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Methods and compositions for the inhibition of gene expressionRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellMethods and compositions for the inhibition of gene expression description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20050287667, Methods and compositions for the inhibition of gene expression. Brief Patent Description - Full Patent Description - Patent Application Claims
FIELD OF THE INVENTION [0001] The present invention relates to methods and compositions for the inhibition of gene expression. In particular, the present invention provides oligonucleotide-based therapeutics for the inhibition of oncogenes involved in cancers. BACKGROUND OF THE INVENTION [0002] Oncogenes have become the central concept in understanding cancer biology and may provide valuable targets for therapeutic drugs. All oncogenes and their products operate inside the cell. This makes protein-based drugs ineffective since their specificity involves ligand-receptor recognition. [0003] Antisense oligodeoxyribonucleotides (oligonucleotides) are under investigation of therapeutic compound for specifically targeting oncogenes (Wickstrom, E. (ed). Prospects for antisense nucleic acid therapy of cancer and Aids. New York: Wiley-Liss, Inc. 1991; Murray, J. A. H. (ed). Antisense RNA and DNA New York: Wiley-Liss, Inc. 1992). Antisense drugs are modified synthetic oligonucleotides that work by interfering with ribosomal translation of the target mRNA. The antisense drugs developed thus far destroy the targeted mRNA by binding to it and triggering ribonuclease H (RNase H) degradation of mRNA. Oligonucleotides have a half-life of about 20 minutes and they are therefore rapidly degraded in most cells (Fisher, T. L. et al., Nucleic Acids Res. 21:3857-3865 (1993)). To increase the stability of oligonucleotides, they are often chemically modified, e.g., they are protected by a sulfur replacing one of the phosphate oxygens in the backbone (phosphorothioate) (Milligan, J. F. et al., J. Med. Chem. 36:1923-1937 (1993); Wagner, R. W. et al., Science 260:1510-1513 (1993)). However, this modification can only slow the degradation of antisense and therefore large dosages of antisense drug are required to be effective. [0004] Despite the optimism surrounding the use of antisense therapies, there are a number of serious problems with the use of antisense drugs such as difficulty in getting a sufficient amount of antisense into the cell, non-sequence-specific effects, toxicity due to the large amount of sulfur containing phosphothioates oligonucleotides and their inability to get into their target cells, and high cost due to continuous delivery of large doses. An additional problem with antisense drugs has been their nonspecific activities. [0005] What is needed are additional non-protein based cancer therapeutics that target oncogenes. Therapeutics that are effective in low doses and that are non-toxic to the subject are particularly needed. SUMMARY OF THE INVENTION [0006] The present invention relates to methods and compositions for the inhibition of gene expression. In particular, the present invention provides oligonucleotide-based therapeutics for the inhibition of oncogenes involved in cancers. [0007] Accordingly, in some embodiments, the present invention provides a composition comprising a first oligonucleotide that hybridizes to the promoter region of a c-ki-Ras gene under physiological conditions (e.g., SEQ ID NOs: 47, 48, 50 or 53). In certain embodiments, at least one of the cytosine bases (e.g., all) of the first oligonucleotide are 5-methylcytosine. In some embodiments, the hybridization of the first oligonucleotide to the promoter region of c-ki-Ras gene inhibits expression of the c-ki-Ras gene. In certain embodiments, the c-ki-Ras gene is on a chromosome of a cell, and the hybridization of the first oligonucleotide to the promoter region of c-ki-Ras reduces proliferation of the cell. In some embodiments, the composition further comprises a second oligonucleotide. In some embodiments, the at least one of the cytosine bases in the second oligonucleotide is 5-methylcytosine. In other embodiments, all of the cytosine bases in the second oligonucleotide are 5-methylcytosine. In some embodiments, the second oligonucleotide comprises SEQ ID NOs: 47, 48, 50 or 53, wherein the second oligonucleotide is different than the first oligonucleotide (e.g., if the second oligonucleotide has the sequence of SEQ ID NO:47, the first oligonucleotide has a sequence other than SEQ ID NO:47, etc.). In certain embodiments, the second oligonucleotide hybridizes to a promoter region of a second gene, wherein the second gene is not c-ki-Ras. In some embodiments, the second gene is an oncogene (e.g., including, but not limited to, c-Ha-Ras, c-myc, Her-2, TGF-.alpha., and bcl-2). [0008] In yet other embodiments, the present invention provides a composition comprising an oligonucleotide that hybridizes to a promoter region of a c-ki-Ras gene at a position comprising between nucleotides 1 and 289 of SEQ ID NO: 46 or between nucleotides 432 and 658 of SEQ ID NO: 46. [0009] In other embodiments, the present invention provides a method, comprising providing an oligonucleotide that hybridizes to the promoter of a c-ki-Ras gene (e.g., an oligonucleotide comprising SEQ ID NOs: 47, 48, 50 or 53); and a cell comprising a c-ki-Ras gene that is capable of being expressed, and wherein the cell is capable of proliferation; and introducing the oligonucleotide to the cell. In some embodiments, the oligonucleotide comprising at least one CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine. In other embodiments, all of the cytosine bases in the CG dinucleotide pairs of the oligonucleotide are 5-methylcytosine. In some embodiments, the oligonucleotide hybridizes to a promoter region of a c-ki-Ras gene at a position comprising between nucleotides 1 and 289 of SEQ ID NO: 46 or between nucleotides 432 and 658 of SEQ ID NO: 46. In some embodiments, the oligonucleotide is between 15 and 30 bases in length. [0010] In some preferred embodiments, the introducing results in the reduction of proliferation of the cell. In some embodiments, the introducing results in inhibition of expression of the c-ki-Ras gene. In some embodiments, the cell is a cancer cell (e.g., including, but not limited to, pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate, lymphoma, ovarian, and melanoma). In some embodiments, the cell is in a host animal. In some embodiments, the host animal is a non-human mammal. In some embodiments, the oligonucleotide is introduced to the host animal at a dosage of between 0.01 .mu.g to 100 g, and preferably between 1 mg to 100 mg per kg of body weight. In some embodiments, the oligonucleotide is introduced to the host animal one or more times per day. In other embodiments, the oligonucleotide is introduced to the host animal continuously (e.g., for a period of between 2 hours and 2 weeks). In other embodiments, the cell is in cell culture. In certain embodiments, the method further comprises the step of introducing a test compound to the cell. In some embodiments, the test compound is a known chemotherapy agent. [0011] In some embodiments, the method further provides a drug delivery system. In some embodiments, the drug delivery system comprises a liposome (e.g., a liposome comprising a neutral lipid or a lipid like compound). In some embodiments, the drug delivery system comprises a cell targeting component (e.g., a ligand or ligand like molecule for a cell surface receptor or a nuclear receptor). In certain embodiments, the drug delivery system is for use in vivo, and the oligonucleotide and the liposome are present in the ratio of from 2:1 to 1:3/1 .mu.g to 100 mg per kg body weight. [0012] In still further embodiments, the present invention provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-Ras gene, wherein at least one (e.g., all) of the cytosine bases in the oligonucleotide is 5-methylcytosine. In some embodiments, the oligonucleotide is completely complementary to the promoter region of the c-ki-Ras gene. In other embodiments, the oligonucleotide is partially complementary to the promoter region of the c-ki-Ras gene. Fore example, in certain embodiments, the oligonucleotide contains one mismatch to the promoter region of the c-ki-Ras gene. In some preferred embodiments, the oligonucleotide is complementary only to the promoter region of the c-ki-Ras gene and is not completely complementary to other regions of the human genome. In some embodiments, the oligonucleotide is between 10 nucleotides and 60, and preferably between 15 and 35 nucleotides in length. [0013] The present invention further provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-Ras gene under conditions such that expression of the c-ki-Ras gene is inhibited. [0014] The present invention additionally provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-Ras gene located on a chromosome of a cell under conditions such that proliferation of the cell is reduced. [0015] The present invention also provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-Ras gene, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine; and a second oligonucleotide, the second oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine. [0016] In certain embodiments, the present invention provides a kit comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-Ras gene, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine; and instructions for using the kit for reducing proliferation of a cell comprising a c-ki-Ras gene on a chromosome of the cell or inhibiting gene expression. In some embodiments, the composition in the kit are used for treating cancer in a subject and the instructions comprise instructions for using the kit to treat cancer in the subject. In some embodiments, the instructions are instructions required by the U.S. Food and Drug Agency for labeling of pharmaceuticals. [0017] The present invention also provides a method, comprising: providing a biological sample from a subject diagnosed with a cancer; and reagents for detecting the present or absence of expression of a oncogene in the sample; and detecting the presence or absence of expression of an oncogene in the sample; administering an oligonucleotide that hybridizes under physiological conditions to the promoter region of an oncogene expressed in the biological sample, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine to the subject. [0018] The present invention additionally provides a method of inhibiting the expression of a gene in a subject (e.g., for the treatment of cancer or other hyperproliferative disorders) comprising providing an oligonucleotide that hybridizes under physiological conditions to the promoter region of a gene involved in cancer or a hyperproliferative disorder expressed in the biological sample, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methylcytosine; and administering the oligonucleotide to the subject under conditions such that expression of the gene is inhibited. In some embodiments, the subject is a human. [0019] In yet further embodiments, the present invention provides a method of screening compounds comprising providing a cell comprising a suspected oncogene; and an oligonucleotide that hybridizes to the promoter region of the gene; and administering the oligonucleotide to the cell; and determining if proliferation of the cell is inhibited in the presence of the oligonucleotide relative to the absence of the oligonucleotide. In some embodiments, the cell is in culture (e.g., a cancer cell line). In other embodiments, the cell is in a host animal (e.g., a non-human mammal). In some embodiments, the method is a high-throughput screening method. DESCRIPTION OF THE FIGURES [0020] FIG. 1 shows the nucleic acid sequence of the bcl-2 gene (SEQ ID NO: 1). Continue reading about Methods and compositions for the inhibition of gene expression... 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