   13   136   acccaggctggagatggcgg   61   85   2                  
 
     [0293] As shown in Table 1, SEQ ID NOs 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 74, 79, 81, 82 and 85 demonstrated at least 60% inhibition of human IL-1 receptor-associated kinase-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 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found.  
                   TABLE 2                          Sequence and position of preferred target regions identified           in IL-1 receptor-associated kinase-1.                                                 TARGET           REV                   SITE   SEQ ID   TARGET       COMP OF       SEQ ID       ID   NO   SITE   SEQUENCE   SEQ ID   ACTIVE IN   NO                                                     66912   4   3139   gtggtggcctgcaagccagg   14     H. sapiens     86                   66913   4   2424   gggcaaggaaggctggcatc   15     H. sapiens     87               66914   4   965   ctggaggaccgtctccactg   16     H. sapiens     88               66915   4   3196   caggcttcggaggccacaag   17     H. sapiens     89               66916   4   2127   gcctgcagctgctgtcgtcc   18     H. sapiens     90               66917   4   1448   caagcaggtctggctgcaga   19     H. sapiens     91               66918   4   1443   cactgcaagcaggtctggct   20     H. sapiens     92               66919   4   3450   ggctgtagcacaggcttcac   21     H. sapiens     93               66920   4   191   gagcccgccgactggtgcca   22     H. sapiens     94               66921   4   2070   ctgcccgacagaagatggtc   23     H. sapiens     95               66922   4   1264   catcaagacgggaaggctgg   24     H. sapiens     96               66923   4   2465   atgttgggaggctgctggct   25     H. sapiens     97               66924   4   2199   gtgatgaatttcagagctga   26     H. sapiens     98               66925   4   1995   ccacagccgtggaaggactg   27     H. sapiens     99               66926   4   1315   ggtagtgctagagaccttgg   28     H. sapiens     100               66927   4   3269   tagctaggcccaagacctag   29     H. sapiens     101               66928   4   766   ggtgatgaggaacacggtgt   30     H. sapiens     102               66930   4   1598   aggaggcctcctatgaccca   32     H. sapiens     103               66932   4   1348   tgtgaagacgcacggtgcca   34     H. sapiens     104               66933   4   146   ccgccctgggtcatgtgccg   35     H. sapiens     105               66935   4   1383   aagacctggtggaagaggag   37     H. sapiens     106               66936   4   2252   agtcaaagttctcatggtca   38     H. sapiens     107               66938   4   1709   gagaactcctacgtgtccag   40     H. sapiens     108               66939   4   1711   gaactcctacgtgtccagca   41     H. sapiens     109               66940   4   2069   cctgcccgacagaagatggt   42     H. sapiens     110               66941   4   1432   cacccagagcacactgcaag   43     H. sapiens     111               66942   4   854   gaggtggagcagctgtccag   44     H. sapiens     112               66943   4   3436   caaaagtagcccagggctgt   45     H. sapiens     113               66944   4   2390   cagagtggaaccctgcctcc   46     H. sapiens     114               66945   4   1446   tgcaagcaggtctggctgca   47     H. sapiens     115               66946   4   3246   cagaggggccactacccaag   48     H. sapiens     116               66947   4   1998   cagccgtggaaggactggcc   49     H. sapiens     117               66948   4   901   tgctggctactgtgctcaga   50     H. sapiens     118               112415   4   341   acgcacctgcagctgctccg   51     H. sapiens     119               112416   4   556   cctggttccaagccctgctt   52     H. sapiens     120               112417   4   715   ctcggaggagctcaagatcg   53     H. sapiens     121               112418   4   840   agagcttcctgaccgaggtg   54     H. sapiens     122               112419   4   933   gcctggtgtacggcttcctg   55     H. sapiens     123               112420   4   1211   gtggcccggacacagacagt   56     H. sapiens     124               112422   4   1574   tgctgcctgcaccgccgggc   58     H. sapiens     125               112423   4   1621   gtacgagaggctagagaagc   59     H. sapiens     126               112425   4   1795   ccaggcagcagagcagctgc   61     H. sapiens     127               112426   4   1848   agagcctaggcggcctctct   62     H. sapiens     128               112428   4   2084   atggtccagaagctggccct   64     H. sapiens     129               112430   4   2577   ccaggcctctggcagcagct   66     H. sapiens     130               112431   4   2994   aaagtgaagtatggctgtaa   67     H. sapiens     131               112432   4   3029   cctagcaagaagcgagaatt   68     H. sapiens     132               112433   4   3115   tggggacctgcgagctggcc   69     H. sapiens     133               112434   4   3332   agcatggcagacagggaagg   70     H. sapiens     134               112435   4   3413   aaccgagtgtcctcttgcgt   71     H. sapiens     135               112436   4   3469   cagtgattttgtgttcagcc   72     H. sapiens     136               112437   4   3477   ttgtgttcagccgtgagtca   73     H. sapiens     137               112438   4   3544   cttgagtaataaaaacgtat   74     H. sapiens     138               112443   11   5276   tgtcttcaagtgggctctcc   79     H. sapiens     139               112445   11   7534   attccaggccttgctctgta   81     H. sapiens     140               112446   11   7694   ccttcacatagagagaagga   82     H. sapiens     141               112449   13   136   ccgccatctccagcctgggt   85     H. sapiens     142                  
 
     [0294] 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 IL-1 receptor-associated kinase-1.  
     Example 16  
     [0295] Western Blot Analysis of IL-1 Receptor-Associated Kinase-1 Protein Levels  
     [0296] 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 IL-1 receptor-associated kinase-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 
         
           
             142  
           
           
             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  
             3590  
             DNA  
             H. sapiens  
             
 
             
               CDS  
               (80)...(2218)  
             
           
            4 

cgcggacccg gccggcccag gcccgcgccc gccgcggccc tgagaggccc cggcaggtcc     60 

cggcccggcg gcggcagcc atg gcc ggg ggg ccg ggc ccg ggg gag ccc gca     112 
                     Met Ala Gly Gly Pro Gly Pro Gly Glu Pro Ala 
                       1               5                  10 

gcc ccc ggc gcc cag cac ttc ttg tac gag gtg ccg ccc tgg gtc atg      160 
Ala Pro Gly Ala Gln His Phe Leu Tyr Glu Val Pro Pro Trp Val Met 
             15                  20                  25 

tgc cgc ttc tac aaa gtg atg gac gcc ctg gag ccc gcc gac tgg tgc      208 
Cys Arg Phe Tyr Lys Val Met Asp Ala Leu Glu Pro Ala Asp Trp Cys 
         30                  35                  40 

cag ttc gcc gcc ctg atc gtg cgc gac cag acc gag ctg cgg ctg tgc      256 
Gln Phe Ala Ala Leu Ile Val Arg Asp Gln Thr Glu Leu Arg Leu Cys 
     45                  50                  55 

gag cgc tcc ggg cag cgc acg gcc agc gtc ctg tgg ccc tgg atc aac      304 
Glu Arg Ser Gly Gln Arg Thr Ala Ser Val Leu Trp Pro Trp Ile Asn 
 60                  65                  70                  75 

cgc aac gcc cgt gtg gcc gac ctc gtg cac atc ctc acg cac ctg cag      352 
Arg Asn Ala Arg Val Ala Asp Leu Val His Ile Leu Thr His Leu Gln 
                 80                  85                  90 

ctg ctc cgt gcg cgg gac atc atc aca gcc tgg cac cct ccc gcc ccg      400 
Leu Leu Arg Ala Arg Asp Ile Ile Thr Ala Trp His Pro Pro Ala Pro 
             95                 100                 105 

ctt ccg tcc cca ggc acc act gcc ccg agg ccc agc agc atc cct gca      448 
Leu Pro Ser Pro Gly Thr Thr Ala Pro Arg Pro Ser Ser Ile Pro Ala 
        110                 115                 120 

ccc gcc gag gcc gag gcc tgg agc ccc cgg aag ttg cca tcc tca gcc      496 
Pro Ala Glu Ala Glu Ala Trp Ser Pro Arg Lys Leu Pro Ser Ser Ala 
    125                 130                 135 

tcc acc ttc ctc tcc cca gct ttt cca ggc tcc cag acc cat tca ggg      544 
Ser Thr Phe Leu Ser Pro Ala Phe Pro Gly Ser Gln Thr His Ser Gly 
140                 145                 150                 155 

cct gag ctc ggc ctg gtt cca agc cct gct tcc ctg tgg cct cca ccg      592 
Pro Glu Leu Gly Leu Val Pro Ser Pro Ala Ser Leu Trp Pro Pro Pro 
                160                 165                 170 

cca tct cca gcc cct tct tct acc aag cca ggc cca gag agc tca gtg      640 
Pro Ser Pro Ala Pro Ser Ser Thr Lys Pro Gly Pro Glu Ser Ser Val 
            175                 180                 185 

tcc ctc ctg cag gga gcc cgc ccc tct ccg ttt tgc tgg ccc ctc tgt      688 
Ser Leu Leu Gln Gly Ala Arg Pro Ser Pro Phe Cys Trp Pro Leu Cys 
        190                 195                 200 

gag att tcc cgg ggc acc cac aac ttc tcg gag gag ctc aag atc ggg      736 
Glu Ile Ser Arg Gly Thr His Asn Phe Ser Glu Glu Leu Lys Ile Gly 
    205                 210                 215 

gag ggt ggc ttt ggg tgc gtg tac cgg gcg gtg atg agg aac acg gtg      784 
Glu Gly Gly Phe Gly Cys Val Tyr Arg Ala Val Met Arg Asn Thr Val 
220                 225                 230                 235 

tat gct gtg aag agg ctg aag gag aac gct gac ctg gag tgg act gca      832 
Tyr Ala Val Lys Arg Leu Lys Glu Asn Ala Asp Leu Glu Trp Thr Ala 
                240                 245                 250 

gtg aag cag agc ttc ctg acc gag gtg gag cag ctg tcc agg ttt cgt      880 
Val Lys Gln Ser Phe Leu Thr Glu Val Glu Gln Leu Ser Arg Phe Arg 
            255                 260                 265 

cac cca aac att gtg gac ttt gct ggc tac tgt gct cag aac ggc ttc      928 
His Pro Asn Ile Val Asp Phe Ala Gly Tyr Cys Ala Gln Asn Gly Phe 
        270                 275                 280 

tac tgc ctg gtg tac ggc ttc ctg ccc aac ggc tcc ctg gag gac cgt      976 
Tyr Cys Leu Val Tyr Gly Phe Leu Pro Asn Gly Ser Leu Glu Asp Arg 
    285                 290                 295 

ctc cac tgc cag acc cag gcc tgc cca cct ctc tcc tgg cct cag cga     1024 
Leu His Cys Gln Thr Gln Ala Cys Pro Pro Leu Ser Trp Pro Gln Arg 
300                 305                 310                 315 

ctg gac atc ctt ctg ggt aca gcc cgg gca att cag ttt cta cat cag     1072 
Leu Asp Ile Leu Leu Gly Thr Ala Arg Ala Ile Gln Phe Leu His Gln 
                320                 325                 330 

gac agc ccc agc ctc atc cat gga gac atc aag agt tcc aac gtc ctt     1120 
Asp Ser Pro Ser Leu Ile His Gly Asp Ile Lys Ser Ser Asn Val Leu 
            335                 340                 345 

ctg gat gag agg ctg aca ccc aag ctg gga gac ttt ggc ctg gcc cgg     1168 
Leu Asp Glu Arg Leu Thr Pro Lys Leu Gly Asp Phe Gly Leu Ala Arg 
        350                 355                 360 

ttc agc cgc ttt gcc ggg tcc agc ccc agc cag agc agc atg gtg gcc     1216 
Phe Ser Arg Phe Ala Gly Ser Ser Pro Ser Gln Ser Ser Met Val Ala 
    365                 370                 375 

cgg aca cag aca gtg cgg ggc acc ctg gcc tac ctg ccc gag gag tac     1264 
Arg Thr Gln Thr Val Arg Gly Thr Leu Ala Tyr Leu Pro Glu Glu Tyr 
380                 385                 390                 395 

atc aag acg gga agg ctg gct gtg gac acg gac acc ttc agc ttt ggg     1312 
Ile Lys Thr Gly Arg Leu Ala Val Asp Thr Asp Thr Phe Ser Phe Gly 
                400                 405                 410 

gtg gta gtg cta gag acc ttg gct ggt cag agg gct gtg aag acg cac     1360 
Val Val Val Leu Glu Thr Leu Ala Gly Gln Arg Ala Val Lys Thr His 
            415                 420                 425 

ggt gcc agg acc aag tat ctg aaa gac ctg gtg gaa gag gag gct gag     1408 
Gly Ala Arg Thr Lys Tyr Leu Lys Asp Leu Val Glu Glu Glu Ala Glu 
        430                 435                 440 

gag gct gga gtg gct ttg aga agc acc cag agc aca ctg caa gca ggt     1456 
Glu Ala Gly Val Ala Leu Arg Ser Thr Gln Ser Thr Leu Gln Ala Gly 
    445                 450                 455 

ctg gct gca gat gcc tgg gct gct ccc atc gcc atg cag atc tac aag     1504 
Leu Ala Ala Asp Ala Trp Ala Ala Pro Ile Ala Met Gln Ile Tyr Lys 
460                 465                 470                 475 

aag cac ctg gac ccc agg ccc ggg ccc tgc cca cct gag ctg ggc ctg     1552 
Lys His Leu Asp Pro Arg Pro Gly Pro Cys Pro Pro Glu Leu Gly Leu 
                480                 485                 490 

ggc ctg ggc cag ctg gcc tgc tgc tgc ctg cac cgc cgg gcc aaa agg     1600 
Gly Leu Gly Gln Leu Ala Cys Cys Cys Leu His Arg Arg Ala Lys Arg 
            495                 500                 505 

agg cct cct atg acc cag gtg tac gag agg cta gag aag ctg cag gca     1648 
Arg Pro Pro Met Thr Gln Val Tyr Glu Arg Leu Glu Lys Leu Gln Ala 
        510                 515                 520 

gtg gtg gcg ggg gtg ccc ggg cat ttg gag gcc gcc agc tgc atc ccc     1696 
Val Val Ala Gly Val Pro Gly His Leu Glu Ala Ala Ser Cys Ile Pro 
    525                 530                 535 

cct tcc ccg cag gag aac tcc tac gtg tcc agc act ggc aga gcc cac     1744 
Pro Ser Pro Gln Glu Asn Ser Tyr Val Ser Ser Thr Gly Arg Ala His 
540                 545                 550                 555 

agt ggg gct gct cca tgg cag ccc ctg gca gcg cca tca gga gcc agt     1792 
Ser Gly Ala Ala Pro Trp Gln Pro Leu Ala Ala Pro Ser Gly Ala Ser 
                560                 565                 570 

gcc cag gca gca gag cag ctg cag aga ggc ccc aac cag ccc gtg gag     1840 
Ala Gln Ala Ala Glu Gln Leu Gln Arg Gly Pro Asn Gln Pro Val Glu 
            575                 580                 585 

agt gac gag agc cta ggc ggc ctc tct gct gcc ctg cgc tcc tgg cac     1888 
Ser Asp Glu Ser Leu Gly Gly Leu Ser Ala Ala Leu Arg Ser Trp His 
        590                 595                 600 

ttg act cca agc tgc cct ctg gac cca gca ccc ctc agg gag gcc ggc     1936 
Leu Thr Pro Ser Cys Pro Leu Asp Pro Ala Pro Leu Arg Glu Ala Gly 
    605                 610                 615 

tgt cct cag ggg gac acg gca gga gaa tcg agc tgg ggg agt ggc cca     1984 
Cys Pro Gln Gly Asp Thr Ala Gly Glu Ser Ser Trp Gly Ser Gly Pro 
620                 625                 630                 635 

gga tcc cgg ccc aca gcc gtg gaa gga ctg gcc ctt ggc agc tct gca     2032 
Gly Ser Arg Pro Thr Ala Val Glu Gly Leu Ala Leu Gly Ser Ser Ala 
                640                 645                 650 

tca tcg tcg tca gag cca ccg cag att atc atc aac cct gcc cga cag     2080 
Ser Ser Ser Ser Glu Pro Pro Gln Ile Ile Ile Asn Pro Ala Arg Gln 
            655                 660                 665 

aag atg gtc cag aag ctg gcc ctg tac gag gat ggg gcc ctg gac agc     2128 
Lys Met Val Gln Lys Leu Ala Leu Tyr Glu Asp Gly Ala Leu Asp Ser 
        670                 675                 680 

ctg cag ctg ctg tcg tcc agc tcc ctc cca ggc ttg ggc ctg gaa cag     2176 
Leu Gln Leu Leu Ser Ser Ser Ser Leu Pro Gly Leu Gly Leu Glu Gln 
    685                 690                 695 

gac agg cag ggg ccc gaa gaa agt gat gaa ttt cag agc tga tgtgttcacc  2228 
Asp Arg Gln Gly Pro Glu Glu Ser Asp Glu Phe Gln Ser 
700                 705                 710 

tgggcagatc ccccaaatcc ggaagtcaaa gttctcatgg tcagaagttc tcatggtgca   2288 

cgagtcctca gcactctgcc ggcagtgggg gtgggggccc atgcccgcgg gggagagaag   2348 

gaggtggccc tgctgttcta ggctctgtgg gcataggcag gcagagtgga accctgcctc   2408 

catgccagca tctgggggca aggaaggctg gcatcatcca gtgaggaggc tggcgcatgt   2468 

tgggaggctg ctggctgcac agacccgtga ggggaggaga ggggctgctg tgcaggggtg   2528 

tggagtaggg agctggctcc cctgagagcc atgcagggcg tctgcagccc aggcctctgg   2588 

cagcagctct ttgcccatct ctttggacag tggccaccct gcacaatggg gccgacgagg   2648 

cctagggccc tcctacctgc ttacaatttg gaaaagtgtg gccgggtgcg gtggctcacg   2708 

cctgtaatcc cagcactttg ggaggccaag gcaggaggat cgctggagcc cagtaggtca   2768 

agaccagcca gggcaacatg atgagaccct gtctctgcca aaaaattttt taaactatta   2828 

gcctggcgtg gtagcgcacg cctgtggtcc cagctgctgg ggaggctgaa gtaggaggat   2888 

catttatgct tgggaggtcg aggctgcagt gagtcatgat tgtatgactg cactccagcc   2948 

tgggtgacag agcaagaccc tgtttcaaaa agaaaaaccc tgggaaaagt gaagtatggc   3008 

tgtaagtctc atggttcagt cctagcaaga agcgagaatt ctgagatcct ccagaaagtc   3068 

gagcagcacc cacctccaac ctcgggccag tgtcttcagg ctttactggg gacctgcgag   3128 

ctggcctaat gtggtggcct gcaagccagg ccatccctgg gcgccacaga cgagctccga   3188 

gccaggtcag gcttcggagg ccacaagctc agcctcaggc ccaggcactg attgtggcag   3248 

aggggccact acccaaggtc tagctaggcc caagacctag ttacccagac agtgagaagc   3308 

ccctggaagg cagaaaagtt gggagcatgg cagacaggga agggaaacat tttcagggaa   3368 

aagacatgta tcacatgtct tcagaagcaa gtcaggtttc atgtaaccga gtgtcctctt   3428 

gcgtgtccaa aagtagccca gggctgtagc acaggcttca cagtgatttt gtgttcagcc   3488 

gtgagtcaca ctacatgccc ccgtgaagct gggcattggt gacgtccagg ttgtccttga   3548 

gtaataaaaa cgtatgttcc ctaaaaaaaa aaaaaggaat tc                      3590 

 
           
             5  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

acttctcgga ggagctcaag atc                                             23 

 
           
             6  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

gcatacaccg tgttcctcat ca                                              22 

 
           
             7  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

cgcccggtac acgcacccaa                                                 20 

 
           
             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  
             13000  
             DNA  
             H. sapiens  
             
 
           
            11 

ctcacatgac agcatggtgc tgcgtttcct cattggatct ggctgtccct ggacacaggt     60 

agctgccttc aggcctgcca cgagcggcca agggaagcct cctccatatg ctggcctcgc    120 

tggcccctca gcttcttcca agccagtgct ctccaggcac actgctccag cgtgtgacgg    180 

gaagggcctg gcatgagtca gcctgcagca caacctccct gctccagacc cgtatggtag    240 

gggcaccccc taggtctgga tgtgctgtgg tgcttttgga cacccccacc cccgcaggct    300 

gtggctcctc ctgtgtctca ttctggccag gaccctcacg tgccctctgt tgactgctaa    360 

cgtggttctc tgaccaggca agggcaggct gaggggtttg cccaaagggg gcccccttgt    420 

tactggcttc cttggctctc aggagcagcc tcaccaggtt ggtaaggggc tggaggagac    480 

aactgctcaa aggagtccag cttcacatgc acatgctaga aggtaccctc ggaaggcctg    540 

gccttcaaag gtagatccca gggttgaaaa gtcaacttgt atgcattgag catctcgtat    600 

gccagccctg ttccgtgagc tgatgggcct ttgtgtgtaa gtaggaccaa gtgcccccgt    660 

ggaggttagc atgggtgtgc agtcatttca gatacttgag ttggtacatc tcagtaaagt    720 

ctgtcccgtg agaagccatg ggtttcatgg tatggttggc atcttccttg ggagtggcca    780 

cagtggtggt ggcttcagga aagagactcc aacaggggcc agctgtgggc cttgggcact    840 

tctcgtttct aggaaaagtc ctaagtctgt agggctaggg gtggggaacc ccttcgctgt    900 

caggatcaag agggcaaggg gaactgtcgc tggaggagac atccagctgg agaaacaaaa    960 

gagtaagtct gcgttgctgc ttgtggggtc ttccccatct cagggcgggg accgggggtg   1020 

gcggtccaga caagtaatca aggacgatgc ccaggagggg acaggtacgg ggtggcagga   1080 

gctctgccgg cgggctcagg aagccttcac cacagctgcc tgagctcacc cttgccaaat   1140 

gagggctggg gcagcagcaa cgcatacact cacggctgtg gcgggcagcg ttctcggcat   1200 

atttcaggac acctaaggag actgaatggc tcaaggctgc tgccgtgtgc agggggctag   1260 

acgtggggcg ggcaggcagg gctcctggta acagccctgc aggccgcagt ggagagcagg   1320 

gttccggcag ggccgcccag gagctttcgg aaggcccggc cccggcccct ttccgagcag   1380 

cccgggcctc cgccctgccc tctgtcccca acgccgggag ccgccgttcg tcctccagag   1440 

ccccgcccgg gcgagcccgg gaggccgatc gccgctcgcg gaacccgccg ggacccgggc   1500 

cctccccggc gcggggcgcc cccgtgtgac ccagcgcgcg gccgcggcgc gcaagatggc   1560 

ggcgggcccg ggcaccgccc cttccgcccc gccgggcgtc gcacgaggcc ggctcgaagg   1620 

ggaagtgagt cagtgtccgc ggacccggcc ggcccaggcc cgcgcccgcc gcggccctga   1680 

gaggccccgg caggtcccgg cccggcggcg gcagccatgg ccggggggcc gggcccgggg   1740 

gagcccgcag cccccggcgc ccagcacttc ttgtacgagg tgccgccctg ggtcatgtgc   1800 

cgcttctaca aagtgatgga cgccctggag cccgccgact ggtgccagtt cggtgggtgg   1860 

cggcgggctg ccggggggcg ggaggcgcgc gggctcctgg cgccgacgcc tgacgccccc   1920 

cgccccgcag ccgccctgat cgtgcgcgac cagaccgagc tgcggctgtg cgagcgctcc   1980 

gggcagcgca cggccagcgt cctgtggccc tggatcaacc gcaacgcccg tgtggccgac   2040 

ctcgtgcaca tcctcacgca cctgcagctg ctccgtgcgc gggacatcat cacagcctgt   2100 

gagcgcggga ctccgggcac cccacggctg ggaggccggc gggccccacg gggctccccc   2160 

acccgggcct caaccttcct ttccttcctt ggcgtcccag ggcaccctcc cgccccgctt   2220 

ccgtccccag gcaccactgc cccgaggccc agcagcatcc ctgcacccgc cgaggccgag   2280 

gcctggagcc cccggaagtt gccatcctca gcctccacct tcctctcccc aggtaagagg   2340 

gcccggttgt taggctcggt ggacccaaag aagagcccac cttgaccacg gccacggctg   2400 

tagaccctgc tgctggtctc tgcctgcctc tcactggtgt ctttatgaag cttttccagg   2460 

ctcccagacc cattcagggc ctgagctcgg cctggtccca agccctgctt ccctgtggcc   2520 

tccaccgcca tctccagccc cttcttctac caaggtaggt gtcccctgcc ccccagggaa   2580 

gattcgagac aaggaggaag gaattcagcc tttgatgtag cgcagagccc cagtcagcca   2640 

agctgggtca gctgggaggc agctgtggtg gggagagcct ggagccttgg gcagaaggga   2700 

agagacaggg accccacctg atccaggctc tcttcccaca gccaggccca gagagctcag   2760 

tgtccctcct gcagggagcc cgcccctttc cgttttgctg gcccctctgt gagatttccc   2820 

ggggcaccca caacttctcg gaggagctca agatcgggga gggtggcttt gggtgcgtgt   2880 

accgggcggt gatgaggaac acggtgtatg ctgtgaagag gctgaaggag gtgagtgtcg   2940 

caccctggca gggaccctgg aaggccatca gataaccctc accacttctc cagcctttcc   3000 

ccctcgcttc cccacacaac tccttcagcc ctcattctgg cgtagggtcc ctggcccctt   3060 

ggtggttctg ggcctcgggt aggtggcact ggtggcccga aggccttcgc ttcgagagcc   3120 

tcacgctgcc cgtcttccct gccccttccc ccaccgcacc ctgggggctg cagaacgctg   3180 

acctggagtg gactgcagtg aagcagagct tcctgaccga ggtggagcag ctgtccaggt   3240 

gaggccagag ggggagccac accaggtccc gtggggcttc agaccgcaca ccacaggacc   3300 

tggctccctt gggcacctga ggcctggcag gcccgggcga gctgaggccc cagccagggc   3360 

tgcccaccca gtctggcctg atggaaagtg ctcccctttt tcaaacaggt ttcgtcaccc   3420 

aaacattgtg gactttgctg gctactgtgc tcagaacggc ttctactgcc tggtgtacgg   3480 

cttcctgccc aacggctccc tggaggaccg tctccactgc caggtaggct cacctggccc   3540 

ggcacgcttc ccaggaccca aagcactcct gacacctggc tggagccggg cgcggggcct   3600 

agggctttca gcctgtgtga gtgggtcctg ccagcaggcc aggcctgcac ttccagctcc   3660 

ccagcagcac ccggctcagg atttggccca cggtggggtc aatttttttt tttttttttt   3720 

tttttttttt tttgaggtgg agtcttgctc tgtcatccag gctggagtgc agtggtgtga   3780 

tctcagctca ccacaacctc cacctcccgg gttcaggcga tcctcctgcc gcagcctccc   3840 

gagtagctgg gactacaggc atgcaccacc acacctgcct aatttttgta tttttagtag   3900 

agatgaggtt ttgccacatt ggccaggcta gtctcgaact cctgacccca ggtggtctgc   3960 

gcgcctcagc ctcccaaagt gctgggatta caggcgtgag ccaccacgcc tggcccgacc   4020 

caatgttttc tatagagctc tttcccaggc ctctcccctt tgcaagcagc gtagctggag   4080 

ggtctcatca gcaagccccg gaggcgaggg ggtctggggc taccagctgg accacctaca   4140 

gctgagggag ggcccccttg cctcctcctg catgctgcgt ttggggagag cgggaagaat   4200 

gccttcaagg acttcccgac caccagggac aaagggatga gccctgggag ccgaagccca   4260 

gcagattcta ttgaacgtgt ccccagccat tgcttaagaa gtgcaggtca cggagacttt   4320 

gctcctcgtt ttccagaagg gggaaactga ggcctagaga gtgaagtggc tgttccaggc   4380 

tgcacggtga caggtagaag gatggttggg atcaaggaac ggccatccag caacctcccc   4440 

tgtccccctt tgccacccca gacccaggcc tgcccacctc tctcctggcc tcagcgactg   4500 

gacatccttc tgggtacagc ccgggcaatt cagtttctac atcaggacag ccccagcctc   4560 

atccatggag acatcaagag gtgaggaggg gcccttgaga actgccgggg cagggcctgc   4620 

agcaaggggg gccccgcgtc ctatcaatgt ggggatcagg catggcctgg gacctcaaca   4680 

ccccggcatc gcacaggtgt gggaacgggc caaggatggg ccctactgat gagcagaggc   4740 

ccccaggcag ctggagcgct cagggcagtg ccagcgcttt ctgtgggcaa ggcaccgggc   4800 

tggcagcctc gagtccagcc ttatctaagc cgggcaggtg taggagctag gaccgggctg   4860 

acgccactgt cttctctccc caagttccaa cgtccttctg gatgagaggc tgacacccaa   4920 

gctgggagac tttggcctgg cccggttcag ccgctttgcc gggtccagcc ccagccagag   4980 

cagcatggtg gcccggacac agacagtgcg gggcaccctg gcctacctgc ccgaggagta   5040 

catcaagacg ggaaggctgg ctgtggacac ggacaccttc agctttgggg tggtgagcca   5100 

ctgacccctc tgctggctca gaggaggaga agccacaggc aggcagaggt gggggctgca   5160 

gagtgcactg cgggccaggg gccatctgcc aagaccccag gaggctgcag ctccagggtc   5220 

cccctccctc cgaggccctc ctcctcaccc tgcacctaac tgtgtgtttg taatttgtct   5280 

tcaagtgggc tctccgagtt gcccgagctt cagtcccata acatctggct ctgcctttgt   5340 

ccacaccctt gtcaggccca atccatgtcc acaccagagg cctcttccct gccaaggcca   5400 

ctgccatgct ctccctcttc cctctctcca ggtagtgcta gagaccttgg ctggtcagag   5460 

ggctgtgaag acgcacggtg ccaggaccaa gtatctggtg agccccttga ggcagggcca   5520 

ggagggacac acagctgctg gcagccagca ggcacagccc cagtggcggg gataactggg   5580 

gcgcagtgcc catggatgcc tctgctgcca cagtggcctc atttttgaaa gtaggcaggg   5640 

ctccaaacaa cttcgtttac cttgccgagg acaaacctgt ctgtcctgca gacactatgg   5700 

gccttgtaca gaccccacct gggctggggg cagggggaag ggcggtccca gggcactgag   5760 

acccaagctg cagtggaact cagaggactc tggccggaga aaggcggtgg tagagaagaa   5820 

gcaggccccg aggaacctcc tgggccccag caggctgcag ctgagctctc cgcaccgtgc   5880 

agggcagcct gagctgcctc acggtcttac tccactcagt ctgcctcacc gtggactgtg   5940 

gtggggccag gagactagag acctgggttt tagccccagc ctgacagtgg ccttccagca   6000 

aattcctgct cccctgtggg cctgttttcc catatgcaaa acagacttca cagaatgtgc   6060 

tcagccagta attgcttcac tgcttctcct cttgtttggg gcggttcctg tgtgctgtgg   6120 

ggtctccgtc aggattcagc cccgctgaga acccaggagc cgggcttgag ccctccttcc   6180 

tttcccttcc ccgtccgtcc atccatgcat cctgctgaag aagcgcacca ggctccttgg   6240 

gggtccttgg acttccccac ttgctcccca ccctgcagcc aaagtgctct tttcaaaggc   6300 

ccctttgcct tttctctgct cttggggtga aggcccagtc ccttatgtgg ttgacccccc   6360 

aacgccccag gtccctggga ctcggggcgc tccctgctgc ctgcttcaca gccttagtat   6420 

gtgccgttcg ctctgcgaga aaagccaccg cccacccagg tggttcctcc tggtctgtct   6480 

gatttcagaa ctggagatgg cctccggtcc tgtttccacc ctgggggcgc ctctctgcgg   6540 

ggcgcctctc tctctggggt acctctttgt ctgtggggca cctctctgtg gggcgctttc   6600 

cttctcggct ctgccctctc aggctacttc ctgccttcag accccagctc catggggctc   6660 

tcccccacca ggaagtcagc tctgtcaaac cgggtcccag ggttctgttt gttcctgtat   6720 

ccctagggcc cagagcacca ctggcccaca gtaggtgttt aataaatctc tagaagctac   6780 

tcgggaatct gaggcaggag gatcgcttga gcctgggagt tggagaccag cctgggcaac   6840 

atagcaagat aggcatggtg gtacacacct gtaatcccag ctgctcggga ggccgaggtg   6900 

ggaggatcac gagcctggga ggttgaggct gcactgagcc atgattgcac cactgcactc   6960 

cagcctgggt gacagaatga gtccctgtct cagaaaaaaa gtaagttgta gaaagaccaa   7020 

gagctgtggc acagtgtctc acacctgtaa caccagcact ttgggaggct gaggcgggag   7080 

gatcacttga gcctacttgg agaccagcca ggccaacaaa gcgagacccc atctcttttt   7140 

tttttttttt tttttttatc aaaacccata cgattgagtg acaaggacct gaggactgca   7200 

gctgcaggtg tggccacctg gtagccatac tgacagtatt tatcccacag aaagacctgg   7260 

tggaagagga ggctgaggag gctggagtgg ctttgagaag cacccagagc acactgcaag   7320 

caggtctggc tgcagatgcc tgggctgctc ccatcgccat gcagatctac aagaagcacc   7380 

tggaccccag gcccgggccc tgcccacctg agctgggcct gggcctgggc cagctggcct   7440 

gctgctgcct gcaccgccgg gccaaaagga ggcctcctat gacccaggta gccagctgcg   7500 

cactgggacg gggtggccag atagaaagcc cgcattccag gccttgctct gtagtgaccc   7560 

catctcagca cctgctaggt ctctctggag tctccacaca tttcttgctt gccctttggt   7620 

tctgtttggg gcagcgcccc tctgaactga ggggccccgg gcagtcctgc tttgcggagc   7680 

ccagctccga cccccttcac atagagagaa ggaaagagct gctgccgcgc cccctgctgg   7740 

gcgcactgca ctactgcatc tgcctttttc tgtcccctcc ctagtacccc acctcttctc   7800 

ctctggtgac agttgaaaat ggagaggccc cgtttgaggg cagcggggca gtgagattca   7860 

ttttgtagaa aagaacgagg ccattctcag tccttgcttt tggcagccgc gcttctcagc   7920 

actccctgtg atgggaacag aggggcgagg ggcagagcgt tcccagctgc agggtatgtc   7980 

attttagagc cctggggcag gtcacggacg gcctggagca gccctgtggt ttgcccacgg   8040 

ggtgaccggc cagggctgcc atctcaccct gagagtccct cttttccact tgcaggtgta   8100 

cgagaggcta gagaagctgc aggcagtggt ggcgggggtg cccgggcatt cggaggccgc   8160 

cagctgcatc cccccttccc cgcaggagaa ctcctacgtg tccagcactg gcagagccca   8220 

cagtggggct gctccatggc agcccctggc agcgccatca ggagccagtg cccaggcagc   8280 

agagcagctg cagagaggcc ccaaccagcc cgtggagagt gacgagagcc taggcggcct   8340 

ctctgctgcc ctgcgctcct ggcacttgac tccaagctgc cctctggacc cagcacccct   8400 

cagggaggcc ggctgtcctc agggggacac ggcaggagaa tcgagctggg ggagtggccc   8460 

aggatcccgg cccacagccg tggaaggtag ctggggagac gggttcccag gagagggacc   8520 

aaggcctctt tgggccaaag cccctgtaag tccccacccc agccttctag aagagaacca   8580 

gggccaaatg ttcagctcac tgtgacctta gcaaccctgg tttcccctcc ccaggccaca   8640 

tccttcccag gtggagcttg ctctccagcc ctccccccac cccattcctg aaggctggga   8700 

acaaggaggg ctctgtctgg tagcctgaga gctgggccct gcccttggac ttctctgagg   8760 

aattcaggcc tgaggccagg gaggcagggt gctaggctgc gggctgggga gccacagcat   8820 

gaggctaagg gagtgccatc tccaccccag gactggccct tggcagctct gcatcatcgt   8880 

cgtcagagcc accgcagatt atcatcaacc ctgcccgaca gaagatggtc cagaagctgg   8940 

ccctgtacga ggatggggcc ctggacagcc tgcagctgct gtcgtccagc tccctcccag   9000 

gtgctgccgc ccaggctggc ctctggggtg ctcaggcgca tccgtgtcag ccccaaagag   9060 

cagagtgtct gtcccgactg cgctgagggc gtggggcagc cgggcaggcc actggctctg   9120 

gcgacctcta gaagcccagc cggccccaca tgcctccctt agcaagaccc tggcccactc   9180 

cttccctcgc ctcctgacag tagcacctcc tttagcccga gggtgcctgc cccactctgt   9240 

gctttcagga aataggaagc cccagcagga attttccatc ccaggtacta tttgaagaac   9300 

cactgcttag gaaccctcag ctgggcgagg tggctcatgc ctataatacc agcacttttg   9360 

gaggccaaga tgggaggatc acttgagccc aggaggtgga ggctacagtg agctgtgatc   9420 

aagccactgc actccagcct gggagacagt tagaccctgt ctcaaaaaca aatgaacaaa   9480 

caaacaaaaa ccctcaattc ccacgaacgc cccaggagat aagggagcat ggcccaggcc   9540 

ttgagccagg gcttctggca gtaggggagc ctcccccatt tgctaagcgg actttcctct   9600 

tccttctgta ggcttgggcc tggaacagga caggcagggg cccgaagaaa gtgatgaatt   9660 

tcagagctga tgtgttcacc tgggcagatc ccccaaatcc ggaagtcaaa gttctcatgg   9720 

tcagaagttc tcatggtgca cgagtcctca gcactctgcc ggcagtgggg gtgggggccc   9780 

atgcccgcgg gggagagaag gaggtggccc tgctgttcta ggctctgtgg gcataggcag   9840 

gcagagtgga accctgcctc catgccagca tctgggggca aggaaggctg gcatcatcca   9900 

gtgaggaggc tggcgcatgt tgggaggctg ctggctgcac agacccgtga ggggaggaga   9960 

ggggctgctg tgcaggggtg tggagtaggg agctggctcc cctgagagcc atgcagggcg  10020 

tctgcagccc aggcctctgg cagcagctct ttgcccatct ctttggacag tggccaccct  10080 

gcacaatggg gccgacgagg cctagggccc tcctacctgc ttacaatttg gaaaagtgtg  10140 

gccgggtgcg gtggctcacg cctgtaatcc cagcactttg ggaggccaag gcaggaggat  10200 

cgctggagcc cagtaggtca agaccagcca gggcaacatg atgagaccct gtctctgcca  10260 

aaaaattttt taaactatta gcctggcgtg gtagcgcacg cctgtggtcc cagctgctgg  10320 

ggaggctgaa gtaggaggat catttatgct tgggaggtcg aggctgcagt gagtcatgat  10380 

tgtatgactg cactccagcc tgggtgacag agcaagaccc tgtttcaaaa agaaaaaccc  10440 

tgggaaaagt gaagtatggc tgtaagtctc atggttcagt cctagcaaga agcgagaatt  10500 

ctgagatcct ccagaaagtc gagcagcacc cacctccaac ctcgggccag tgtcttcagg  10560 

ctttactggg gacctgcgag ctggcctaat gtggtggcct gcaagccagg ccatccctgg  10620 

gcgccacaga cgagctccga gccaggtcag gcttcggagg ccacaagctc agcctcaggc  10680 

ccaggcactg attgtggcag aggggccact acccaaggtc tagctaggcc caagacctag  10740 

ttacccagac agtgagaagc ccctggaagg cagaaaagtt gggagcatgg cagacaggga  10800 

agggaaacat tttcagggaa aagacatgta tcacatgtct tcagaagcaa gtcaggtttc  10860 

atgtaaccga gtgtcctctt gcgtgtccaa aagtagccca gggctgtagc acaggcttca  10920 

cagtgatttt gtgttcagcc gtgagtcaca ctacatgccc ccgtgaagct gggcattggt  10980 

gacgtccagg ttgtccttga gtaataaaaa cgtatgttgc aatctcgggc tctacttgtg  11040 

gactttgttg caccgaaagc cttgagcttt cctgatgcct tacacttcag ggttcttgag  11100 

cgtccagggt cttgttacta ctctgggctg gccacaccca gcacttcccg tgtcaggttt  11160 

ttcctgatgt agtccatgtt ttttatgcta ttctaaatgg tatctttgat tttctagttc  11220 

atcatgatat tatacagaaa tgcaattgat gctgggcacg gtggctcacg cctgtgattc  11280 

cagcgctttg ggaagctaag gcgggcagat cacttgaggc caggagtttg agaccagcct  11340 

gggcaacatg gcgaaacccc gtctctacaa aaagtacaaa aattagccag gcatggtggt  11400 

gcatgcctgt agtttgagct actcaggagg ctgacccagg aggatagttt gagcccagga  11460 

cgttgaggct gcagtgagcc atgattccac cactgcactc cagcccgggc aacagaggga  11520 

gaccttgcct caaaaaaaaa aaaaaaaaaa aaaaaagcgg ttgagttttg catatgaacc  11580 

gtatattctg tgaccttgtt taaattcttt tttttttttc tttttttgag atggagtttt  11640 

gctcttgttg cccaggctgc agtgcaatgg cgctatctca gctcactgca acctctgcct  11700 

cctaggttca agtgattctc ctgcctcagc ctcccgagta gctgggatta caggtgccca  11760 

ccaccacacc cggctaattt ttttgtattt ttaatagaga cagggtttcc acatgttgac  11820 

caggctggtc tcgaactcct gacctccagt gatccgcccg cctcggcctc ccaaagtgct  11880 

agattacagg tgtgagccac tgcacctgtc cctggctgtc tgtatattta cttttttttt  11940 

tgagatggag tttcgctctt gtcacccagg ctgcagtgca atggtgcgat ctcggctcat  12000 

tgtaacctct gcctcccagg ttcaggtgat tctcctgcct cagtctcccg agtagctggg  12060 

attacaggcg tccgctacca cgcccgactg atttttctat ttttagtaga gacggggttt  12120 

caacatgttg gccagtctga tctcgaactc ctgacctcag gtgattcacc cacctcagcc  12180 

tcccaaagtg ctgggattac aggtatgagg cactgtgccc ggcttttttt tttttttttt  12240 

ttttcttcag acaagagtct tactctgtca cccaggctga agtgcagtgg tgcaatcttg  12300 

gctcactgca acctccgcct cccaggttca agcgattctt ctgcctcagc ctccatagta  12360 

gctgggacta caggtgtgtg ccaccacgcc cagctaattt ttatattttt atttagtaga  12420 

gacaaggttt caccatgttg gccaagctgg tctcgaactc ctgacctcaa gtgatctgcc  12480 

cgcctcagcc tcccaaagtg ctgggattac aggtgtgagc cgtggcaccc agcccagcct  12540 

tattctttta aacaatctga caatctctgc ctttagttgg tctgtttaat ccatttccat  12600 

ttaatggttg gagttaagtc tatcatcttg ttatttgttt tctattaccc catctgtttt  12660 

gacttttgga ttaattacat atttctggga ttctgttttt tctctgctat tggcttggtc  12720 

gctctagtaa ttcagtgaga ctgctggttc cgctcaggcc cctttgctga accatggtgt  12780 

gaaagtgcct ccaggcagaa actcagggta cttgtaaggc tcaccttctt tgttttctct  12840 

ctggtcacag ccctgcacag cctattgtcc gatatctaaa aatagttgcc cagtgtttta  12900 

ggtgtttaca actggcatca gttattccac tgtggccaga attgcaagtt tctcctcttt  12960 

tctgaggact tcttcactca taatgtcacc cgacatgatc                        13000 

 
           
             12  
             754  
             DNA  
             H. sapiens  
             
               unsure  
               625  
               unknown  
             
           
            12 

ggagaccttg gctggtcaga gggctgtgaa gacgcacggt gccaggacca agtatctgaa     60 

agacctggtg gaagaggagg ctgaggaggc tggagtggct ttgagaagca cccagagcac    120 

actgcaagca ggtctggctg cagatgcctg ggctgctccc atcgccatgc agatctacaa    180 

gaagcgcctg ggccagctgg cctgctgctg cctgcaccgc cgggccaaaa ggaggcctcc    240 

tatgacccag gtgtacgaga ggctagagaa gctgcaggca gtggtggcgg gggtgcccgg    300 

gcatttggag gccgccagct gcatcccccc ttccccgcag gagaactcct acgtgtccag    360 

cactggcaga gcccacagtg gggctgctcc atggcagccc ctggcagcgc catcaggagc    420 

cagtgcccag gcagcagagc agctgcagag aggccccaac cagcccgtgg agagtgacga    480 

gagcctaggc ggcctctctg ctgccctgcg ctcctggcac ttgactccaa gctgccctct    540 

ggacccagca cccctcaggc aggccggctg tcctcagggg gacacggcag gagaatcgag    600 

ctgggggagt ggcccaggat cccgngccac agccgtggaa ggactggtcc ttggcagctc    660 

tgcatcatcg tcgtcagagc caccgcagat tatcatcaac cctgcccgac agaagatggt    720 

ccagaagctg gccctgtacg aggatggtgc cctg                                754 

 
           
             13  
             577  
             DNA  
             H. sapiens  
             
 
           
            13 

gaggccgagg cctggagccc ccggaagttg ccatcctcag ccyccacctt cctctcccca     60 

gcttttccag gctcccagac ccattcaggg cctgagctcg gcctggttcc aagccctgct    120 

tccctgtggc ctccaccgcc atctccagcc tgggtgacag agcaagaccc tgtttcaaaa    180 

agaaaaaccc tgggaaaagt gaagtatggc tgtaagtctc atggttcagt cctagcaaga    240 

agcgagaatt ctgagatcct ccagaaagtc gagcagcacc cacctccaac ctcgggccag    300 

tgtcttcagg ctttactggg gacctgcgag ctggcctaat gtggtggcct gcaagccagg    360 

ccatccctgg gcgccacaga cgagctccga gccaggtcag gcttcggagg ccacaagctc    420 

agcctcaggc ccaggcactg attgtggcag aggggccact acccaaggtc tagctaggcc    480 

caagacctag ttacccagac agtgagaagc ccctggaagg cagaaaagtt gggagcatgg    540 

cagacaggga agggaaamat tttcagggaa aagacat                             577 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

cctggcttgc aggccaccac                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

gatgccagcc ttccttgccc                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

cagtggagac ggtcctccag                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

cttgtggcct ccgaagcctg                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

ggacgacagc agctgcaggc                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

tctgcagcca gacctgcttg                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

agccagacct gcttgcagtg                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

gtgaagcctg tgctacagcc                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

tggcaccagt cggcgggctc                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

gaccatcttc tgtcgggcag                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

ccagccttcc cgtcttgatg                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

agccagcagc ctcccaacat                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

tcagctctga aattcatcac                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

cagtccttcc acggctgtgg                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

ccaaggtctc tagcactacc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

ctaggtcttg ggcctagcta                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

acaccgtgtt cctcatcacc                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

ctctgaaatt catcactttc                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

tgggtcatag gaggcctcct                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

gctcggagct cgtctgtggc                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

tggcaccgtg cgtcttcaca                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

cggcacatga cccagggcgg                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

tgaccagcca aggtctctag                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

ctcctcttcc accaggtctt                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

tgaccatgag aactttgact                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

aagggccagt ccttccacgg                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

ctggacacgt aggagttctc                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

tgctggacac gtaggagttc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

accatcttct gtcgggcagg                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

cttgcagtgt gctctgggtg                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

ctggacagct gctccacctc                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

acagccctgg gctacttttg                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

ggaggcaggg ttccactctg                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

tgcagccaga cctgcttgca                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

cttgggtagt ggcccctctg                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

ggccagtcct tccacggctg                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

tctgagcaca gtagccagca                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

cggagcagct gcaggtgcgt                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

aagcagggct tggaaccagg                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

cgatcttgag ctcctccgag                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

cacctcggtc aggaagctct                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

caggaagccg tacaccaggc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

actgtctgtg tccgggccac                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

cagctggccc aggcccaggc                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

gcccggcggt gcaggcagca                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

gcttctctag cctctcgtac                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

ggctgccatg gagcagcccc                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

gcagctgctc tgctgcctgg                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

agagaggccg cctaggctct                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

cagagggcag cttggagtca                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

agggccagct tctggaccat                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

ggtgaacaca tcagctctga                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

agctgctgcc agaggcctgg                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

ttacagccat acttcacttt                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

aattctcgct tcttgctagg                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

ggccagctcg caggtcccca                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

ccttccctgt ctgccatgct                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

acgcaagagg acactcggtt                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

ggctgaacac aaaatcactg                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

tgactcacgg ctgaacacaa                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

atacgttttt attactcaag                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

agggaacata cgtttttatt                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

cctggaaaag cttcataaag                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

ctggcctcac ctggacagct                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

agaccctcca gctacgctgc                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

ggagagccca cttgaagaca                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

agctggctac ctgggtcata                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

tacagagcaa ggcctggaat                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

tccttctctc tatgtgaagg                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

aggcccaagc ctacagaagg                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

aggccagctg gcccaggcgc                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

acccaggctg gagatggcgg                                                 20 

 
           
             86  
             20  
             DNA  
             H. sapiens  
             
 
           
            86 

gtggtggcct gcaagccagg                                                 20 

 
           
             87  
             20  
             DNA  
             H. sapiens  
             
 
           
            87 

gggcaaggaa ggctggcatc                                                 20 

 
           
             88  
             20  
             DNA  
             H. sapiens  
             
 
           
            88 

ctggaggacc gtctccactg                                                 20 

 
           
             89  
             20  
             DNA  
             H. sapiens  
             
 
           
            89 

caggcttcgg aggccacaag                                                 20 

 
           
             90  
             20  
             DNA  
             H. sapiens  
             
 
           
            90 

gcctgcagct gctgtcgtcc                                                 20 

 
           
             91  
             20  
             DNA  
             H. sapiens  
             
 
           
            91 

caagcaggtc tggctgcaga                                                 20 

 
           
             92  
             20  
             DNA  
             H. sapiens  
             
 
           
            92 

cactgcaagc aggtctggct                                                 20 

 
           
             93  
             20  
             DNA  
             H. sapiens  
             
 
           
            93 

ggctgtagca caggcttcac                                                 20 

 
           
             94  
             20  
             DNA  
             H. sapiens  
             
 
           
            94 

gagcccgccg actggtgcca                                                 20 

 
           
             95  
             20  
             DNA  
             H. sapiens  
             
 
           
            95 

ctgcccgaca gaagatggtc                                                 20 

 
           
             96  
             20  
             DNA  
             H. sapiens  
             
 
           
            96 

catcaagacg ggaaggctgg                                                 20 

 
           
             97  
             20  
             DNA  
             H. sapiens  
             
 
           
            97 

atgttgggag gctgctggct                                                 20 

 
           
             98  
             20  
             DNA  
             H. sapiens  
             
 
           
            98 

gtgatgaatt tcagagctga                                                 20 

 
           
             99  
             20  
             DNA  
             H. sapiens  
             
 
           
            99 

ccacagccgt ggaaggactg                                                 20 

 
           
             100  
             20  
             DNA  
             H. sapiens  
             
 
           
            100 

ggtagtgcta gagaccttgg                                                 20 

 
           
             101  
             20  
             DNA  
             H. sapiens  
             
 
           
            101 

tagctaggcc caagacctag                                                 20 

 
           
             102  
             20  
             DNA  
             H. sapiens  
             
 
           
            102 

ggtgatgagg aacacggtgt                                                 20 

 
           
             103  
             20  
             DNA  
             H. sapiens  
             
 
           
            103 

aggaggcctc ctatgaccca                                                 20 

 
           
             104  
             20  
             DNA  
             H. sapiens  
             
 
           
            104 

tgtgaagacg cacggtgcca                                                 20 

 
           
             105  
             20  
             DNA  
             H. sapiens  
             
 
           
            105 

ccgccctggg tcatgtgccg                                                 20 

 
           
             106  
             20  
             DNA  
             H. sapiens  
             
 
           
            106 

aagacctggt ggaagaggag                                                 20 

 
           
             107  
             20  
             DNA  
             H. sapiens  
             
 
           
            107 

agtcaaagtt ctcatggtca                                                 20 

 
           
             108  
             20  
             DNA  
             H. sapiens  
             
 
           
            108 

gagaactcct acgtgtccag                                                 20 

 
           
             109  
             20  
             DNA  
             H. sapiens  
             
 
           
            109 

gaactcctac gtgtccagca                                                 20 

 
           
             110  
             20  
             DNA  
             H. sapiens  
             
 
           
            110 

cctgcccgac agaagatggt                                                 20 

 
           
             111  
             20  
             DNA  
             H. sapiens  
             
 
           
            111 

cacccagagc acactgcaag                                                 20 

 
           
             112  
             20  
             DNA  
             H. sapiens  
             
 
           
            112 

gaggtggagc agctgtccag                                                 20 

 
           
             113  
             20  
             DNA  
             H. sapiens  
             
 
           
            113 

caaaagtagc ccagggctgt                                                 20 

 
           
             114  
             20  
             DNA  
             H. sapiens  
             
 
           
            114 

cagagtggaa ccctgcctcc                                                 20 

 
           
             115  
             20  
             DNA  
             H. sapiens  
             
 
           
            115 

tgcaagcagg tctggctgca                                                 20 

 
           
             116  
             20  
             DNA  
             H. sapiens  
             
 
           
            116 

cagaggggcc actacccaag                                                 20 

 
           
             117  
             20  
             DNA  
             H. sapiens  
             
 
           
            117 

cagccgtgga aggactggcc                                                 20 

 
           
             118  
             20  
             DNA  
             H. sapiens  
             
 
           
            118 

tgctggctac tgtgctcaga                                                 20 

 
           
             119  
             20  
             DNA  
             H. sapiens  
             
 
           
            119 

acgcacctgc agctgctccg                                                 20 

 
           
             120  
             20  
             DNA  
             H. sapiens  
             
 
           
            120 

cctggttcca agccctgctt                                                 20 

 
           
             121  
             20  
             DNA  
             H. sapiens  
             
 
           
            121 

ctcggaggag ctcaagatcg                                                 20 

 
           
             122  
             20  
             DNA  
             H. sapiens  
             
 
           
            122 

agagcttcct gaccgaggtg                                                 20 

 
           
             123  
             20  
             DNA  
             H. sapiens  
             
 
           
            123 

gcctggtgta cggcttcctg                                                 20 

 
           
             124  
             20  
             DNA  
             H. sapiens  
             
 
           
            124 

gtggcccgga cacagacagt                                                 20 

 
           
             125  
             20  
             DNA  
             H. sapiens  
             
 
           
            125 

tgctgcctgc accgccgggc                                                 20 

 
           
             126  
             20  
             DNA  
             H. sapiens  
             
 
           
            126 

gtacgagagg ctagagaagc                                                 20 

 
           
             127  
             20  
             DNA  
             H. sapiens  
             
 
           
            127 

ccaggcagca gagcagctgc                                                 20 

 
           
             128  
             20  
             DNA  
             H. sapiens  
             
 
           
            128 

agagcctagg cggcctctct                                                 20 

 
           
             129  
             20  
             DNA  
             H. sapiens  
             
 
           
            129 

atggtccaga agctggccct                                                 20 

 
           
             130  
             20  
             DNA  
             H. sapiens  
             
 
           
            130 

ccaggcctct ggcagcagct                                                 20 

 
           
             131  
             20  
             DNA  
             H. sapiens  
             
 
           
            131 

aaagtgaagt atggctgtaa                                                 20 

 
           
             132  
             20  
             DNA  
             H. sapiens  
             
 
           
            132 

cctagcaaga agcgagaatt                                                 20 

 
           
             133  
             20  
             DNA  
             H. sapiens  
             
 
           
            133 

tggggacctg cgagctggcc                                                 20 

 
           
             134  
             20  
             DNA  
             H. sapiens  
             
 
           
            134 

agcatggcag acagggaagg                                                 20 

 
           
             135  
             20  
             DNA  
             H. sapiens  
             
 
           
            135 

aaccgagtgt cctcttgcgt                                                 20 

 
           
             136  
             20  
             DNA  
             H. sapiens  
             
 
           
            136 

cagtgatttt gtgttcagcc                                                 20 

 
           
             137  
             20  
             DNA  
             H. sapiens  
             
 
           
            137 

ttgtgttcag ccgtgagtca                                                 20 

 
           
             138  
             20  
             DNA  
             H. sapiens  
             
 
           
            138 

cttgagtaat aaaaacgtat                                                 20 

 
           
             139  
             20  
             DNA  
             H. sapiens  
             
 
           
            139 

tgtcttcaag tgggctctcc                                                 20 

 
           
             140  
             20  
             DNA  
             H. sapiens  
             
 
           
            140 

attccaggcc ttgctctgta                                                 20 

 
           
             141  
             20  
             DNA  
             H. sapiens  
             
 
           
            141 

ccttcacata gagagaagga                                                 20 

 
           
             142  
             20  
             DNA  
             H. sapiens  
             
 
           
            142 

ccgccatctc cagcctgggt                                                 20