Patent Publication Number: US-2004043957-A1

Title: Antisense modulation of urokinase plasminogen activator expression

Description:
INTRODUCTION  
     [0001] This application is a continuation of U.S. Ser. No. 09/821,972 filed Mar. 30, 2001, which is herein incorporated by reference in its entirety. 
    
    
     
       FIELD OF THE INVENTION  
       [0002] The present invention provides compositions and methods for modulating the expression of urokinase plasminogen activator. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding urokinase plasminogen activator. Such compounds have been shown to modulate the expression of urokinase plasminogen activator.  
       BACKGROUND OF THE INVENTION  
       [0003] Cell-cell and cell-extracellular matrix interactions provide cells with information essential for controlling morphogenesis, cell fate specification, gain or loss of tissue specific functions, cell migration, tissue repair and cell death. By concentrating proteolytic events at or near the cell surface, these processes can be very effective even in the presence of high concentrations of inhibitors (Werb,  Cell,  1997, 91, 439-442.).  
       [0004] An important serine protease involved in matrix degradation and integral member of the plasminogen activation system is urokinase plasminogen activator (uPA, also known as PLAU and URK). The gene for human uPA was first isolated and sequenced in 1985 (Riccio et al.,  Nucleic Acids Res.,  1985, 13, 2759-2771). It has since been demonstrated that uPA acts as a specific trigger in the plasminogen proteolytic cascade (Andreasen et al.,  Cell Mol Life Sci.,  2000, 57, 25-40.). Its immediate target is plasminogen, an inactive protease precursor. When uPA specifically cleaves plasminogen, an active protease known as plasmin is generated. Plasmin has a broad specificity for matrix degradation and cleaves a variety of proteins such as fibrin, fibronectin and laminin (Parry et al.,  Trends Biochem. Sci.,  2000, 25, 53-59.).  
       [0005] uPA has a specific cell-surface receptor (uPA-R, also known as CD87) which serves to localize uPA to specific regions on migrating cell surfaces (Schmitt et al.,  Thromb. Haemost.,  1997, 78, 285-296). During tumor invasion and metastasis, penetrating tumor cells take advantage of the uPA/uPA-R interaction to focus proteolytic activity on the points of invasion. The uPA/uPA-R system is one of the strongest cancer prognostic markers described to date. Strong prognostic value to predict disease recurrence and overall survival has been documented for patients with cancer of the breast, ovary, cervix, endometrium, stomach, colon, lung, bladder, kidney, brain, and soft tissue (Schmitt et al.,  Thromb. Haemost.,  1997, 78, 285-296). This has prompted clinicians and basic researchers to explore new tumor biology-oriented concepts in order to suppress expression and synthesis of factors of the plasminogen activation system (Schmitt et al.,  Thromb. Haemost.,  1997, 78, 285-296).  
       [0006] Various approaches to interfere with the expression and reactivity of uPA or its receptor uPA-R at the gene and protein level have been investigated. These include antibodies, soluble recombinant uPA or uPA-R analogues, synthetic inhibitors and antisense oligonucleotides and vectors targeting uPA and/or uPA-R (Schmitt et al.,  Thromb. Haemost.,  1997, 78, 285-296).  
       [0007] Expression of human uPA in murine B16-F1 cells transfected with the human gene for the prepro-form of urokinase has been inhibited by transfection with a plasmid containing 265 nucleotides of the 5′ end of preprourokinase in the antisense direction (Yu and Schultz,  Cancer Res.,  1990, 50, 7623-7633). Antisense inhibition of uPA was also demonstrated in a human osteosarcoma cell line by transfection with a vector encoding 1,021 bases of the 3′ end of uPA cDNA in antisense orientation (Haeckel et al.,  Verh. Dtsch. Ges. Pathol.,  1998, 82, 170-177).  
       [0008] uPA expression has been inhibited in quail ( Coturnix coturnix japonica ) embryonic endocardial-derived mesenchymal cells in vitro by a 15-mer antisense phosphorothioate oligonucleotide targeting and centered on the initiation codon of uPA mRNA (McGuire and Alexander,  Development,  1993, 118, 931-939). Furthermore, similar oligonucleotides have been shown to suppress uPA expression in human ovarian cancer cells (Wilhelm et al.,  Clin. Exp. Metastasis,  1995, 13, 296-302). In this study, 16- and 18-mer antisense phosphorothioate oligonucleotides targeting the start codon of human uPA were used.  
       [0009] An antisense phosphorothioate oligodeoxynucleotide targeting the first seven codons (including the AUG start codon) of human uPA was shown to cause a decrease in uPA expression in four human gliboblastoma cell lines and the C6 rat cell line (Engelhard,  Cancer Control,  1998, 5, 163-170). A longer 22-mer antisense oligonucleotide targeting bases 368-389 of human uPA mRNA caused inhibition of uPA expression in three human esophageal carcinoma cell lines (Morrissey et al.,  Clin. Exp. Metastasis,  1999, 17, 77-85). The expression of human uPA was inhibited in human colon cancer cells with a 19-mer antisense oligonucleotide containing two phosphorothioate modifications at each end and targeting bases 426-444 of human uPA cDNA (Wilson and Gibson, Gut, 2000, 47, 105-111).  
       [0010] Disclosed in U.S. Pat. No. 5,552,390 are antisense oligonucleotides targeting human and murine uPA as well as methods of using same to inhibit tumor invasion and metastasis (Scholar and Iversen, 1996).  
       [0011] Antisense technology is emerging as an effective means of reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic and research applications involving modulation of uPA expression.  
       [0012] The present invention provides compositions and methods for modulating uPA expression.  
       SUMMARY OF THE INVENTION  
       [0013] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding urokinase plasminogen activator, and which modulate the expression of urokinase plasminogen activator. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of urokinase plasminogen activator 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 urokinase plasminogen activator 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  
       [0014] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding urokinase plasminogen activator, ultimately modulating the amount of urokinase plasminogen activator produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding urokinase plasminogen activator. As used herein, the terms “target nucleic acid” and “nucleic acid encoding urokinase plasminogen activator” encompass DNA encoding urokinase plasminogen activator, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of urokinase plasminogen activator. 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.  
       [0015] 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 urokinase plasminogen activator. 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 urokinase plasminogen activator, regardless of the sequence(s) of such codons.  
       [0016] 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.  
       [0017] 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.  
       [0018] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. It has also been found that introns can also be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0019] 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.  
       [0020] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.  
       [0021] Antisense and other compounds of the invention which hybridize to the target and inhibit expression of the target are identified through experimentation, and the sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The target sites to which these preferred sequences are complementary are hereinbelow referred to as “active sites” and are therefore preferred sites for targeting. Therefore another embodiment of the invention encompasses compounds which hybridize to these active sites.  
       [0022] 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.  
       [0023] 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.  
       [0024] 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.  
       [0025] 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).  
       [0026] 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.  
       [0027] 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.  
       [0028] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 50 nucleobases (i.e. from about 8 to about 50 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.  
       [0029] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.  
       [0030] 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.  
       [0031] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates 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 21 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.  
       [0032] 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.  
       [0033] 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.  
       [0034] 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.  
       [0035] 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.  
       [0036] 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.  
       [0037] 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 )ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O-CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta,  1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples hereinbelow.  
       [0038] 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.  
       [0039] Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.  
       [0040] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in  The Concise Encyclopedia Of Polymer Science And Engineering,  pages 858-859, Kroschwitz, J. I., ed. John Wiley &amp; Sons, 1990, those disclosed by Englisch et al.,  Angewandte Chemie,  International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15 , Antisense Research and Applications,  pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 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.  
       [0041] 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.  
       [0042] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res.,  1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &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.  
       [0043] 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.  
       [0044] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.  
       [0045] 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.  
       [0046] 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.  
       [0047] The antisense compounds of the invention are synthesized in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.  
       [0048] 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.  
       [0049] 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.  
       [0050] 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.  
       [0051] 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.  
       [0052] 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.  
       [0053] 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.  
       [0054] 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 urokinase plasminogen activator 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.  
       [0055] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding urokinase plasminogen activator, 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 urokinase plasminogen activator 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 urokinase plasminogen activator in a sample may also be prepared.  
       [0056] 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.  
       [0057] 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.  
       [0058] 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, sodium glycodihydrofusidate,. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications 08/886,829 (filed Jul. 1, 1997), 09/108,673 (filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624 (filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of which is incorporated herein by reference in their entirety.  
       [0059] 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.  
       [0060] 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.  
       [0061] 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.  
       [0062] 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.  
       [0063] 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.  
       [0064] Emulsions  
       [0065] 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 of two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be either water-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous provides an o/w/o emulsion.  
       [0066] 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).  
       [0067] 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).  
       [0068] 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.  
       [0069] 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).  
       [0070] 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.  
       [0071] 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.  
       [0072] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of reasons of ease of formulation, efficacy from an absorption and bioavailability standpoint. (Rosoff, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in  Pharmaceutical Dosage Forms,  Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.  
       [0073] 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).  
       [0074] 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.  
       [0075] 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.  
       [0076] 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.  
       [0077] 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.  
       [0078] Liposomes  
       [0079] 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.  
       [0080] 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.  
       [0081] 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.  
       [0082] 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.  
       [0083] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes. As the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.  
       [0084] 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.  
       [0085] 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.  
       [0086] 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).  
       [0087] 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).  
       [0088] 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.  
       [0089] 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).  
       [0090] 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).  
       [0091] 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).  
       [0092] 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.).  
       [0093] 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.  
       [0094] 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.  
       [0095] 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.  
       [0096] 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).  
       [0097] 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.  
       [0098] 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.  
       [0099] 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.  
       [0100] 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.  
       [0101] 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).  
       [0102] Penetration Enhancers  
       [0103] 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.  
       [0104] 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.  
       [0105] 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).  
       [0106] 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).  
       [0107] 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).  
       [0108] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett,  J. Chromatogr.,  1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; Buur et al.,  J. Control Rel.,  1990, 14, 43-51).  
       [0109] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,  J. Pharm. Pharmacol.,  1987, 39, 621-626).  
       [0110] 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.  
       [0111] 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.  
       [0112] Carriers  
       [0113] 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).  
       [0114] Excipients  
       [0115] 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.).  
       [0116] 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.  
       [0117] 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.  
       [0118] 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.  
       [0119] Other Components  
       [0120] 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.  
       [0121] 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.  
       [0122] 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.  
       [0123] 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.  
       [0124] 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.  
       [0125] 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  
     [0126] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0127] 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, the standard cycle for unmodified oligonucleotides was utilized, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds.  
     [0128] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me—C) 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.).  
     [0129] 2′-Fluoro Amidites  
     [0130] 2′-Fluorodeoxyadenosine Amidites  
     [0131] 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. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and by modifying literature procedures whereby the 2′-alpha-fluoro atom is introduced by a S N 2-displacement of a 2′-beta-trityl 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 and standard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.  
     [0132] 2′-Fluorodeoxyguanosine  
     [0133] 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 diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give diisobutyryl 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.  
     [0134] 2′-Fluorouridine  
     [0135] 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.  
     [0136] 2′-Fluorodeoxycytidine  
     [0137] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0138] 2′-O-(2-Methoxyethyl) Modified Amidites  
     [0139] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared as follows, or alternatively, as per the methods of Martin, P.,  Helvetica Chimica Acta,  1995, 78, 486-504.  
     [0140] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine] 
     [0141] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for 24 h) to give a solid that was crushed to a light tan powder (57 g, 85% crude yield). The NMR spectrum was consistent with the structure, contaminated with phenol as its sodium salt (ca. 5%). The material was used as is for further reactions (or it can be purified further by column chromatography using a gradient of methanol in ethyl acetate (10-25%) to give a white solid, mp 222-4° C.)  
     [0142] 2′-O-Methoxyethyl-5-methyluridine  
     [0143] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160° C. After heating for 48 hours at 155-160° C., the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH 3 CN (600 mL) and evaporated. A silica gel column (3 kg) was packed in CH 2 Cl 2 /acetone/MeOH (20:5:3) containing 0.5% Et 3 NH. The residue was dissolved in CH 2 Cl 2  (250 mL) and adsorbed onto silica (150 g) prior to loading onto the column. The product was eluted with the packing solvent to give 160 g (63%) of product. Additional material was obtained by reworking impure fractions.  
     [0144] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine  
     [0145] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH 3 CN (200 mL) The residue was dissolved in CHCl 3  (1.5 L) and extracted with 2×500 mL of saturated NaHCO 3  and 2×500 mL of saturated NaCl. The organic phase was dried over Na 2 SO 4 , filtered and evaporated. 275 g of residue was obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et 3 NH. The pure fractions were evaporated to give 164 g of product. Approximately 20 g additional was obtained from the impure fractions to give a total yield of 183 g (57%).  
     [0146] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine  
     [0147] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by TLC by first quenching the TLC sample with the addition of MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL) was added and the mixture evaporated at 35° C. The residue was dissolved in CHCl 3  (800 mL) and extracted with 2×200 mL of saturated sodium bicarbonate and 2×200 mL of saturated NaCl. The water layers were back extracted with 200 mL of CHCl 3 . The combined organics were dried with sodium sulfate and evaporated to give 122 g of residue (approx. 90% product). The residue was purified on a 3.5 kg silica gel column and eluted using EtOAc/hexane(4:1). Pure product fractions were evaporated to yield 96 g (84%). An additional 1.5 g was recovered from later fractions.  
     [0148] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine  
     [0149] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH 3 CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M) in CH 3 CN (1 L), cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl 3  was added dropwise, over a 30 minute period, to the stirred solution maintained at 0-10° C., and the resulting mixture stirred for an additional 2 hours. The first solution was added dropwise, over a 45 minute period, to the latter solution. The resulting reaction mixture was stored overnight in a cold room. Salts were filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solids were removed by filtration. The filtrate was washed with 1×300 mL of NaHCO 3  and 2×300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.  
     [0150] 2′-O-Methoxyethyl-51-O-dimethoxytrityl-5-methylcytidine  
     [0151] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH 4 OH (30 mL) was stirred at room temperature for 2 hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2×200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. MeOH (400 mL) saturated with NH 3  gas was added and the vessel heated to 100° C. for 2 hours (TLC showed complete conversion). The vessel contents were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated to give 85 g (95%) of the title compound.  
     [0152] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine  
     [0153] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, TLC showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl 3  (700 mL) and extracted with saturated NaHCO 3  (2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO 4  and evaporated to give a residue (96 g). The residue was chromatographed on a 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et 3 NH as the eluting solvent. The pure product fractions were evaporated to give 90 g (90%) of the title compound.  
     [0154] N4-Benzoyl-21-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite  
     [0155] N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in CH 2 Cl 2  (1 L) Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) were added with stirring, under a nitrogen atmosphere. The resulting mixture was stirred for 20 hours at room temperature (TLC showed the reaction to be 95% complete). The reaction mixture was extracted with saturated NaHCO 3  (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes were back-extracted with CH 2 Cl 2  (300 mL), and the extracts were combined, dried over MgSO 4  and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) as the eluting solvent. The pure fractions were combined to give 90.6 g (87%) of the title compound.  
     [0156] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0157] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0158] 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.  
     [0159] 5′-O-tert-Butyldiphenylsilyl-O 2 -2′-anhydro-5-methyluridine  
     [0160] 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, ethyl acetate) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) and the solution was cooled to −10° C. The resulting crystalline product was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMR were consistent with pure product.  
     [0161] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0162] In a 2 L stainless steel, unstirred pressure reactor was added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) was added cautiously at first until the evolution of hydrogen gas subsided. 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 and opened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T side product, ethyl acetate) indicated about 70% conversion to the product. In order to avoid additional side product formation, the reaction was stopped, 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 low boiling solvent is gone, the remaining solution can be partitioned between ethyl acetate and water. The product will be in the organic phase.] The residue was purified by column chromatography (2 kg silica gel, ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, stripped and dried to product as a white crisp foam (84 g, 50%), contaminated starting material (17.4 g) and pure reusable starting material 20 g. The yield based on starting material less pure recovered starting material was 58%. TLC and NMR were consistent with 99% pure product.  
     [0163] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0164] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (209, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.249, 44.36 mmol). It was then dried over P 2 O 5  under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture. The rate of addition is maintained such that resulting deep red coloration is just discharged before adding the next drop. After the addition was complete, the reaction was stirred for 4 hrs. By that time TLC showed the completion of the reaction (ethylacetate:hexane, 60:40). The solvent was evaporated in vacuum. Residue obtained was placed on a flash column and eluted with ethyl acetate:hexane (60:40), to get 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%).  
     [0165] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0166] 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 was washed with ice cold CH 2 Cl 2  and the combined organic phase was washed with water, brine and dried over anhydrous Na 2 SO 4 . The solution was concentrated to get 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was strirred for 1 h. Solvent was removed under vacuum; residue chromatographed to get 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%).  
     [0167] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine  
     [0168] 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). Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at 10° C. under inert atmosphere. The reaction mixture was stirred for 10 minutes at 10° C. After that the reaction vessel was removed from the ice bath and stirred at room temperature for 2 h, the reaction monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20 mL). Ethyl acetate phase was dried over anhydrous Na 2 SO 4 , evaporated to dryness. Residue was dissolved in a solution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reaction mixture was stirred at room temperature for 10 minutes. Reaction mixture cooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reaction mixture stirred at 10° C. for 10 minutes. After 10 minutes, the reaction mixture was removed from the ice bath and stirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO 3  (25 mL) solution was added and extracted with ethyl acetate (2×25 mL). Ethyl acetate layer was dried over anhydrous Na 2 SO 4  and evaporated to dryness. The residue obtained was purified by flash column chromatography and eluted with 5% MeOH in CH 2 Cl 2  to get 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%).  
     [0169] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0170] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF was then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Solvent was removed under vacuum and the residue placed on a flash column and eluted with 10% MeOH in CH 2 Cl 2  to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).  
     [0171] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0172] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 5  under high vacuum overnight at 40° C. It was then co-evaporated with anhydrous pyridine (20 mL). The residue obtained was dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the mixture and the reaction mixture was stirred at room temperature until all of the starting material disappeared. Pyridine was removed under vacuum and the residue chromatographed and eluted with 10% MeOH in CH 2 Cl 2  (containing a few drops of pyridine) to get 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).  
     [0173] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0174] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and dried over P 2 O 5  under high vacuum overnight at 40° C. Then the reaction mixture 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 hrs under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated, then the residue was dissolved in ethyl acetate (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). Ethyl acetate layer was dried over anhydrous Na 2 SO 4  and concentrated. Residue obtained was chromatographed (ethyl acetate as eluent) to get 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%).  
     [0175] 2′-(Aminooxyethoxy) Nucleoside Amidites  
     [0176] 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.  
     [0177] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0178] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may 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].  
     [0179] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0180] 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.  
     [0181] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine  
     [0182] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the solid dissolves. O 2 -, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oil bath and heated to 155° C. for 26 hours. The bomb is cooled to room temperature and opened. The crude solution is concentrated and the residue partitioned between water (200 mL) and hexanes (200 mL). The excess phenol is extracted into the hexane layer. The aqueous layer is extracted with ethyl acetate (3×200 mL) and the combined organic layers are washed once with water, dried over anhydrous sodium sulfate and concentrated. The residue is columned on silica gel using methanol/methylene chloride 1:20 (which has 2% triethylamine) as the eluent. As the column fractions are concentrated a colorless solid forms which is collected to give the title compound as a white solid.  
     [0183] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl Uridine  
     [0184] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reaction mixture is poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers are washed with saturated NaHCO 3  solution, followed by saturated NaCl solution and dried over anhydrous sodium sulfate. Evaporation of the solvent followed by silica gel chromatography using MeOH:CH 2 Cl 2 :Et 3 N (20:1, v/v, with 1% triethylamine) gives the title compound.  
     [0185] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0186] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are 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 is stirred overnight and the solvent evaporated. The resulting residue is purified by silica gel flash column chromatography with ethyl acetate as the eluent to give the title compound.  
     Example 2  
     [0187] Oligonucleotide Synthesis  
     [0188] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine.  
     [0189] Phosphorothioates (P═S) are synthesized as for the phosphodiester oligonucleotides except the standard oxidation bottle was replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation wait step was increased to 68 sec and was followed by the capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides were purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.  
     [0190] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.  
     [0191] 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.  
     [0192] Phosphoramidite oligonucleotides are prepared as described in U.S. Patent, 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0193] 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.  
     [0194] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0195] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0196] 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  
     [0197] Oligonucleoside Synthesis  
     [0198] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethyl-hydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked 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.  
     [0199] 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.  
     [0200] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0201] PNA Synthesis  
     [0202] 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  
     [0203] Synthesis of Chimeric Oligonucleotides  
     [0204] 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”.  
     [0205] [2′-O—Me]—[2′-deoxy]—[2′-O—Me] Chimeric Phosphorothioate Oligonucleotides  
     [0206] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 380B, 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 increasing the wait step after the delivery of tetrazole and base to 600 s repeated four times for RNA and twice for 2′-o-methyl. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Treatment in methanolic ammonia for 24 hrs at room temperature is then done to deprotect all bases and sample was again lyophilized to dryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at room temperature to deprotect the 2′ positions. The reaction is then quenched with 1M TEAA and the sample is then reduced to ½ volume by rotovac before being desalted on a G25 size exclusion column. The oligo recovered is then analyzed spectrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0207] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0208] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)] 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.  
     [0209] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0210] [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, oxidization 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.  
     [0211] 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  
     [0212] Oligonucleotide Isolation  
     [0213] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides or oligonucleosides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by  31 P nuclear magnetic resonance spectroscopy, and 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  
     [0214] Oligonucleotide Synthesis—96 Well Plate Format  
     [0215] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a standard 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-cyanoethyldiisopropyl 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 known literature or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.  
     [0216] 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  
     [0217] Oligonucleotide Analysis—96 Well Plate Format  
     [0218] 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  
     [0219] Cell Culture and Oligonucleotide Treatment  
     [0220] 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 5 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.  
     [0221] T-24 Cells:  
     [0222] 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 (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.  
     [0223] 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.  
     [0224] A549 Cells:  
     [0225] 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 (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.  
     [0226] NHDF Cells:  
     [0227] 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.  
     [0228] HEK Cells:  
     [0229] 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.  
     [0230] b.END Cells:  
     [0231] The mouse brain endothelial cell line b.END was obtained from Dr. Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cells were routinely cultured in DMEM, high glucose (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000 cells/well for use in RT-PCR analysis.  
     [0232] For Northern blotting or other analyses, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.  
     [0233] Treatment with Antisense Compounds:  
     [0234] When cells reached 80% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Gibco BRL) 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.  
     [0235] 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 ISIS 13920, TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to human H-ras. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, 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.  
     Example 10  
     [0236] Analysis of Oligonucleotide Inhibition of Urokinase Plasminogen Activator Expression  
     [0237] Antisense modulation of urokinase plasminogen activator expression can be assayed in a variety of ways known in the art. For example, urokinase plasminogen activator 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. 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.  
     [0238] Protein levels of urokinase plasminogen activator 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 urokinase plasminogen activator 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.  
     [0239] 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  
     [0240] Poly(A)+ mRNA Isolation  
     [0241] 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.  
     [0242] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0243] Total RNA Isolation  
     [0244] 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 mL cold PBS. 100 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 100 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 15 seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum again applied for 15 seconds. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 15 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 10 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 60 μL water into each well, incubating 1 minute, and then applying the vacuum for 30 seconds. The elution step was repeated with an additional 60 μL water.  
     [0245] 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  
     [0246] Real-time Quantitative PCR Analysis of Urokinase Plasminogen Activator mRNA Levels  
     [0247] Quantitation of urokinase plasminogen activator mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;s instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR, in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., JOE, FAM, or VIC, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) 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.  
     [0248] 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.  
     [0249] PCR reagents were obtained from PE-Applied Biosystems, Foster City, Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail (1×TAQMAN™ buffer A, 5.5 mM MgCl 2 , 300 μM each of DATP, dCTP and dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer, and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5 Units MuLV reverse transcriptase) to 96 well plates containing 25 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 AMPLITAQ GOLD™, 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).  
     [0250] 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.  
     [0251] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 25 uL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0252] Probes and primers to human urokinase plasminogen activator were designed to hybridize to a human urokinase plasminogen activator sequence, using published sequence information (GenBank accession number M15476, incorporated herein as SEQ ID NO:3). For human urokinase plasminogen activator the PCR primers were:  
     [0253] forward primer: GAATGGTCACTTTTACCGAGGAA (SEQ ID NO: 4)  
     [0254] reverse primer: GGCATGGTACGTTTGCTGAA (SEQ ID NO: 5) and the PCR probe was: FAM-CCTGCCCTGGAACTCTGCCACTGT-TAMRA  
     [0255] (SEQ ID NO: 6) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For human GAPDH the PCR primers were:  
     [0256] forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 7)  
     [0257] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.  
     [0258] Probes and primers to mouse urokinase plasminogen activator were designed to hybridize to a mouse urokinase plasminogen activator sequence, using published sequence information (GenBank accession number X02389, incorporated herein as SEQ ID NO:10). For mouse urokinase plasminogen activator the PCR primers were:  
     [0259] forward primer: TCCGCTGCAGTCACCGA (SEQ ID NO:11)  
     [0260] reverse primer: GCCAGCCAGACTTTCATGGTA (SEQ ID NO: 12) and the PCR probe was: FAM-TGCTGTCTAGAGCCCAGCGGCA-TAMRA  
     [0261] (SEQ ID NO: 13) where FAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye. For mouse GAPDH the PCR primers were:  
     [0262] forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)  
     [0263] reverse primer: GGGTCTCGCTCCTGGAAGAT (SEQ ID NO: 15) and the PCR probe was: 5′ JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ 
     [0264] (SEQ ID NO: 16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.  
     Example 14  
     [0265] Northern Blot Analysis of Urokinase Plasminogen Activator mRNA Levels  
     [0266] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer&#39;s recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer&#39;s recommendations for stringent conditions.  
     [0267] To detect human urokinase plasminogen activator, a human urokinase plasminogen activator specific probe was prepared by PCR using the forward primer GAATGGTCACTTTTACCGAGGAA (SEQ ID NO: 4) and the reverse primer GGCATGGTACGTTTGCTGAA (SEQ ID NO: 5). 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.).  
     [0268] To detect mouse urokinase plasminogen activator, a mouse urokinase plasminogen activator specific probe was prepared by PCR using the forward primer TCCGCTGCAGTCACCGA (SEQ ID NO:11) and the reverse primer GCCAGCCAGACTTTCATGGTA (SEQ ID NO: 12). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0269] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.  
     Example 15  
     [0270] Antisense Inhibition of Human Urokinase Plasminogen Activator Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap  
     [0271] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human urokinase plasminogen activator RNA, using published sequences (GenBank accession number M15476, incorporated herein as SEQ ID NO: 3, and GenBank accession number X02419, genomic sequence of human urokinase plasminogen activator incorporated herein as SEQ ID NO: 17). 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 urokinase plasminogen activator mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.  
               TABLE 1                          Inhibition of human urokinase plasminogen activator mRNA levels       by chimeric phosphorothioate oligonucleotides       having 2′-MOE wings and a deoxy gap                                                 TARGET                               SEQ ID   TARGET           SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   % INHIB   NO                                                 136420   5′ UTR   3   24   ctcggtggcctgcggcagga   61   18       136422   Exon:Exon   3   124   cattgctgcctttggagtcg   86   19           Junction       136423   Exon:Exon   3   152   tcacagttcgatggaacttg   67   20           Junction       136424   Coding   3   188   tacttgttggacacacatgt   68   21       136425   Coding   3   193   agaagtacttgttggacaca   72   22       136426   Coding   3   233   cctccgaatttctttgggca   80   23       136427   Exon:Exon   3   257   gacttatctatttcacagtg   72   24           Junction       136429   Coding   3   392   agagcatcagatctgtgggc   61   25       136430   Exon:Exon   3   436   tgtctgggttcctgcagtaa   72   26           Junction       136431   Coding   3   460   catagcaccagggtcgcctc   73   27       136432   Coding   3   465   ctgcacatagcaccagggtc   77   28       136433   Coding   3   565   ggccacactgaaattttaat   69   29       136435   Coding   3   657   gtgcctcctgtagatggccg   83   30       136436   Coding   3   662   ccccggtgcctcctgtagat   57   31       136437   Exon:Exon   3   747   tgggtaatcaatgaagcagt   61   32           Junction       136438   Coding   3   791   ttaagccttgagcgacccag   88   33       136439   Coding   3   800   gtgttggagttaagccttga   75   34       136440   Coding   3   806   ccttgcgtgttggagttaag   79   35       136441   Coding   3   837   gatgaggttttccacctcaa   0   36       136442   Coding   3   890   aaggcaatgtcgttgtggtg   69   37       136443   Exon:Exon   3   896   ttcagcaaggcaatgtcgtt   72   38           Junction       136444   Coding   3   933   ggatggctgcgcacacctgc   70   39       136445   Coding   3   939   agtccgggatggctgcgcac   75   40       136446   Coding   3   955   ggcagatggtctgtatagtc   56   41       136447   Coding   3   959   ggcaggcagatggtctgtat   57   42       136448   Coding   3   1012   caaagccagtgatctcacag   85   43       136449   Coding   3   1018   cttttccaaagccagtgatc   74   44       136450   Coding   3   1025   gaattctcttttccaaagcc   64   45       136451   Exon:Exon   3   1037   agatagtcggtagaattctc   79   46           Junction       136452   Coding   3   1069   tcacaacagtcattttcagc   75   47       136453   Intron 1   17   1090   cggtgaccaggctccccagc   79   48       136454   Coding   3   1121   acttcagagccgtagtagtg   79   49       136455   Coding   3   1177   cctggcaggaatctgtttc   48   50       136456   Exon:Exon   3   1186   ctgagtctccctggcaggaa   64   51           Junction       136457   Coding   3   1216   ggccttggagggaacagacg   69   52       136458   Coding   3   1261   gggcacatccacggccccag   68   53       136459   Coding   3   1270   tgtccttcagggcacatcca   64   54       136460   Stop   3   1359   ggaccctcagagggccaggc   59   55           Codon       136462   Intron 2   17   1561   cgtatctagttctcaaatgg   60   56       136463   3′ UTR   3   1627   tgcaggcttcagtccagaaa   78   57       136464   3′ UTR   3   1640   cctttttaactcctgcaggc   80   58       136465   Intron 2   17   1646   cccatgctccagggatccag   87   59       136466   3′ UTR   3   1652   gagatgccctgcccttttta   69   60       136467   3′ UTR   3   1750   tacacttcctaattgggaaa   60   61       136468   Intron 3   17   1766   ccctgaagccttcccaactt   62   62       136469   3′ UTR   3   1775   gctccctcaagagacctcag   88   63       136470   3′ UTR   3   1834   ccctgccctgaagtcgttag   89   64       136471   3′ UTR   3   1862   ttcctgatacattcatggaa   74   65       136473   3′ UTR   3   1940   aatcagacaccagctcttac   65   66       136475   3′ UTR   3   2085   aggtcatgctgcagaataag   85   67       136476   3′ UTR   3   2117   tgtgaaagtgaaactgagac   70   68       136477   3′ UTR   3   2158   ggctaaaaggaagggataac   79   69       136478   Intron 4   17   2166   actggacagggtctccttcc   69   70       136479   3′ UTR   3   2172   gattggatgaactaggctaa   70   71       136480   3′ UTR   3   2176   tgaggattggatgaactagg   90   72       136481   3′ UTR   3   2178   agtgaggattggatgaacta   68   73       136482   3′ UTR   3   2183   cacccagtgaggattggatg   68   74       136483   Intron 4   17   2198   ttttaaagatgtcgtttcag   63   75       136484   3′ UTR   3   2201   aaggagtggtcctcacccca   76   76       136485   3′ UTR   3   2208   tcagtgtaaggagtggtcct   81   77       136486   3′ UTR   3   2278   aatcacattttattgatcac   69   78       136487   3′ UTR   3   2280   aaaatcacattttattgatc   6   79       136488   3′ UTR   3   2285   tcagaaaaatcacattttat   36   80       136489   Intron 7   17   3538   cttgtccccaaggccatggg   52   81       136490   Intron 8   17   4015   ggctgtttccactcgcaaga   78   82       136491   Intron 8   17   4105   acagacagaggctgcatccc   69   83       136492   Intron 8   17   4148   gaatccatcccaaggctcca   47   84       136493   Intron 8   17   4378   tcccctccaagtgccgagga   59   85       136494   Intron 9   17   4628   gtcaaaacactcctcaggac   76   86       136495   Intron 9   17   4890   atttatctgcctccacgtgg   2   87       136496   Intron 10   17   5222   gactggtttaaaccctcaaa   78   88       136497   Intron 10   17   5857   aaacttgcctcctgtgctca   61   89                  
 
     [0272] As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 81, 82, 83, 85, 86, 88 and 89 demonstrated at least 50% inhibition of human urokinase plasminogen activator expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.  
     Example 16  
     [0273] Antisense Inhibition of Mouse Urokinase Plasminogen Activator Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap.  
     [0274] In accordance with the present invention, a second series of oligonucleotides were designed to target different regions of the mouse urokinase plasminogen activator RNA, using published sequences (GenBank accession number X02389, incorporated herein as SEQ ID NO: 10, and GenBank accession number M17922, representing genomic sequence of mouse urokinase plasminogen activator incorporated herein as SEQ ID NO: 90). The oligonucleotides are shown in Table 2. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on mouse urokinase plasminogen activator mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments. If present, “N.D.” indicates “no data”.  
               TABLE 2                          Inhibition of mouse urokinase plasminogen activator mRNA levels       by chimeric phosphorothioate oligonucleotides       having 2′-MOE wings and a deoxy gap                                                 TARGET                               SEQ ID   TARGET           SEQ ID       ISIS #   REGION   NO   SITE   SEQUENCE   % INHIB   NO                                                 136342   5′ UTR   10   17   cagcagttcggtgactgcag   49   91       136343   5′ UTR   10   23   tctagacagcagttcggtga   0   92       136344   Start   10   51   ccagactttcatggtagtgc   72   93           Codon       136345   Coding   10   74   gcgcagaggaacaggctcgc   83   94       136346   Coding   10   94   cagagtttttcaccaccaag   64   95       136347   Coding   10   107   acactgccaccttcagagtt   71   96       136348   Exon:Exon   10   136   cacagtttgattcatcagga   42   97           Junction       136349   Exon:Exon   10   138   gccacagtttgattcatcag   52   98           Junction       136350   Coding   10   144   ctgacagccacagtttgatt   72   99       136351   Coding   10   150   tccgttctgacagccacagt   68   100       136352   Coding   10   163   acacgcatacacctccgttc   87   101       136353   Coding   10   206   cttgggcagctgcatcggcg   81   102       136354   Exon:Exon   10   245   tttgatgcatctatctcaca   66   103           Junction       136355   Coding   10   258   atgatagcaggtttttgatg   35   104       136356   Coding   10   276   gtaagagtcaccatttccat   68   105       136357   Coding   10   313   agggccgacctttggtatca   86   106       136358   Coding   10   396   tttccccaggcctaggctaa   59   107       136359   Coding   10   501   aagagagcagtcatgcacca   65   108       136360   Exon:Exon   10   512   ggctttttgctaagagagca   75   109           Junction       136361   Coding   10   536   ccttgttggtctacagacga   63   110       136362   Coding   10   648   tcccttgttcttctggtaga   0   111       136363   Coding   10   674   ccacatttaaaggagggagg   66   112       136364   Coding   10   680   ctcccaccacatttaaagga   53   113       136365   Coding   10   690   actgatgagactcccaccac   65   114       136366   Exon:Exon   10   736   ttgggagttgaatgaagcag   71   115           Junction       136367   Coding   10   769   actgacccaggtagacaacg   55   116       136368   Coding   10   774   cttcgactgacccaggtaga   72   117       136369   Coding   10   791   ggattataggagctctcctt   78   118       136370   Coding   10   799   tctctccaggattataggag   58   119       136371   Coding   10   826   agatgagctgctccacctca   79   120       136372   Coding   10   833   tcgtgcaagatgagctgctc   25   121       136373   Coding   10   842   ctgtagtattcgtgcaagat   60   122       136374   Coding   10   898   tgctggtacgtatcttcagc   76   123       136375   Coding   10   904   ggcccgtgctggtacgtatc   87   124       136376   Coding   10   979   ctgaaccaaacggagcatca   24   125       136377   Coding   10   985   cacagtctgaaccaaacgga   60   126       136378   Coding   10   991   tgatctcacagtctgaacca   87   127       136379   Exon:Exon   10   1025   agatagtcactttcagactc   63   128           Junction       136380   Coding   10   1113   attaatttcagagccatagt   67   129       136381   Coding   10   1119   tttataattaatttcagagc   46   130       136382   Coding   10   1127   cacagcattttataattaat   44   131       136383   Coding   10   1132   cagcacacagcattttataa   61   132       136384   Exon:Exon   10   1175   ccagaatcgcccttgcagga   16   133           Junction       136385   Coding   10   1187   ataagcggtcctccagaatc   59   134       136386   Coding   10   1262   ggcttgtttttctctgcaca   38   135       136387   Coding   10   1325   ttctcttctccaatgtggga   63   136       136388   Stop   10   1349   gagggccatcagaaggccag   53   137           Codon       136389   3′ UTR   10   1419   acgactctgttgcagagagg   75   138       136390   3′ UTR   10   1440   tcagcttcttccctccattt   71   139       136391   3′ UTR   10   1458   aatgcaaaacctgtcttttc   66   140       136392   3′ UTR   10   1560   ctttccatcctggtcgggag   62   141       136393   3′ UTR   10   1578   atcctgagtcaggaccaact   76   142       136394   3′ UTR   10   1618   atggctttagtccataaaaa   70   143       136395   3′ UTR   10   1624   ctgcagatggctttagtcca   89   144       136396   3′ UTR   10   1677   ccattaagggaaacagctct   57   145       136397   3′ UTR   10   1695   gcagatctcatgaatgaccc   87   146       136398   3′ UTR   10   1711   catttatttcccaacagcag   53   147       136399   3′ UTR   10   1747   caatacctcagctgttgcac   23   148       136400   3′ UTR   10   1768   catattggacaagcaccctc   73   149       136401   3′ UTR   10   1939   tagacttgcagttagataca   54   150       136402   3′ UTR   10   1945   aatacctagacttgcagtta   66   151       136403   3′ UTR   10   2025   tcacgcccgggaatgataca   63   152       136404   3′ UTR   10   2043   tttagtgctagtcacgggtc   76   153       136405   3′ UTR   10   2072   ggacatctatataaaaagtg   66   154       136406   3′ UTR   10   2077   gaagtggacatctatataaa   22   155       136407   3′ UTR   10   2146   gaactaggctaattagtaaa   27   156       136408   3′ UTR   10   2261   attgatcacctttactcaga   73   157       136409   3′ UTR   10   2271   aatcacatttattgatcacc   64   158       136410   Intron B   90   2924   tttattcctagccagcaccc   56   159       136411   Intron D   90   3622   cagtgttcttaatcactcac   35   160       136412   Intron E   90   4273   gtctgtgctcttaactgctg   76   161       136413   Intron E   90   4315   acgagctgccctgggaatca   62   162       136414   Intron G   90   5128   ccatagggaaggaggaagga   61   163       136415   Intron H   90   5668   cctactttgtacaaacaggg   69   164       136416   Intron H   90   5816   tatttcatcagatgacagat   46   165       136417   Intron I   90   6238   gtttccagtcctgtttccac   64   166       136418   Intron I   90   6411   tgtgtctataaatgagactc   31   167       136419   Intron J   90   7488   gttcatagtctctttaagac   73   168                  
 
     [0275] As shown in Table 2, SEQ ID NOs 93, 94, 95, 96, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, 112, 113, 114, 115, 116, 117, 118, 119, 120, 122, 123, 124, 126, 127, 128, 129, 132, 134, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154, 157, 158, 159, 161, 162, 163, 164, 166 and 168 demonstrated at least 50% inhibition of mouse urokinase plasminogen activator expression in this experiment and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “active sites” and are therefore preferred sites for targeting by compounds of the present invention.  
     Example 17  
     [0276] Western Blot Analysis of Urokinase Plasminogen Activator Protein Levels  
     [0277] 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 urokinase plasminogen activator is used, with a radiolabelled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).  
    
     
       
         1 
         
           
             168  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

atgcattctg cccccaagga                                                 20 

 
           
             3  
             2304  
             DNA  
             Homo sapiens  
             
               CDS  
               (77)...(1372)  
             
           
            3 

gtccccgcag cgccgtcgcg ccctcctgcc gcaggccacc gaggccgccg ccgtctagcg     60 

ccccgacctc gccacc atg aga gcc ctg ctg gcg cgc ctg ctt ctc tgc gtc    112 
                  Met Arg Ala Leu Leu Ala Arg Leu Leu Leu Cys Val 
                    1               5                  10 

ctg gtc gtg agc gac tcc aaa ggc agc aat gaa ctt cat caa gtt cca      160 
Leu Val Val Ser Asp Ser Lys Gly Ser Asn Glu Leu His Gln Val Pro 
         15                  20                  25 

tcg aac tgt gac tgt cta aat gga gga aca tgt gtg tcc aac aag tac      208 
Ser Asn Cys Asp Cys Leu Asn Gly Gly Thr Cys Val Ser Asn Lys Tyr 
     30                  35                  40 

ttc tcc aac att cac tgg tgc aac tgc cca aag aaa ttc gga ggg cag      256 
Phe Ser Asn Ile His Trp Cys Asn Cys Pro Lys Lys Phe Gly Gly Gln 
 45                  50                  55                  60 

cac tgt gaa ata gat aag tca aaa acc tgc tat gag ggg aat ggt cac      304 
His Cys Glu Ile Asp Lys Ser Lys Thr Cys Tyr Glu Gly Asn Gly His 
                 65                  70                  75 

ttt tac cga gga aag gcc agc act gac acc atg ggc cgg ccc tgc ctg      352 
Phe Tyr Arg Gly Lys Ala Ser Thr Asp Thr Met Gly Arg Pro Cys Leu 
             80                  85                  90 

ccc tgg aac tct gcc act gtc ctt cag caa acg tac cat gcc cac aga      400 
Pro Trp Asn Ser Ala Thr Val Leu Gln Gln Thr Tyr His Ala His Arg 
         95                 100                 105 

tct gat gct ctt cag ctg ggc ctg ggg aaa cat aat tac tgc agg aac      448 
Ser Asp Ala Leu Gln Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn 
    110                 115                 120 

cca gac aac cgg agg cga ccc tgg tgc tat gtg cag gtg ggc cta aag      496 
Pro Asp Asn Arg Arg Arg Pro Trp Cys Tyr Val Gln Val Gly Leu Lys 
125                 130                 135                 140 

ccg ctt gtc caa gag tgc atg gtg cat gac tgc gca gat gga aaa aag      544 
Pro Leu Val Gln Glu Cys Met Val His Asp Cys Ala Asp Gly Lys Lys 
                145                 150                 155 

ccc tcc tct cct cca gaa gaa tta aaa ttt cag tgt ggc caa aag act      592 
Pro Ser Ser Pro Pro Glu Glu Leu Lys Phe Gln Cys Gly Gln Lys Thr 
            160                 165                 170 

ctg agg ccc cgc ttt aag att att ggg gga gaa ttc acc acc atc gag      640 
Leu Arg Pro Arg Phe Lys Ile Ile Gly Gly Glu Phe Thr Thr Ile Glu 
        175                 180                 185 

aac cag ccc tgg ttt gcg gcc atc tac agg agg cac cgg ggg ggc tct      688 
Asn Gln Pro Trp Phe Ala Ala Ile Tyr Arg Arg His Arg Gly Gly Ser 
    190                 195                 200 

gtc acc tac gtg tgt gga ggc agc ctc atc agc cct tgc tgg gtg atc      736 
Val Thr Tyr Val Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ile 
205                 210                 215                 220 

agc gcc aca cac tgc ttc att gat tac cca aag aag gag gac tac atc      784 
Ser Ala Thr His Cys Phe Ile Asp Tyr Pro Lys Lys Glu Asp Tyr Ile 
                225                 230                 235 

gtc tac ctg ggt cgc tca agg ctt aac tcc aac acg caa ggg gag atg      832 
Val Tyr Leu Gly Arg Ser Arg Leu Asn Ser Asn Thr Gln Gly Glu Met 
            240                 245                 250 

aag ttt gag gtg gaa aac ctc atc cta cac aag gac tac agc gct gac      880 
Lys Phe Glu Val Glu Asn Leu Ile Leu His Lys Asp Tyr Ser Ala Asp 
        255                 260                 265 

acg ctt gct cac cac aac gac att gcc ttg ctg aag atc cgt tcc aag      928 
Thr Leu Ala His His Asn Asp Ile Ala Leu Leu Lys Ile Arg Ser Lys 
    270                 275                 280 

gag ggc agg tgt gcg cag cca tcc cgg act ata cag acc atc tgc ctg      976 
Glu Gly Arg Cys Ala Gln Pro Ser Arg Thr Ile Gln Thr Ile Cys Leu 
285                 290                 295                 300 

ccc tcg atg tat aac gat ccc cag ttt ggc aca agc tgt gag atc act     1024 
Pro Ser Met Tyr Asn Asp Pro Gln Phe Gly Thr Ser Cys Glu Ile Thr 
                305                 310                 315 

ggc ttt gga aaa gag aat tct acc gac tat ctc tat ccg gag cag ctg     1072 
Gly Phe Gly Lys Glu Asn Ser Thr Asp Tyr Leu Tyr Pro Glu Gln Leu 
            320                 325                 330 

aaa atg act gtt gtg aag ctg att tcc cac cgg gag tgt cag cag ccc     1120 
Lys Met Thr Val Val Lys Leu Ile Ser His Arg Glu Cys Gln Gln Pro 
        335                 340                 345 

cac tac tac ggc tct gaa gtc acc acc aaa atg cta tgt gct gct gac     1168 
His Tyr Tyr Gly Ser Glu Val Thr Thr Lys Met Leu Cys Ala Ala Asp 
    350                 355                 360 

ccc caa tgg aaa aca gat tcc tgc cag gga gac tca ggg gga ccc ctc     1216 
Pro Gln Trp Lys Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu 
365                 370                 375                 380 

gtc tgt tcc ctc caa ggc cgc atg act ttg act gga att gtg agc tgg     1264 
Val Cys Ser Leu Gln Gly Arg Met Thr Leu Thr Gly Ile Val Ser Trp 
                385                 390                 395 

ggc cgt gga tgt gcc ctg aag gac aag cca ggc gtc tac acg aga gtc     1312 
Gly Arg Gly Cys Ala Leu Lys Asp Lys Pro Gly Val Tyr Thr Arg Val 
            400                 405                 410 

tca cac ttc tta ccc tgg atc cgc agt cac acc aag gaa gag aat ggc     1360 
Ser His Phe Leu Pro Trp Ile Arg Ser His Thr Lys Glu Glu Asn Gly 
        415                 420                 425 

ctg gcc ctc tga gggtccccag ggaggaaacg ggcaccaccc gctttcttgc         1412 
Leu Ala Leu 
    430 

tggttgtcat ttttgcagta gagtcatctc catcagctgt aagaagagac tgggaagata   1472 

ggctctgcac agatggattt gcctgtggca ccaccagggt gaacgacaat agctttaccc   1532 

tcacggatag gcctgggtgc tggctgccca gaccctctgg ccaggatgga ggggtggtcc   1592 

tgactcaaca tgttactgac cagcaacttg tctttttctg gactgaagcc tgcaggagtt   1652 

aaaaagggca gggcatctcc tgtgcatggg ctcgaaggga gagccagctc ccccgaccgg   1712 

tgggcatttg tgaggcccat ggttgagaaa tgaataattt cccaattagg aagtgtaagc   1772 

agctgaggtc tcttgaggga gcttagccaa tgtgggagca gcggtttggg gagcagagac   1832 

actaacgact tcagggcagg gctctgatat tccatgaatg tatcaggaaa tatatatgtg   1892 

tgtgtatgtt tgcacacttg ttgtgtgggc tgtgagtgta agtgtgagta agagctggtg   1952 

tctgattgtt aagtctaaat atttccttaa actgtgtgga ctgtgatgcc acacagagtg   2012 

gtctttctgg agaggttata ggtcactcct ggggcctctt gggtccccca cgtgacagtg   2072 

cctgggaatg tacttattct gcagcatgac ctgtgaccag cactgtctca gtttcacttt   2132 

cacatagatg tccctttctt ggccagttat cccttccttt tagcctagtt catccaatcc   2192 

tcactgggtg gggtgaggac cactccttac actgaatatt tatatttcac tatttttatt   2252 

tatatttttg taattttaaa taaaagtgat caataaaatg tgatttttct ga           2304 

 
           
             4  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            4 

gaatggtcac ttttaccgag gaa                                             23 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

ggcatggtac gtttgctgaa                                                 20 

 
           
             6  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            6 

cctgccctgg aactctgcca ctgt                                            24 

 
           
             7  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            7 

gaaggtgaag gtcggagtc                                                  19 

 
           
             8  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            8 

gaagatggtg atgggatttc                                                 20 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            9 

caagcttccc gttctcagcc                                                 20 

 
           
             10  
             2299  
             DNA  
             Mus musculus  
             
               CDS  
               (59)...(1360)  
             
           
            10 

tgccagcgcc cttccgctgc agtcaccgaa ctgctgtcta gagcccagcg gcactacc       58 

atg aaa gtc tgg ctg gcg agc ctg ttc ctc tgc gcc ttg gtg gtg aaa      106 
Met Lys Val Trp Leu Ala Ser Leu Phe Leu Cys Ala Leu Val Val Lys 
  1               5                  10                  15 

aac tct gaa ggt ggc agt gta ctt gga gct cct gat gaa tca aac tgt      154 
Asn Ser Glu Gly Gly Ser Val Leu Gly Ala Pro Asp Glu Ser Asn Cys 
             20                  25                  30 

ggc tgt cag aac gga ggt gta tgc gtg tcc tac aag tac ttc tcc aga      202 
Gly Cys Gln Asn Gly Gly Val Cys Val Ser Tyr Lys Tyr Phe Ser Arg 
         35                  40                  45 

att cgc cga tgc agc tgc cca agg aaa ttc cag ggg gag cac tgt gag      250 
Ile Arg Arg Cys Ser Cys Pro Arg Lys Phe Gln Gly Glu His Cys Glu 
     50                  55                  60 

ata gat gca tca aaa acc tgc tat cat gga aat ggt gac tct tac cga      298 
Ile Asp Ala Ser Lys Thr Cys Tyr His Gly Asn Gly Asp Ser Tyr Arg 
 65                  70                  75                  80 

gga aag gcc aac act gat acc aaa ggt cgg ccc tgc ctg gcc tgg aat      346 
Gly Lys Ala Asn Thr Asp Thr Lys Gly Arg Pro Cys Leu Ala Trp Asn 
                 85                  90                  95 

gcg cct gct gtc ctt cag aaa ccc tac aat gcc cac aga cct gat gct      394 
Ala Pro Ala Val Leu Gln Lys Pro Tyr Asn Ala His Arg Pro Asp Ala 
            100                 105                 110 

att agc cta ggc ctg ggg aaa cac aat tac tgc agg aac cct gac aac      442 
Ile Ser Leu Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp Asn 
        115                 120                 125 

cag aag cga ccc tgg tgc tat gtg cag att ggc cta agg cag ttt gtc      490 
Gln Lys Arg Pro Trp Cys Tyr Val Gln Ile Gly Leu Arg Gln Phe Val 
    130                 135                 140 

caa gaa tgc atg gtg cat gac tgc tct ctt agc aaa aag cct tct tcg      538 
Gln Glu Cys Met Val His Asp Cys Ser Leu Ser Lys Lys Pro Ser Ser 
145                 150                 155                 160 

tct gta gac caa caa ggc ttc cag tgt ggc cag aag gct cta agg ccc      586 
Ser Val Asp Gln Gln Gly Phe Gln Cys Gly Gln Lys Ala Leu Arg Pro 
                165                 170                 175 

cgc ttt aag att gtt ggg gga gaa ttc act gag gtg gag aac cag ccc      634 
Arg Phe Lys Ile Val Gly Gly Glu Phe Thr Glu Val Glu Asn Gln Pro 
            180                 185                 190 

tgg ttc gca gcc atc tac cag aag aac aag gga gga agt cct ccc tcc      682 
Trp Phe Ala Ala Ile Tyr Gln Lys Asn Lys Gly Gly Ser Pro Pro Ser 
        195                 200                 205 

ttt aaa tgt ggt ggg agt ctc atc agt cct tgc tgg gtg gcc agt gcc      730 
Phe Lys Cys Gly Gly Ser Leu Ile Ser Pro Cys Trp Val Ala Ser Ala 
    210                 215                 220 

gca cac tgc ttc att caa ctc cca aag aag gaa aac tac gtt gtc tac      778 
Ala His Cys Phe Ile Gln Leu Pro Lys Lys Glu Asn Tyr Val Val Tyr 
225                 230                 235                 240 

ctg ggt cag tcg aag gag agc tcc tat aat cct gga gag atg aag ttt      826 
Leu Gly Gln Ser Lys Glu Ser Ser Tyr Asn Pro Gly Glu Met Lys Phe 
                245                 250                 255 

gag gtg gag cag ctc atc ttg cac gaa tac tac agg gaa gac agc ctg      874 
Glu Val Glu Gln Leu Ile Leu His Glu Tyr Tyr Arg Glu Asp Ser Leu 
            260                 265                 270 

gcc tac cat aat gat att gcc ttg ctg aag ata cgt acc agc acg ggc      922 
Ala Tyr His Asn Asp Ile Ala Leu Leu Lys Ile Arg Thr Ser Thr Gly 
        275                 280                 285 

caa tgt gca cag cca tcc agg tcc ata cag acc atc tgc ctg ccc cca      970 
Gln Cys Ala Gln Pro Ser Arg Ser Ile Gln Thr Ile Cys Leu Pro Pro 
    290                 295                 300 

agg ttt act gat gct ccg ttt ggt tca gac tgt gag atc act ggc ttt     1018 
Arg Phe Thr Asp Ala Pro Phe Gly Ser Asp Cys Glu Ile Thr Gly Phe 
305                 310                 315                 320 

gga aaa gag tct gaa agt gac tat ctc tat cca aag aac ctg aaa atg     1066 
Gly Lys Glu Ser Glu Ser Asp Tyr Leu Tyr Pro Lys Asn Leu Lys Met 
                325                 330                 335 

tcc gtc gta aag ctt gtt tct cat gaa cag tgt atg cag ccc cac tac     1114 
Ser Val Val Lys Leu Val Ser His Glu Gln Cys Met Gln Pro His Tyr 
            340                 345                 350 

tat ggc tct gaa att aat tat aaa atg ctg tgt gct gcg gac cca gag     1162 
Tyr Gly Ser Glu Ile Asn Tyr Lys Met Leu Cys Ala Ala Asp Pro Glu 
        355                 360                 365 

tgg aaa aca gat tcc tgc aag ggc gat tct gga gga ccg ctt atc tgt     1210 
Trp Lys Thr Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro Leu Ile Cys 
    370                 375                 380 

aac atc gaa ggc cgc cca act ctg agt ggg att gtg agc tgg ggc cga     1258 
Asn Ile Glu Gly Arg Pro Thr Leu Ser Gly Ile Val Ser Trp Gly Arg 
385                 390                 395                 400 

gga tgt gca gag aaa aac aag ccc ggt gtc tac acg agg gtc tca cac     1306 
Gly Cys Ala Glu Lys Asn Lys Pro Gly Val Tyr Thr Arg Val Ser His 
                405                 410                 415 

ttc ctg gac tgg att caa tcc cac att gga gaa gag aaa ggt ctg gcc     1354 
Phe Leu Asp Trp Ile Gln Ser His Ile Gly Glu Glu Lys Gly Leu Ala 
            420                 425                 430 

ttc tga tggccctcag gtagctgagg gaagaaacag atgggtcact tgttcccatg      1410 
Phe 

ctgaccgtcc tctctgcaac agagtcgtca aatggaggga agaagctgaa aagacaggtt   1470 

ttgcattgat cctctgctgt gctgcccacc agggtgagcg ccaatagcat taccctcaga   1530 

cacaggcctg ggtgctggcc atccagaccc tcccgaccag gatggaaagt tggtcctgac   1590 

tcaggatgct atagaccagg agttgccttt ttatggacta aagccatctg cagtttagaa   1650 

aacatctcct gggcaagtgt aggaggagag ctgtttccct taatgggtca ttcatgagat   1710 

ctgctgttgg gaaataaatg atttcccaat taggaagtgc aacagctgag gtattgtgag   1770 

ggtgcttgtc caatatgaga acggtagctt gaggagtaga gacactaacg gcttgaggga   1830 

acagctctag catcccatga atggatcagg aaatgttata tttgtgtgta tgtttgttca   1890 

ctctgcacag gctgtgagta taagcctgag caaaagctgg tgtatttctg tatctaactg   1950 

caagtctagg tatttcccta actccagact gtgatgcggg gccatttggt cttccatgtg   2010 

atgctccacg tgaatgtatc attcccgggc gtgacccgtg actagcacta aatgtcggtt   2070 

tcacttttta tatagatgtc cacttcttgg ccagttatct tttttttttt tttttttttt   2130 

tttttttttt ttttttttac taattagcct agttcatcca atcctcactg ggtggggtaa   2190 

ggaccacttc tacatactta atatttaata attatgttct gctattttta tttatatcta   2250 

tttttataat tctgagtaaa ggtgatcaat aaatgtgatt tttctgaag               2299 

 
           
             11  
             17  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            11 

tccgctgcag tcaccga                                                    17 

 
           
             12  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            12 

gccagccaga ctttcatggt a                                               21 

 
           
             13  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            13 

tgctgtctag agcccagcgg ca                                              22 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            14 

ggcaaattca acggcacagt                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            15 

gggtctcgct cctggaagat                                                 20 

 
           
             16  
             27  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            16 

aaggccgaga atgggaagct tgtcatc                                         27 

 
           
             17  
             7258  
             DNA  
             Homo sapiens  
             
 
           
            17 

ttcaatagga agcaccaaca gtttatgccc taggactttg ttcccacaat cctgtaacat     60 

catatcacga cacctaaccc aatccttatc aagccctgtc aaaaacggac tttaaaccaa    120 

gctgcaaatt ttcagtaatc tggccttgcc tttccccctc tgatagcacc atcaaacaaa    180 

cccccttact gccgaaagca ataagcccgg ctttgttcca tccactggtt gtgttggtga    240 

tatctgggga ctgccactga acagacgcac agagggagcc cctacaggca ggggtttttc    300 

tgtctgtgct tcttgggaga gtatgtctcg tacatttgtc gcgtgatgaa gacttcacag    360 

ctccatccag cgaccagact cacagctcca tccagctgcg gcaagggggt ctgaggcagt    420 

cttaggcaag ttggggccca gcgggagaag ttgcagaaga actgattaga ggacccagga    480 

ggcttcagag ctgggctgag gtagagagtc tcctgtgcgc cttctctcct ctctgcaatt    540 

cggggactcc ttgcactggg gcaggccccg gcaggtgcat gggaggaagc acggagaatt    600 

tacaagcctc tcgattcctc agtccagacg ctgttgggtc ccctccgctg gagatcgcgc    660 

ttcccccaaa tctttgtgag cgttgcggaa gcacgcgggg tccgggtcgc tgagcgctgc    720 

aagacagggg agggagccgg gcgggagagg gaggggcggc gccggggcgg gccctgatat    780 

agagcaggcg ccgcgggtcg cagcacagtc ggagaccgca gcccggagcc cgggccaggg    840 

tccacctgtc cccgcagcgc cggctcgcgc cctcctgccg cagccaccgg tgagtgccgc    900 

ggtcctgaga tccccgggcc ggatgcgcgg cggccccagc tcccgagcgt ctgcctgccc    960 

cgccctgggc tgcccgggct ccctgggctc cccggcggct gcacggagtc aaggcgcccc   1020 

gtcccgggcg tcccccgcgg gtgccgatcc aggctgcccg gagtccggag cccatagagg   1080 

agagagacag ctggggagcc tggtcaccgc gggcatctcc cctgcgctgc agtcgcccgc   1140 

ctggcctgcc ttcccgttcc tccgcctctt gccctgactt ctccttcctt tgcagagccg   1200 

ccgtctagcg ccccgacctc gccaccatga gagccctgct ggcgcgcctg cttctctgcg   1260 

tcctggtcgt gagcgactcc aaagtgagtg cgctcttgct ttgactgatg ctgcccaagg   1320 

acctctgatc agcaccaggg gagaggaggg gctgctcagg gagctggggt ctccggattc   1380 

catccacagc agggccagac tctccccagg aaatgggaca gggtggcagc ggaggcttga   1440 

gaaccacggg ggttggcact ggctggcaag ggaggaagag ggccaccggg actgccccag   1500 

cctgcgggca tctggtagat gaagcttaat ccatttctcc tggctggaaa ccatggtctt   1560 

ccatttgaga actagatacg aacagggtga ggcgagaggg agagggaaga gtgggttttg   1620 

ggattggggc cagtttaccc tcaccctgga tccctggagc atgggacctt tgatgaagcc   1680 

tcctcccgaa tctcttccag ggcagcaatg aacttcatca agttccatgt gagtatccac   1740 

ccctacaaca gttggctgca cagacaagtt gggaaggctt caggggacac tcccctccct   1800 

gccctctgct gcagcgtgcg ccacccctta ccacttccac tccccctcgc ttaccccacc   1860 

tttgttctct ccagcgaact gtgactgtct aaatggagga acatgtgtgt ccaacaagta   1920 

cttctccaac attcactggt gcaactgccc aaagaaattc ggagggcagc actgtgaaat   1980 

aggtatgggg atctccactg caactgggag agaaatttgg ggacagggag ggatgggtgg   2040 

gaggcaagag caggcaggag ttaggagctg gaggtagggt gggtgacatc ttcatcccta   2100 

tgtgacaagc ataaacacac acacacgctc acgaaacagt ggccacacaa atgtgaggtg   2160 

gggttggaag gagaccctgt ccagtcttct ggcaggtctg aaacgacatc tttaaaatgt   2220 

ccgttggcag ccgggcatgg tggctcacgc ttgtaatccc agcattttga gaggtcaagt   2280 

ttgagtggat catttaggtc aggagttcaa gaccagcctg gacaacatgg tgtaaccctg   2340 

cctctactaa aaatgcaaaa atcagcctgg catggtggtg gatgcctgta gtcccagcta   2400 

cttgggaggc tgaggcagga gaattgcttg aacatgggag gccagatctc agtgagctga   2460 

gatcacacca ctgcactcca actgggcgac agagcaagac tccatctcaa aaaaaaaaaa   2520 

aaataaaagt tagttggaat gttcttctct ttctcatatt ctctcatcct cctgtcccct   2580 

tgtagataag tcaaaaacct gctatgaggg gaatggtcac ttttaccgag gaaaggccag   2640 

cactgacacc atgggccggc cctgcctgcc ctggaactct gccactgtcc ttcagcaaac   2700 

gtaccatgcc cacagatctg atgctcttca gctgggcctg gggaaacata attactgcag   2760 

gtgaggtggg ggcaacaagg accaaaagcc ctccctacag cttcccagaa accttgttac   2820 

catccccttc tcccagaggg ctggccatag cacaagagaa gtgcggcctc tggttgagtc   2880 

ttccctgagg ggaggaggca gggaaggccc tctgggttgg aatgacatcc cctatctttc   2940 

tgtgttgtgc caggaaccca gacaaccgga ggcgaccctg gtgctatgtg caggtgggcc   3000 

taaagccgct tgtccaagag tgcatggtgc atgactgcgc agatggtgag catcactgac   3060 

ctgctgatga caggtgggtg gaaggggaca aacttacatg tccccttatt ccatcacagg   3120 

aggactgagg aggtgggggg tgcccgagag ggatgctttc tcctacctgc ctccctaaga   3180 

catccctctg tttgtcctcc aggaaaaaag ccctcctctc ctccagaaga attaaaattt   3240 

cagtgtggcc aaaagactct gaggccccgc tttaagatta ttgggggaga attcaccacc   3300 

atcgagaacc agccctggtt tgcggccatc tacaggaggc accggggggg ctctgtcacc   3360 

tacgtgtgtg gaggcagcct catgagccct tgctgggtga tcagcgccac acactgcttc   3420 

atgtacggcc ctgggtttct cctcttcgac tcttctgccc caccccaagc acatcccttt   3480 

ctccttccca gcaaagtgtt ccgcctcatt tctccctcat ctgcccctgt ccatgcgccc   3540 

atggccttgg ggacaagtcg tgctttgagg cctctaggga gggaaggaag aagtggcatg   3600 

atttcatggg actaagctgt ttgatgggta tcttcttcca cagtgattac ccaaagaagg   3660 

aggactacat cgtctacctg ggtcgctcaa ggcttaactc caacacgcaa ggggagatga   3720 

agtttgaggt ggaaaacctc atcctacaca aggactacag cgctgacacg cttgctcacc   3780 

acaacgacat tggtgagggg gaacgcccgc gactactgtg gccataatgg cttggggaga   3840 

gtgggaccca gggagagact ggagctgagt tgaagctgcc ggtggggcag gggtggggcg   3900 

agggaccttg aagcctcgat atacatgaca aaggatggca gggaagagtt ccatgaagtc   3960 

tgaggggcct ggtgctcctc tggagagacc ctgaatttcc ccaacaagta gccctcttgc   4020 

gagtggaaac agccctgtgg gtatatggct tgggctggga aggccctgtt tatatgaatt   4080 

agaaaaagac acaccttcct ttgtgggatg cagcctctgt ctgtgctagg atatagaact   4140 

tggagaatgg agccttggga tggattccag cctaactacc tcagggggat cctctagagt   4200 

gcagctggga gtttttgcag aaacgacctg tacagctgta tgcagtggct ctggccatcc   4260 

aagccttttt caacacctgg aacaaagccc ttggggcatg gggcagggga ggtttccagg   4320 

tgataagcga ccagcagacc tccctggatg actgacctag ggataggcat agctacttcc   4380 

tcggcacttg gaggggacag atggggaccg cctaaccagt agtgatcttt ctcctctgac   4440 

cctctgtcct cccccagcct tgctgaagat ccgttccaag gagggcaggt gtgcgcagcc   4500 

atcccggact atacagacca tctgcctgcc ctcgatgtat aacgatcccc agtttggcac   4560 

aagctgtgag atcactggct ttggaaaaga gaattctagt aagtgacaat tgcgactgac   4620 

ttagaaggtc ctgaggagtg ttttgacctg aaaatgagcc cagtgtgatc aagggaagac   4680 

tgcagagtta gaggtgggag cactgaggcg gtggcagatg ggtccaggga tggatgaaga   4740 

gtgttgttta gggagcgatg ggctgcaaag gtaaatagat ggtaggggct ataggtggag   4800 

gtaaatggct cagatttgca tggagagaga ataatgggcc tctccctggg tgatgatact   4860 

ttatggtgtc ccctctctgg cgagacgtcc cacgtggagg cagataaatc ttgatgcaaa   4920 

cgcctccctg ttttctccac ctagccgact atctctatcc ggagcagctg aaaatgactg   4980 

ttgtgaagct gatttcccac cgggagtgtc agcagcccca ctactacggc tctgaagtca   5040 

ccaccaaaat gctgtgtgct gctgacccac agtggaaaac agattcctgc caggtgagtg   5100 

ttccaagcat ctctctccac ctcttccata tctccccaga gctcctgggc ttgttccagc   5160 

cagcttaagg gtgtctctct ctagccaaag ccctaagtag ccagaatcag gagctcaggt   5220 

ctttgagggt ttaaaccagt ccttatgtgt ttgccagaca ttaccaaaaa aatcccagct   5280 

ctgcgctagt cacttcagac tgggggcacg agatcctaga aagaggaaac agtaaaagac   5340 

aatgtaactc agtgcccagg gtgtgttgtg aactataaat gatcaggtgt tcaggagagg   5400 

gaggtgagtg ccaacctgag ggtcagggag gggaggcttt aaaggaaatg tgacttgata   5460 

ggcatttgaa gaggcagagg gaagaaagga aggtgtttca gttgaaagat acaaaactga   5520 

gaaggaggct ggcatattcc gggtggggag gagaactagg gtctgggagt gtggatggaa   5580 

tagtggcaga tgacagggct tttaaagcca agcaggggat tttccaactt cgatgtggta   5640 

gaaatggggc tgcgtcaggc acagtggctc atgcctgtaa tcccagcatt gggctaggcc   5700 

gtagtcgatg gatcattgag gccagagttg agaccggcct ggaccaacat ggtgaaaccc   5760 

tgtgtctact aaaaaatgca aaaaaaaaaa ttagccaggt gtggtggtgc ctgcctgtaa   5820 

tcccagctaa tcaggaggct gagacatgga atcgcttgag cacaggaggc aagtttgacg   5880 

tgagctgaga tcacgtcatt gcacgccagc ctgggcgaca gagcgagatt ctgtcctccc   5940 

gccgaaaaaa gaaagaaaat gggaagtcgc taaggacttt gactgggaaa ctcttccctc   6000 

tctctggtat ggttgggtga tgggatcaga aatcccctcc tcacttctct agggctcatc   6060 

ttttgtatct ttggcgtcac agggagactc agggggaccc ctcgtctgtt ccctccaagg   6120 

ccgcatgact ttgactggaa ttgtgagctg gggccgtgga tgtgccctga aggacaagcc   6180 

aggcgtctac acgagagtct cacacttctt accctggatc cgcagtcaca ccaaggaaga   6240 

gaatggcctg gccctctgag ggtccccagg gaggaaacgg gcaccacccg ctttcttgct   6300 

ggttgtcatt tttgcagtag agtcatctcc atcagctgta agaagagact gggaagatag   6360 

gctctgcaca gatggatttg cctgtgccac ccaccagggt gaacgacaat agctttaccc   6420 

tcaggcatag gcctgggtgc tggctgccca gacccctctg gccaggatgg aggggtggtc   6480 

ctgactcaac atgttactga ccagcaactt gtctttttct ggactgaagc ctgcaggagt   6540 

taaaaagggc agggcatctc ctgtgcatgg gtgaagggag agccagctcc cccgacggtg   6600 

ggcatttgtg aggcccatgg ttgagaaatg aataatttcc caattaggaa gtgtaacagc   6660 

tgaggtctct tgagggagct tagccaatgt gggagcagcg gtttggggag cagagacact   6720 

aacgacttca gggcagggct ctgatattcc atgaatgtat caggaaatat atatgtgtgt   6780 

gtatgtttgc acacttgtgt gtgggctgtg agtgtaagtg tgagtaagag ctggtgtctg   6840 

attgttaagt ctaaatattt ccttaaactg tgtggactgt gatgccacac agagtggtct   6900 

ttctggagag gttataggtc actcctgggg cctcttgggt cccccacgtg acagtgcctg   6960 

ggaatgtact tattctgcag catgacctgt gaccagcact gtctcagttt cactttcaca   7020 

tagatgtccc tttcttggcc agttatccct tccttttagc ctagttcatc caatcctcac   7080 

tgggtggggt gaggaccact ccttacactg aatatttata tttcactatt tttatttata   7140 

tttttgtaat tttaaataaa agtgatcaat aaaatgtgat ttttctgatg acaaatctcc   7200 

ctggtgcttg tatgggaagg agttggagta cataaaaagg agaaaataac aaaggtgg     7258 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

ctcggtggcc tgcggcagga                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

cattgctgcc tttggagtcg                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

tcacagttcg atggaacttg                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

tacttgttgg acacacatgt                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

agaagtactt gttggacaca                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

cctccgaatt tctttgggca                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

gacttatcta tttcacagtg                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

agagcatcag atctgtgggc                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

tgtctgggtt cctgcagtaa                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

catagcacca gggtcgcctc                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

ctgcacatag caccagggtc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

ggccacactg aaattttaat                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

gtgcctcctg tagatggccg                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

ccccggtgcc tcctgtagat                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

tgggtaatca atgaagcagt                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

ttaagccttg agcgacccag                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

gtgttggagt taagccttga                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

ccttgcgtgt tggagttaag                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

gatgaggttt tccacctcaa                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

aaggcaatgt cgttgtggtg                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

ttcagcaagg caatgtcgtt                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

ggatggctgc gcacacctgc                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

agtccgggat ggctgcgcac                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

ggcagatggt ctgtatagtc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

ggcaggcaga tggtctgtat                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

caaagccagt gatctcacag                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

cttttccaaa gccagtgatc                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

gaattctctt ttccaaagcc                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

agatagtcgg tagaattctc                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

tcacaacagt cattttcagc                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

cggtgaccag gctccccagc                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

acttcagagc cgtagtagtg                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

cctggcagga atctgttttc                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

ctgagtctcc ctggcaggaa                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

ggccttggag ggaacagacg                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

gggcacatcc acggccccag                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

tgtccttcag ggcacatcca                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

ggaccctcag agggccaggc                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

cgtatctagt tctcaaatgg                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

tgcaggcttc agtccagaaa                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

cctttttaac tcctgcaggc                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

cccatgctcc agggatccag                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

gagatgccct gcccttttta                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

tacacttcct aattgggaaa                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

ccctgaagcc ttcccaactt                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

gctccctcaa gagacctcag                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

ccctgccctg aagtcgttag                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

ttcctgatac attcatggaa                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

aatcagacac cagctcttac                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

aggtcatgct gcagaataag                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

tgtgaaagtg aaactgagac                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

ggctaaaagg aagggataac                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

actggacagg gtctccttcc                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

gattggatga actaggctaa                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

tgaggattgg atgaactagg                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

agtgaggatt ggatgaacta                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

cacccagtga ggattggatg                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

ttttaaagat gtcgtttcag                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

aaggagtggt cctcacccca                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

tcagtgtaag gagtggtcct                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

aatcacattt tattgatcac                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

aaaatcacat tttattgatc                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

tcagaaaaat cacattttat                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

cttgtcccca aggccatggg                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

ggctgtttcc actcgcaaga                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

acagacagag gctgcatccc                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

gaatccatcc caaggctcca                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

tcccctccaa gtgccgagga                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

gtcaaaacac tcctcaggac                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

atttatctgc ctccacgtgg                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

gactggttta aaccctcaaa                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

aaacttgcct cctgtgctca                                                 20 

 
           
             90  
             9950  
             DNA  
             Mus musculus  
             
 
           
            90 

tatggaatct ctgattcagt ggtcctgtgg gttgctagca gctcggctgc gcaaggaaaa     60 

cgtattctga agaacgatgg tcacctgcct acccaaagct gggtattcaa tgtgtacttt    120 

cctatccaga ggttggcatt ctgggccact ggctggggta aagtcaagca gcctcctcct    180 

tcctacctcc tggcattctt ttccaaggtc caggttgaca gtaaaatgtt gtaggctcag    240 

gcttatggaa ataaagcaaa gctggggcac taatgaatgg tgcgggctca ggcacctctt    300 

ctgtggaagg agcatcaaaa tgaccactgg tccttggctc tgaggcacat gttgcagtga    360 

ctgacccttg gaatcttttc tgagcactcc ctactttgcc tgaggggtgt gtgcatgtgt    420 

gcatgtgtgc aagtgtgtgc atgagggagg gtgctttgtc ttcaagatgt tcagggctca    480 

tttacacatg acctgtcttt atataagcct attttattag ataaattatt agataaataa    540 

ttgttttcgt cttaacatga tgatgtaaaa tgctgaacac aagtgcattt acagtgtgga    600 

attggcaaca gaagagatgg ctaggctcac acccaaaaat aaaatgtgta attatttttt    660 

tagaatgact gatacgagga cgtgtttaaa gtttttaaaa cagtgtgaaa tagtattcag    720 

tgaaaaataa acattccttc atctcttcct tcagacccac agcctctcca ctacaaggga    780 

agagcagaca cccccactgt tgttgcatgt tgatagccca cactaacgag gtttgagtat    840 

tccttgcttt tttttcctat cctgttacaa tgtactgttc atgttactgt tcacatcact    900 

gactgctctt ccagaggtcc tgagttcaat tcccagcgcc acatggtggc tcatgaccat    960 

ctacaatggg atctggtact cttttctggt gtgtctgaag agaggactgt gcactcgcat   1020 

acataaaata aataaatctt taaaaaaaag acacccccct tgattgctta tttgtgtctg   1080 

tgtgcaacca caataccagt gaggtatgca ctcagtcctg atctatgatc ctctgtattg   1140 

gttgtattct tttccagaca aataaataga ttgttcatta taggcatttt tcaaaagacc   1200 

cattgacact tccatagcag ctgctgtgcc aagactttgc tcccacaatc ctaacatagt   1260 

aaccattact gcttttttaa aaatcataat tatttttgag gatttcacta tgtaaattag   1320 

tctgggcttg aactcgcaga gatccacctg cctcttcctc cctagtgttt agataaaaga   1380 

cgtgcaccat tacgtctggc ccttgccata aattttaaaa ttgtctttca aagattgacc   1440 

ttaaaccaaa cagcaaatct gaataaaatc tgaccttgcc tttcaccttc tgatgacagt   1500 

attacactcc ctgacgacaa acttcactct tgtcttctga ttcactgctt gcattagtgg   1560 

catttggaga actcagcatt tgacatgtgg gagcctttgt tagtaggtat tttttattgt   1620 

aaaggaactg cgacttatac ccctctatca gacatctgaa tcagtgtcgt aggcaggtag   1680 

ggggacaggt tggagaagaa ctgattaaac cactaaggaa gaggcttgag atcagcgagc   1740 

caatggctgg agcgccttca ccacgctaca ctggggccgc actaggtgaa tgaaagaaag   1800 

gaagaatgtt caagccgccg gatcatcgct cgatccagac agactgcgtt aagtttgctc   1860 

agctgaaatt ccgtgacttc gtcaaagttg ggaagcaagc gcggtccagt tgcggtggga   1920 

tgcaggaaaa ggaaaaggag agagagagag agagagagag agagagagag agagagagag   1980 

agagggaggg agggagggag ggagggaggg agggagggag ggagggaggg agggagggag   2040 

ggagggagag agagagagag agagagagag agagagagag agagagagag agagagagag   2100 

agccgccctc cagggaacct gggcggggcc agggctctgg cgggccctaa taaagggcga   2160 

gcagcgccga gcagagcctg tagccccaga gctctgtctg tcatccaacc agtccttgcg   2220 

tgtctgccag cgcccttccg ctgcagtcac cggtgagtgc tgttggtctg aagcaagcct   2280 

ggcgggatga ggcagccagg gctcccgcat gcctcccttc cccctacctt ggctggcgga   2340 

actgtgggca aggtcaccac tccagccctt cgcgcccctc tacagagagg ttccatggtg   2400 

ttgtgcggat tcagagcccg cagaggggag agactgcccg gcttggggag gttggtcact   2460 

gatggcttgc cccgcagggt acctggagtg gcttccttcc cttggtctgt agtaactctg   2520 

ccaccttcga gctgctccgc ttcttgtcct gacttctcct tcctttgcag aactgctgtc   2580 

tagagcccag cggcactacc atgaaagtct ggctggcgag cctgttcctc tgcgccttgg   2640 

tggtgaaaaa ctctgaagtg agtggtctcg ctgctttagc accatcagga aggggcttgc   2700 

aggatccctt aagcagcatc aggggaaaaa tgggggctgc acggggaact taggcatcaa   2760 

aggcaggtcc aggctttccc aggaaatagg acaatgtatc agtggagggc ttgtgcaccc   2820 

aaagaggttt gcactatctg gcaagggagg aagaagccac ggggagtacc ttagcccaag   2880 

ggcacctggt ttgtgtgaag tttgcttaag tcagtccatg tctgggtgct ggctaggaat   2940 

aaacagaaag gggagagaca gacaggggtg gggtgggaga aagagagaga gagagagaga   3000 

gagagagaga gagagagaga gagagagaga gagagagaga atattatgag tgaatgaata   3060 

tcactggaag ggattttgag gtggggacct gtttatcctg aacatgaatt ccctagagca   3120 

tgtcaccttc atctcttgca gggtggcagt gtacttggag ctcctgatga atgtgagtat   3180 

ctgcttcctt gcacaatagt tggctgcaca gagacccttg aaaaacctta ggagacatac   3240 

cctccctgtc cctgctgaaa ggctggctcc ccacttgatc cttgcttacc cctcctttgc   3300 

atttgctagc aaactgtggc tgtcagaacg gaggtgtatg cgtgtcctac aagtacttct   3360 

ccagaattcg ccgatgcagc tgcccaagga aattccaggg ggagcactgt gagataggta   3420 

tggggatttg gacttggaat gtgggagtgg gggaggacca gagatcttag aacagggaca   3480 

gatgggtggg atgcagaagc aggcagaagc tggccttgga ggtgtgggtc tgtgagccca   3540 

gcacttagga ggagactgaa gcctggcctc catagtaagt ccttgtctca aaaggcgggc   3600 

gaggcgcagc tagagagaca ggtgagtgat taagaacact ggctgctctt ctagacatcc   3660 

tatagtttga gtttcagcac ccacagaggt gggttacagc catctgtacc cccagtccca   3720 

gggaatctga tgccctcttc tgacctcttc cagcccaagt gacatacatg gtgtacaagt   3780 

atacataaag gaaaaacact catacacata aaataagcaa acaaacaaac aaaacaggca   3840 

gcagaagttg ggagtcacac acacacacac acacacacac acacacacac acacacactc   3900 

atgaagcagt ggctatacaa gtgtgaaaaa aagggtgaat ctccctcata tcacctgaca   3960 

ggtctgaaac cgtgtcacct ctgaaatgcc tgtccaaacc tcatcccttt tctaatactc   4020 

tgcactcctc aaatcatttc tagatgcatc aaaaacctgc tatcatggaa atggtgactc   4080 

ttaccgagga aaggccaaca ctgataccaa aggtcggccc tgcctggcct ggaatgcgcc   4140 

tgctgtcctt cagaaaccct acaatgccca cagacctgat gctattagcc taggcctggg   4200 

gaaacacaat tactgcaggt aggtggtgac tgagtaccaa gaatccttcc caagggggat   4260 

agggaggtgg ctcagcagtt aagagcacag actgcctttc cagagcacct agtttgattc   4320 

ccagggcagc tcgtgacagt ctttaacacc tgttctagag gatccgatgc cctcttctgg   4380 

cctcactggg caggcatgca ctgtcatgaa tataggaaaa cacttataca cattaaaaac   4440 

aacatccctt cccccatcgt ggcctcttag aaacctttgt tatcaccatg gtatacctgg   4500 

gatgggaatc ctggcacaag aatccaggtc tctggttgag cctttgttgg aagggaggat   4560 

acagagaaga cattcgggct tggcatgaca ttccctatct ctttgtgtta ccaggaaccc   4620 

tgacaaccag aagcgaccct ggtgctatgt gcagattggc ctaaggcagt ttgtccaaga   4680 

atgcatggtg catgactgct ctcttagtga gtgtcgctga ctgcttatga caacggggtg   4740 

ggaagagaca aactctattg tcactgcagg agggatgaga agtgaggttg gcctcagaga   4800 

ctcttcatca ttgctgtctc ccccaaacat gtgtctcttt cttttctagg caaaaagcct   4860 

tcttcgtctg tagaccaaca aggcttccag tgtggccaga aggctctaag gccccgcttt   4920 

aagattgttg ggggagaatt cactgaggtg gagaaccagc cctggttcgc agccatctac   4980 

cagaagaaca agggaggaag tcctccctcc tttaaatgtg gtgggagtct catcagtcct   5040 

tgctgggtgg ccagtgccgc acactgcttc atgtacgtcc atccctttgt cccttctctc   5100 

tgactcttcc acccaacccc aagactgtcc ttcctccttc cctatggacg gttacaatgt   5160 

cattctcctg ctaaccctct aaccatgcag cttgtggtct tgggtacaag taatactttg   5220 

aggcctctgg ggtggagtgg agagagtgac cggactttgt gagaccaggc tgacatgttt   5280 

catttctcat agtcaactcc caaagaagga aaactacgtt gtctacctgg gtcagtcgaa   5340 

ggagagctcc tataatcctg gagagatgaa gtttgaggtg gagcagctca tcttgcacga   5400 

atactacagg gaagacagcc tggcctacca taatgatatt ggtgagcaga aagcttagtt   5460 

atcagaaagg ctaaagtagt ggtgggaaat gttgggggac ttgaagcccg ggatttatat   5520 

aacgagacgg atgaggaaga gtgcagaatg agatacatga gaagctgagg ggtgtgggga   5580 

tcctctgtgg agaccttgaa tttcccaaac agatagattc ttctaagtag aaacaatctt   5640 

acaggcatac ggcttaggct gagaatgccc tgtttgtaca aagtaggatg gatgcttctt   5700 

ctctgtatac cagaatatag aaggtataaa gcaaagcctt ggctggattt cagctcagct   5760 

ccctcagcag gaaacaacct gttcagctgt atatggtaga ttttgttgcc cgaacatctg   5820 

tcatctgatg aaataaagca tttggagaat gtggcagggg aggcttcagg gtaacaagat   5880 

accagcagac cttttggatc tctgtgactc ccatgccacg agtatagatc aatgctcagc   5940 

attggtaggg gagagatgat gaccatctga cacagtgata acctttcccc tttgaccttt   6000 

cccttcccca cccagccttg ctgaagatac gtaccagcac aggccaatgt gcacagccat   6060 

ccaggtccat acagaccatc tgcctgcccc caaggtttac tgatgctccg tttggttcag   6120 

actgtgagat cactggcttt ggaaaagagt ctgaaagtag tgacagatga agctcactga   6180 

gagagtctgg gggagtgtta tggtccagag caaagagcag actatcaaag gaagactgtg   6240 

gaaacaggac tggaaacatt atggagggcc agggatagag tagggggaga tgggcaagca   6300 

agtcaaacag ggtgtgaaca attgtgagtg aagtaaaaga ctcagattgg agaaacaaga   6360 

acaagagctt ttcatagctg ggatatgttt tttatcttca cccctgcaga gagtctcatt   6420 

tatagacaca tcttaatgca aacatctgtt tgttccatct aggtgactat ctctatccaa   6480 

agaacctgaa aatgtctgtt gtaaagcttg tttctcatga acagtgtatg cagccccact   6540 

actatggctc tgaaattaat tataaaatgc tgtgtgctgc ggacccagag tggaaaacag   6600 

attcctgcaa ggtaagactc tcaagcaccc ctctttatca ccccaactcc ccagagctct   6660 

tggatttgat ctaacaaccc tggggagtct ctttccagcc aacaatctaa gaatcaagga   6720 

cttaggtctt tgggagcttg tcccaatact tataggttca aacgttgggc atgagtccct   6780 

gtgctatatg cgttttagac taaaagggac caagactgct aaaaaaaaaa taacccagac   6840 

atggtagagc atacctataa cctagcactc ttagcatttt gaatgctgag acaggaggat   6900 

catgagttta aggccagccc aaactacaga gtgagaattc aaggcctgtg ccacatagca   6960 

agatcctttc tcaaataaaa caaggaaagc acaaaccata aaaaccaaga caatagcaac   7020 

aaagggatgg gtgcctagct cagtggttta gtgcttgctt actatgctca aagtcctgag   7080 

ttcaagtctc aactcaggga ctggagatca taagaaaaac taagagtctg gggacgtggc   7140 

ttagttggta gagtacttac ccagcatgaa agaagccctg gatccagtcc tcggcactgt   7200 

atgtaatgac ccaggcctgc agtatcagcc cttgaggggg tcagaatcag tttataagtt   7260 

cttcagttac agagtgagtt caaagccagc ctgaaagaca tgggcttatg agactctgtc   7320 

ccaatctgaa agaacacaac caaccaacca accaccacca ccaacagcaa aatatagata   7380 

ctattcaaat cacttctggg cctttggcaa gacaagtgaa atcaacataa ttctattgtt   7440 

caggatcgca gtgaattacc aaagatcagg taggaaagga aggagaagtc ttaaagagac   7500 

tatgaactgg taaataaaga gacggaagga aaaaggaagc atgtgtcagt tggaaaaaac   7560 

aaaactaaga ctgagcatgc tgtgtgccaa ggcgaggaat agcagggtct gggaaagcac   7620 

tggagagtgg gagaggaaag ctaagacttt ttactcttga ttcggtagaa aatggggagt   7680 

tgcgaatgtc tctgactctg ggaacctctc cccgttctct cccgtggctg ggtagtggcc   7740 

cttccctcag ttcttccagg gcttcacctc tttatctttg gcttcccagg gcgattctgg   7800 

aggaccgctt atctgtaaca tcgaaggccg cccaactctg agtgggattg tgagctgggg   7860 

ccgaggatgt gcagagaaaa acaagcccgg tgtctacacg agggtctcac acttcctgga   7920 

ctggattcaa tcccacattg gagaagagaa aggtctggcc ttctgatggc cctcaggtag   7980 

ctgagggaag aaacagatgg gtcacttgtt cccatgctga ccgtcctctc tgcaacagag   8040 

tcgtcaaatg gagggaagaa gctgaaaaga caggttttgc attgatcctc tgctgtgctg   8100 

cccaccaggg tgagcgccaa tagcattacc ctcagacaca ggcctgggtg ctggccatcc   8160 

agaccctccc gaccaggatg gaaagttggt cctgactcag gatgctatag accaggagtt   8220 

gcctttttat ggactaaagc catctgcagt ttagaaaaca tctcctgggc aagtgtagga   8280 

ggagagctgt ttcccttaat gggtcattca tgagatctgc tgttgggaaa taaatgattt   8340 

cccaattagg aagtgcaaca gctgaggtat tgtgagggtg cttgtccaat atgagaacgg   8400 

tagcttgagg agtagagaca ctaacggctt gagggaacag ctctagcatc ccatgaatgg   8460 

atcaggaaat gttatatttg tgtgtatgtt tgttcactct gcacaggctg tgagtataag   8520 

cctgagcaaa agctggtgta tttctgtatc taactgcaag tctaggtatt tccctaactc   8580 

cagactgtga tgcggggcca tttggtcttc catgtgatgc tccacgtgaa tgtatcattc   8640 

ccgggcgtga cccgtgacta gcactaaatg tcggtttcac tttttatata gatgtccact   8700 

tcttggccag ttatcttttt tttttttttt tttttttttt actaattagc ctagttcatc   8760 

caatcctcac tgggtggggt aaggaccact tctacatact taatatttaa taattatgtt   8820 

ctgctatttt tatttatatc tatttttata attctgagta aaggtgatca ataaatgtga   8880 

tttttctgaa gattctggtt tctccatgat tcttgtgtga cagggaagag ggggacatta   8940 

aaaggaagaa aataatgagg gctacgtgca tcttagtttc atttggggtt tgcttggact   9000 

ttttttggat gagaatgcat ggatgaggct gctgatccaa gccaggcacg gtcctagtcc   9060 

acctgaaggc taaatgaaga ttggtgcaaa ttcaaggtca gcctgacgat gtggttattt   9120 

caaggccagc taggctacat agcaagacat tgtctttaaa aaaaaatgcg caagaaagaa   9180 

aagaaaaaaa tctgattcaa acaaagcagc tgagtcggtg ctgtcgacgg ggtcaggtaa   9240 

tgaagatact tgtgtttgca gctcttggtc ccccgctgaa actacttgta acgcttctgg   9300 

cctctgtagg caccaacacc catgcacaca cacagatgat tacaaataag tcttacagaa   9360 

gaaaacatga aaaaaatcag tgtctcacac ctgtcatccc agcaagtgag aggctgaggc   9420 

aagaagactc ctgtgagttt gaagccaatt ttgctacaaa gctttagtct taaaacagac   9480 

aaaataaaac aaaaagtggg gtggtagtgg tatgccttta atctcagcag aggcagaggt   9540 

tcgaggcctg acttgtctac agagtgagtt caggacagcc aggagctaca catagaaacc   9600 

ttgtctcaaa ataacaataa aataataata acaacaaaac caataaaact aaaccattgt   9660 

gaatctggga ttccagaaag caaacatact tttccatcat ctgtgtgtag gctgatgcta   9720 

aatttccgct gtgctaatgg agcttatctg cacttaatgt ggccttggga aggtacagaa   9780 

ggagagttcc agggttggcc ttcatagcac ctaagttaca aaacaggcca caggctgcgg   9840 

cttggtaagc ggtgttcggg ttgagctgca gctcacaggt gcttcctcag cctggtgcta   9900 

ttgggcagag tacctcgttt attattaatt aattaattaa ttaattaatt              9950 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

cagcagttcg gtgactgcag                                                 20 

 
           
             92  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            92 

tctagacagc agttcggtga                                                 20 

 
           
             93  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            93 

ccagactttc atggtagtgc                                                 20 

 
           
             94  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            94 

gcgcagagga acaggctcgc                                                 20 

 
           
             95  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            95 

cagagttttt caccaccaag                                                 20 

 
           
             96  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            96 

acactgccac cttcagagtt                                                 20 

 
           
             97  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            97 

cacagtttga ttcatcagga                                                 20 

 
           
             98  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            98 

gccacagttt gattcatcag                                                 20 

 
           
             99  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            99 

ctgacagcca cagtttgatt                                                 20 

 
           
             100  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            100 

tccgttctga cagccacagt                                                 20 

 
           
             101  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            101 

acacgcatac acctccgttc                                                 20 

 
           
             102  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            102 

cttgggcagc tgcatcggcg                                                 20 

 
           
             103  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            103 

tttgatgcat ctatctcaca                                                 20 

 
           
             104  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            104 

atgatagcag gtttttgatg                                                 20 

 
           
             105  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            105 

gtaagagtca ccatttccat                                                 20 

 
           
             106  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            106 

agggccgacc tttggtatca                                                 20 

 
           
             107  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            107 

tttccccagg cctaggctaa                                                 20 

 
           
             108  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            108 

aagagagcag tcatgcacca                                                 20 

 
           
             109  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            109 

ggctttttgc taagagagca                                                 20 

 
           
             110  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            110 

ccttgttggt ctacagacga                                                 20 

 
           
             111  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            111 

tcccttgttc ttctggtaga                                                 20 

 
           
             112  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            112 

ccacatttaa aggagggagg                                                 20 

 
           
             113  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            113 

ctcccaccac atttaaagga                                                 20 

 
           
             114  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            114 

actgatgaga ctcccaccac                                                 20 

 
           
             115  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            115 

ttgggagttg aatgaagcag                                                 20 

 
           
             116  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            116 

actgacccag gtagacaacg                                                 20 

 
           
             117  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            117 

cttcgactga cccaggtaga                                                 20 

 
           
             118  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            118 

ggattatagg agctctcctt                                                 20 

 
           
             119  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            119 

tctctccagg attataggag                                                 20 

 
           
             120  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            120 

agatgagctg ctccacctca                                                 20 

 
           
             121  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            121 

tcgtgcaaga tgagctgctc                                                 20 

 
           
             122  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            122 

ctgtagtatt cgtgcaagat                                                 20 

 
           
             123  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            123 

tgctggtacg tatcttcagc                                                 20 

 
           
             124  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            124 

ggcccgtgct ggtacgtatc                                                 20 

 
           
             125  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            125 

ctgaaccaaa cggagcatca                                                 20 

 
           
             126  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            126 

cacagtctga accaaacgga                                                 20 

 
           
             127  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            127 

tgatctcaca gtctgaacca                                                 20 

 
           
             128  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            128 

agatagtcac tttcagactc                                                 20 

 
           
             129  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            129 

attaatttca gagccatagt                                                 20 

 
           
             130  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            130 

tttataatta atttcagagc                                                 20 

 
           
             131  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            131 

cacagcattt tataattaat                                                 20 

 
           
             132  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            132 

cagcacacag cattttataa                                                 20 

 
           
             133  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            133 

ccagaatcgc ccttgcagga                                                 20 

 
           
             134  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            134 

ataagcggtc ctccagaatc                                                 20 

 
           
             135  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            135 

ggcttgtttt tctctgcaca                                                 20 

 
           
             136  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            136 

ttctcttctc caatgtggga                                                 20 

 
           
             137  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            137 

gagggccatc agaaggccag                                                 20 

 
           
             138  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            138 

acgactctgt tgcagagagg                                                 20 

 
           
             139  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            139 

tcagcttctt ccctccattt                                                 20 

 
           
             140  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            140 

aatgcaaaac ctgtcttttc                                                 20 

 
           
             141  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            141 

ctttccatcc tggtcgggag                                                 20 

 
           
             142  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            142 

atcctgagtc aggaccaact                                                 20 

 
           
             143  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            143 

atggctttag tccataaaaa                                                 20 

 
           
             144  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            144 

ctgcagatgg ctttagtcca                                                 20 

 
           
             145  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            145 

ccattaaggg aaacagctct                                                 20 

 
           
             146  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            146 

gcagatctca tgaatgaccc                                                 20 

 
           
             147  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            147 

catttatttc ccaacagcag                                                 20 

 
           
             148  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            148 

caatacctca gctgttgcac                                                 20 

 
           
             149  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            149 

catattggac aagcaccctc                                                 20 

 
           
             150  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            150 

tagacttgca gttagataca                                                 20 

 
           
             151  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            151 

aatacctaga cttgcagtta                                                 20 

 
           
             152  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            152 

tcacgcccgg gaatgataca                                                 20 

 
           
             153  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            153 

tttagtgcta gtcacgggtc                                                 20 

 
           
             154  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            154 

ggacatctat ataaaaagtg                                                 20 

 
           
             155  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            155 

gaagtggaca tctatataaa                                                 20 

 
           
             156  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            156 

gaactaggct aattagtaaa                                                 20 

 
           
             157  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            157 

attgatcacc tttactcaga                                                 20 

 
           
             158  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            158 

aatcacattt attgatcacc                                                 20 

 
           
             159  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            159 

tttattccta gccagcaccc                                                 20 

 
           
             160  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            160 

cagtgttctt aatcactcac                                                 20 

 
           
             161  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            161 

gtctgtgctc ttaactgctg                                                 20 

 
           
             162  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            162 

acgagctgcc ctgggaatca                                                 20 

 
           
             163  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            163 

ccatagggaa ggaggaagga                                                 20 

 
           
             164  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            164 

cctactttgt acaaacaggg                                                 20 

 
           
             165  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            165 

tatttcatca gatgacagat                                                 20 

 
           
             166  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            166 

gtttccagtc ctgtttccac                                                 20 

 
           
             167  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            167 

tgtgtctata aatgagactc                                                 20 

 
           
             168  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            168 

gttcatagtc tctttaagac                                                 20