AU2015213349A1 - Methods and compositions for the inhibition of gene expression - Google Patents

Abstract

METHODS AND COMPOSITIONS FOR THE INHIBITION OF GENE The present invention relates to methods and compositions for the inhibition of gene expression. In particular, the present invention provides oligonucleotides-based therapeutics for the inhibition of oncogenes involved in cancers.

Description

MIETUODS AND COMPOSITIONS FOR THE INHIBITION OF GENE EXPRESSION This Application claims priority to copending patent appications serial numbers 5 10/858,464, filed 6h/14, 10/858,341b filed 6/1/04, 10/858,145, filed 6/1/04, 10/858,094, filed6/l/04, 10/858013, filed 6i/1/04, 10/858,146, filed 6//04, 60/611,974 filed 09/22/04, and 60/637,212 filed 12/17/04, FIELD OF THE INVENTION 10 The present invention relates to methods and compositions for the inhibition of gcne expression. in particular, the present invention provides oigonucleoide-based therapeutics for the inhibition of oncogenes involved in cancers. BACKGROUND OF T IE INVENTION 15 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 cel. This makes protein-ased drugs ineffective since their specificity involves ligandreceptor recognition, Antisense oligodeoxyribonucleotides (oligonucleoides) are under investigation of 20 therapeutic compound for specifcally targeting oncogers (Wickstron, E. (ed). Prospects for antisense nucleic acid therapy of cancer and Aids. New York: Wiiey-Liss, Inc. 1991: Murray, S A. H. (ed), Antisense RNA and DNA New York: Wiley-Liss, Inc. 1992), Antisense drugs are modified synthetic oligonucleotides that work by interftrmg with ribosomsal translation of the target mRNA. The antisense drugs developed thus far destroy 25 the targeted nRNA by bmding to it and triggering ribonuclease H (RNase R) degradation of mRNA. Oligonucleotides have a half-life of about 20 minutes and they are therefore rapidly degraded in most cells fisherr T . et aL, Nucleic Acids Res. 2:3857~3865 (19)3. To increase the stability of oligonucIotides, they are often chemically modified, e.g, they are protected by a sulfur replacing one of the phophate oxygens in the backbone 30 (phosphorothioate) (Milligan, J, F. et~ at. J. Med, Choe, 36:1923-1937 (1993); Wagner, R. W. et at Science 260:1510-15)3 (199.3)) However, this modification can only slow the degradation of antisense and therefore large dosages of antsense drug are required to he effective.

2 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. 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 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. In one aspect the invention provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to a promoter region of a bcl-2 gene, wherein said first olieonucleotide is SEQ ID NO: 1438 and complements thereof. In another aspect the invention provides a method, comprising a) providing i) a composition of the invention; and ii) a cell capable of expressing a bcl-2 gene, wherein said cell is capable of proliferation; and b) introducing said oligonucleotide to said cell, wherein said administering results in a reduction of proliferation of said cell. In another aspect the invention provides a pharmaceutical composition comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 1438 and a liposome. In some embodiments, the present invention provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a bc-3 gene (e.g., SEQ ID NOs: 1438, 3, 7, 8 or 9). In some embodiments, at least one of the cytosine bases in the first oligonucleotide is 5-methylcytosine. In some embodiments, all of the cytosine bases in the first oligonucleotide are 5-methylcytosine. In 2a some preferred embodiments, the hybridization of the first oligonucleotide to the promoter region of a bcl-2 gene inhibits expression of the bcl-2 gene. In some embodiments, the bcl 2 gene is on a chromosome of a cell, and the hybridization of the first oligonucleotide to the promoter region of bcl-2 gene reduces proliferation of the cell. In some embodiments, the composition further comprises a second oligonucleotide. In some embodiments, the at least one (e.g. all) of the cytosines in the second oligonucleotide are 5-methylcytosine. In some embodiments, the second oligonucleotide comprises SEQ ID NOs: 3, 7, 8 or 9, and is different than the first oligonucleotide (e.g., if the second oligonucleotide has the sequence of SEQ ID NO:3, the first oligonucleotide has a sequence other than SEQ ID NO:3, etc.). In some embodiments, the second oligonucleotide hybridizes to a promoter region of a second gene, wherein the second gene is not bcl-2. In some embodiments, the second gene is an oncogene (e.g., c-ki-Ras, c-Ha-Ras, c-myc, Her-2, or TGF-o). In other embodiments, the present invention provides a composition comprising an oligonucleotide that hybridizes to a promoter region of a bcl-2 gene at a position comprising between nucleotides 1-40 of SEQ ID NO:1, between nucleotides 161-350 of SEQ ID NO:1, between nucleotides 401-590 of SEQ ID NO: I or between nucleotides 1002-1260 of SEQ 1D) N: 1. In yet othe embodiments, the present invention provides a method, comprising; providing an oligonucleotide (e.g., SEQ ID NOs: 1438, 3, 7, 8, or 9); and a cell capable of proliferation, and comprising a bDl-2 gene capable of being expressed; and introducing the oligonucleotide to the cel. In son enbodments the introducing results in the reduction of proliferation of the cel. In certain embodiments, the introducing results in inhibition of expression Af the bcl~2 gene. In some embodiments, the cel is a cancer cell in other embodimets, the cell is in a host animal (eg, a non-human mammal or a hfman), In some 10 embodiments, the oligonucleotide is introduced to the host animal at a dosage of between 0.01 pg to 100 g, and preferably at a dosage of between Img to 100 mg per kg ofbody weight. In some enbodiments, the oigonucleotide is introduced to the host animal one or more times per day, hn other embodiments, the oligonucleotide is introduced to the host animal continuously, in still further embodiments, the cell is in cell culture. in some I5 embodiments, the method further comprises the step of introducing a test compound to the clL In some embodiments, the test compound is a known chemotherapy agent. In some embodiments, the cancer is pancreatic cancer, colon cancer, breast cancer, bladder cancer. lung cancer, leukemia, prostate, lymphoma, ovarian, or melanoma. inl some embodiments, at least one (e.g, al) of the cytosine bases in the oligonucleotide are 5-methylcytosine, 20 In some embodiments, the method further provides a drug delivery system, In sonic embodiments, the drug delivery system comprises a liposome (eg, a liposome comprising a neutral lipid or a lpid like compound), li 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 25 for use In vio, and the olgonucleotide and the liposome ate present m the ratio of from 2:1 to 1:3/i pg to 100 mg per kg body weight, The present invention further provides a method, comprising; providing an oligonucleotide tat hybridizes to the promoter region of a bel-2 gene: and a cell comprising a bcl-2 gene; and introducing the oligonucleotide to the cell. In some embodiments, the 30 oligonucleotide comprises at least one CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinuceotide pair is Wmethyicytosine, In some embodiments, he olinonucleotide is comnpletly complementary to the promoter region of the bcl-2 gene. In other embodiments, the oligonucleotide s partially complementary to the promoter region of the bel-2 gene. For example, in certain enbodiments, the oligonucleotide contains one 3 mismatch to the promoter region of the bcl-2 gene, hi somc preferred embodiments, the oligonucleotide is complementary only to the promoter region of the bc~2 gene and is not completely complementary to other regions of the human genome tn some embodiments, the oligonucleotde is between 10 nucleotides and 60, and preferably between 13 and .f5 5 nucleotides in length, In some embodiments, the method further provides a drug delivery system, I some embodiments, the drug delivery system comprises a liposome (e-g, a liposorne 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 10 surface receptor or a nuclear receptor ), n certain embodiments, the drug delivery system is for use in vivo, and the oligocnuceotide and the liposome are present in the ratio of from 2: to 1:3/1 ig to 100 mg per kg body weight. In still further embodiments, the present invention provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the 15 promoter region of a bN- gene. In some embodiments, the oligonucleotide comprises at least one CG dinueleotide pair, wherein at least one of the cytosine bases in the CG dinucleotde pair is S-methylcytosine, Tn some embodiments, the oligonucleotide is completely complementary to the promoter region of the bcl-2 gene, ha other embodiments, the oligonucleotide is partially complemetary to the promoter region of the bcl-2 gene> For 20 example, in certain embodiments, the oligonucleotide contains one mismatch to the proroer region of the bcV2 gene. In some preferred embodiments, the eligonucleotide is complemenary only to the promoter region of die bWf-2 gene and is not completely complemrentay to other regions of the humangenome, i some embodiments, the oligonucleotide is between 10 nucleotides and 60, and preferably between 15S and 35 12 5 nucleotides m length. The present invention further provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a bcl-2 gene under eonditions such that expression of the bei-2 gene is inhibited. The present invention additionally provides a composition comprising an 30 oligonucleotide that hybridizes under physiological conditions to the promoter region of a bel-2 gene located on a chromosome of a cell under conditions such that proliferation of the cell is reduced, The present invention also provides a composition comprising a first oligonucleotide that hybridiues ander physiological conditions to the promoter region of a bcl~2 gene, the 4 oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises $-methylcytosine; and a second oligonucieotide, the second oligonucleotide comprising at least on CG dinucleotide pamr wherein at least one of the cytosine bases in the CG dinueleotide pair comprises 5 5 methylcytosine, In certain embodiments, the present invention provides a kit comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a bel-2 gene, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotde pair is 5-methylcytosine; and instructions 10 for 'sing the kit for reducing proliferation of a cell comprising a bcl-2 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 usig the kit to treat cancer in the subject In some embodiments, the instructions are instructions required by the US. Food and Drug Agency for labeling of 15 phannaceuticals. 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' introducing an oigonucleotide that hybridizes 20 under physiological condhions to the promoter region of an oncogene expressed in the biological sample, the oligonucleotide comprising at least on C() dinuceotide pair wherein at least one of the cytosine bases in the CC dinucleotide pair is $-methyklytosine to the subject. The present inventon additionally provides a method of inhibiting the expression of 25 a gene in a subject (eg, for the treatment of cancer or other hyperproliferative disorders) comprising provding 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 30 methylcytosine; and admmistering the oligonucleotide to the subject under conditions such that expression of the gene is inhibited, In some embodimens; the subject is a human, In yet fuher embodiments, the present invention provides a method of screenmg compounds comprising providing a cell comprising a suspected oncogene; and an oligonucleotide that hybridizes to the promoter region of the gene; and introducing the oligonucleotide to the cell; and detemining if proliferation of the cell is nhibited 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-iuman nanmab. In some embodiments, the method is a 5 high-throughput screening method, Accordingly, in some embodiments, the present invention provides a conposition comprising a first oligonucleotide that hybridizes to the promoter region ofa e-kt-ias gene under physiological conditions (e.g, SEQ U) NOs: 47, 48, 50 or $3), In certain embodiments, at least one of the cytosine bases (eg, all) oC the first oligomacleotide are 5 10 methyleytosine. in some embodiments, the hybridization of the first oligonucleotide to the promoter region of Ai-Ras gene inhibits expression of the c-ki-Ras gene, In certain embodiments, the e-ki-R as gene is on a chromosome of a cit and the hybridization of the first oligonucieotide to the promoter region of c-ki-Ras reduces proliferation of the cell, in some embodiments, the composition further comprises a second oligonucleoide. In some 15 embodiments, the at least one of the cytosine bases in the second &lgonucieotide is 5 methylcytosine, in other embodiments, all of theosinesine bases in the second olgonucleotide are 5-methylcytosine. In some embodimenms. the second oligonucleotide comprises SEQ D NOs: 47, 48, 50 or 53, wherein the second oligonucleotide is ditThrent than the first oligonucleotide (eg, if the second oligonucleotide has the sequence of SEQ 20 Dz NO:47, the first oligonucleotide has a sequence other than SEQ ID NO:47, etc.). In certain embodiments, the second oligontcleotide hybridizes to a promoter region of a second gene, wherein the second gene is not c-ki-Ras. In seome embodiments, the second gene is an oncogene (e.g, including, but not limited to c-Ha-Ras, c-myc, Her~2, TGF-a, and b2), 25 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 j and 289 of SEQ ID NO: 46 or between nucleotides 432 and 658 of SEQ 1) NO: 4 6 in other embodiments, the present invention provides a method, composing 30 providing an oligonucleotide that hybridizes to the promoter of a c-ki-Ras gene (eg, 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 introducmg the oligonucleotide to the cell in sonm embodiments, the oligonucleotide comprising at least one CG dinucleotide pair, where at least one of the cytosine bases in 6 the CG dinucleotide pair comprises 5-methyicytosine, in other embodimens, all of the cvtosinc bases in the CC dinucleotide pairs of the oligonucleotidare 5-methylcytosine, in some embodiments, the oligonucleotide hybridizes to a promoter region of a e-ki-Ras gene at a position compnssng between nucleotides aid 289 of SEQ ID NO: 46 or between $ nucteotides 432 and 658 of SEQ ID NO: 46. in some embodiments, the oligonucleotide is between .S and 30 bases in length. h some preferred embodimnents, the introducing results in the reduction of proliferation of the cell In some embodiments, the introducing results in inhibition of expression of the cki-Ras gene. In some embodiments, the cell is a cancer cell (eg., 10 including, but not limited to, pancre atic cancer, colon cancer, breast cancer, bladder cancer, lung cance-, leukemia, prostate, lymphoma, ovarian, and melanoma) in somne embodiments, the cell is n a host anima. 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 p.g to 100 g, and preferably between I ig to 100 mg per 15 kg of body weight, in some embodiments, the oligonucieotide is introduced to the host animal one or more tines per day. In other embodiments, the oligonucleotide is introduced to the host animal continuously (eg., 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 sonie embodiments, the 20 test compound is a known chemotherapy agent, In some embodinens, the method further provides a drug delivery system in sonic embodiments, the drug delivery system comprises a liposome (eg. 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 2$ 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 jg to I00 mg per kg body weight, in still further embodiments, the preset invention provides a composition comprisng an oligonucleotide that hybridizes under physiological conditions to the 30 promoter region of a c-kiRas gene, wherein at least one (e.g, all) of the cytosine bases in the ohgonucleoide is 5-methyieytosine. in some embodiments the oligonucleotide is completely complementary to the promoter region oftthe c-ki-Ras gene, hi otter embodiments, the oligonucleotide is partially complementary to the promoter region of the c-kiRas gene. Fore example, in cenain embodiments, the oligonucleotide contains one mismatch to the promoter region of the ckiRas gene. In some preferred embodiments, the oligonucleotide is complementary only 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. The present invention further provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-iRas geneo under conditions such that expression of the e-ki-Ras gene is inhibited, The present invention additionally provides a composition comprising an 10 oigonmleotide 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. The present invention also provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-ki-R as gene, the 1 $ oligonucleotlde comprising at least on CO dinucleotide pair, wherein at least one of the cyrosine bases in the CG dnucleotide 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. 20 In certain enbodimrents, 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 CO dinucLeotide pair comprises 5-methylcytosine; and instructions for using the kit for reducing proliferation of a cell omprising a e-i-Ras gene 25 on a chromosome of the cell or inhibitmg 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 US. Food and Drug Agency for labeling of pharmaceuticals, 30 The present invention also provides a method, comprising: providing a biological sample from a subject diagnosed with a cancer; and reagents fr 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 8 biological sample, the oigonucleotde comprising at least on CG dinacleotide pair, wherein at least one of the cytosine bases in the CG dinuceotide pair comprises 3-methylcytosine to the subject. The present invention additionally provides a method of inhibiting die expression of 5 a gene in a subject (eg, for the treatment of cancer or other hyperproliferative disorders) comprising providing an oligonucieotide 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 a. least on CC dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5 10 methyleytosine; and administering the oligonucleotide to the subject uder conditions such that expression of the gene is inhibited in some embodiments, the subject is a human, in yet further embodiments, the present invention provides a method of screenmg compounds comprising providing a cell comprising a suspected oncogene; and an oigonucleotide that hybridizes to the promoter region ofth gene; and administering the 15 oligonucleotide to the celt and determining if proliferation o rthe cell is inhibited in the presence of the oligonucleotide relative to the absence of the oligonucleotide, In some embodiments, the cell is in culture (eg, a ancer cell line), il other embodiments, the cell is in a host animal (eg, a nothnunan ianma). in some embodiments, the method is a high-throughput screening method. 20 in some embodiments, the present invention provides a composition comprising a first oligunucleotide that hybridizes to the promoter region of a -myc gene inder physiologal conditions (e.g, SEQ 1 NOs: 110, 111, 112, 113, 114 or 115), In some embodiments, at least one (e g, all) of the cytosine bases in the first ofigonudeotide is methylcytosine, In some preferred embodriments, the hybndization of the first 25 oligonucleotide to the promoter region of a yc gene inhibits exprssion ofthe C-wyo gene. in sonc embodiments, the c-nye gene is on a chromosome of a cell and the hybridization of thc first ohgonucieotide to the promoter region of the c-myc gene reduces proliferation of the ceL in some embodimens, the composition fUrther comprises a second ohgonucleotide. in some enbodinens, at least one (eg, al) of the cytosine bases in the 30 second oligonucleotide are 5-methylkytosine, in some embodimens, the second oigonucleotide comprises SEQ ID NOs: i10, 111, 112, 113, 114 or 115, wherein the second oligonucleotidc is different from the first oligonucleotide (e.g, if the second oligonucleotide has the sequence of SEQ ID NO: 110, the frst oligonucleotide has a sequence other than SEQ D NO: I 10, etc.). In some embodiments, the second 9 oigonucleotide hybridizes to a promoter region of a second gene, wherein the second gene is not c-mye In some embodiments, the second gene is an oncogene (e.g, c-ki-Ras, c-Ha Ras, bcl-2, Her-2, or TGF-a), The present invention further provides a composition comprising an ligonucleoide 5that hybridizes to a promoter region of a c-nyc gene at a position comprising between nucleotides 3-124 of SEQ ID NO:108 or between nucleotides 165-629 of SEQ PD NO:108. The present invention further provides a method, comprising: providing an oligonuclotide that hybridizes to the promoter region of a -mnye gene under physiological conditions (e.g, SEQ L) Ntis: 110, 11 L 112,11 , 114 or 115); and a cell comprising a c 1t mye gene capable of expressing the e-mye gene, and wherein the cell is capable of proliferation; and introducing the oligonucieotide to the cell In some embodiments, the introducing results in the reduction of proliferation of the cell. In certain embodimens, the iroducing results in inhibition of expression of the e-ny gene- In some embodiments, the cell is a cancer cell In other embodiments, the cel a in a host animal (e,g. a non-human 15 mamnd or a human). In some embodiment, the oligonucleotide is introduced to the host animal at a dosage of between 0.01 pg to 100 g, and preferably at a dosage of between lmg 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. In still fther embodiments, the cell is in cell 20 culture. in some 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. In some embodiments, the cancer is pan creatic cancer, colon cancer, breast cancer, bladder cancer lung cancer, leukemia, prostate, lymphoma, ovarian, or mnelaneoma. In some embodiments, the method further provides a drug delivery system, In some 25 embodiments, the drug delivery system comprises a liposome (eg,. a liposome comprising a neutral lpid 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 m vivo, and the oligonucleotide and the liposome are presem in the ratio of from 2:1 30 to 1:3/1 jg to 100 mg per kg body weight. In some embodimens, at least one, and preferably all, of the cytosine bases in the oligoncleotide are 5-nethylcytosine. In some enbodimens the oligonucleoutide is between 10 nucleorides and 60, and preferably between 15 and 35 nucleotides in length. in some embodiments, the oligonucleotide hybidizes to a promoter region of a c-nyc gene at I Q a position compnsing between nucleotides 3i 24 of SEQ ID NO:108 or between nucleoides 165-629 of SEQ ID NO: 108. I still further embodiments, the present mvention provides a composition compnsing an oligonucleotide that hybridizes under physiological conditions to the 5 promoter region of a c-myc gene, the oligonucleotide comprising at least on CG dinucleotide pait, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methykytosine, in some embodiments, the oligonueleotide is completely complementary to the promoter region of the c-myc gene. In other embodiments, the oligonucleotide is partialy complementary to the promoter region of the c-myc gene. For 1 Q example, in certain embodments, the oligonueleot id contains one mismatch to the promoter region of the -mye gene, In some preferred embodiments, the ohgonucleotide is complementary only to the promoter region of the c-myc gene and is not completely cnnplementary to other regions of the human genome. in some enhodiments, the oligonuceotide is between 10 nucleotides and 60, and preferably between 15 and 35 15 nucleotides in length. T he present invention further provides a composition comprsing an oligonucieotide that hybridizes under physiological conditions to the promoter region of a -myc gene under conditions such that expression of the c-mye gene is inhibited. The present invention additionally provides a composition compising an 20 oligonucleotide that hybridizes under physiological conditions to the promoter region of a C myc gene located on a chromosome of a cell under conditions such that proliferation of the cell is reduced. The present invention also provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a c-my gene, the 25 oligonucleotide comprising at least on CG dinucleotde pair where at least one of the cytosine bases in the C dinuceotide pair comprises 5-nethyleytosine; and a second oligonucleotide, the second oligonucleotide comprising at least on CC dinucleotide pair, wherein at least one of te cytosine bases in the CG dinucleotide pair comprises 5 methyleytosme. 30hi certain embodiments, the present invention provides a kit comparing an oligonueleotide that hybridizes under physiological conditions to the promoter region of a C~ mye gent, the oligonucleotide comprising at least on CG dinueleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5-methyteytosine; and instructions for using the kt for reducing proliferation of a cell comprismg a C-myc gene on i a chromosome ofthe cell or inhibiting gene expression. ht 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 ifnstructions are instructions required by the U.S, Food and Drug Agency for labeing of Pharmaceuticals. The present invention also provides a method, comprising: providing a biological sample ferm a subject diagnosed with a cancer; and reagents for detecting the present or absence of expression of a oncogene in the sample; and detecting tIe presence or absence of expression of an oncogene in the sample; administering an oligonucleotide that hybridizes 10 under physiological cnditions to the promoter region of an oncogene expressed in the biological sample, the oiigonicleotide compnsmg at least on CG dinucleotide par, wherein at least one of the cytosme bases in the CG diinucCotide pair comprises 5-methyicytosine to the subject. The present invention additionally provides a method of inhibiting the expression of 15 a gene in a subject (e.g., for the treatment of cancer or other hyperproliferative disorders) composing providing an oligonucleotide that hybridizes under physiological conditions to the promoter region of a gene involved in cancer or a hyperprliferative 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 20 methylcytosine; and administering the olgonucleutide to the subject under conditions such that expression of the gene is inhibited. In some embodiments, the subJect is a human, In yet father embodiments, te present invention provides a method of screening compounds comprising providing a cCl comprising a suspected oncogene, and an obgonucleotde that hybridizes to the promoter region of the gene; and administering the 25 oiigonucleotide to the cell; and determining if proliferation of the cell is inhbited in the presence of the oligonucleotide relative to the absence of the oligonucleotide. In some embodmients, the cell is in culture (e.g., a cancer cell Wie) In other embodiments, the cell is in a host animal (e.U, a non-human mammab. In some embodiments, the method is a high-throughput screening method, .30 In some embodments, the present invention provides a composition comprising a first oligonucleotide that hybridizes to the prmoter region of a c-Ha-ras gene under physiological conditions (eg, SEQ ID NOs: 67, 68, 69, 71 73, 74, 76, 78, 84, 160, 16, or 162) In some embodiments, at least one of the cytosine bases in the first oligonucleotide is 5-methylcytosin tI some embodiments, all of the cytosine bases in the tist 12 oligonucleotide are 5-methylcytosine, hi certain embodiments, the frst oligonucleotide hybridizes under physiological conditions to the promoter region of a c-Ha-ras gene. In sonme preferred embodiments, the hybridization of the first oligonucleotide to the promoter region of a ,da-ras gene inhibits expression of'the e-Ha-ras gene In some embodiments, 5 the cHaras gene is on a chromosome of a cell, and wherein the hybridization of the First oligonueleotide to the promoter region of c-Ia-ras gene reduces proliferation of the cell, in some embodiments, the composition further comprises a second oligonucleotide. In some embodinents, the at least one (e.g, all) of the cytosines in the second oigomuciotidc are 5 inethykcytosine, in some embodiments, the second oligonucleotide comprises SEQ ID 10 NOs: 67, 68, 69, 71, 73, 74, 76, 78, 84, 160, 161, or 162, wherein the first oligonucleodde is different from the second oligonucleotide (eNg, i f the second oligonucleotide has the sequence of SEQ ID NO:T, the first oligonucleotide has a sequence different than SEQ ID NO:6? ctc,). Jn sonc cmbodiments, the second oligonucleotide hybridizes to a promoter region of a second gene, wherein the second gene is not c-Ha-as In soni embodiments, 15 'he second gene is an oncogene (eg, c-ki-Ras, c-nye, bet-2, Her-2, or TGFo). In other embodiments, the present invention provides a composition comprising an oligonucleotde that hybridizes to a promoter region of a e-Ha-ras gene at a position comprising between nucleotides 21-220 of SEQ ID NO:66, 233-860 of SEQ ID NO:66, 1411 -1530 of SEQ ID NO:66 or between nucleotides 1631-1722 of SEQ ID NO:66, 20 In yet other embodiments, the present invention provides a method, comprising: providing an oligonucleotide that hybridizes to the promoter region of a c-Ha-Ras gene under physiological conditions (c.g, SEQ ID NOs: 67, 8, 69 71,"73, 74, 76, 78 84, 160, 161, or 162); and a cell comprising a c4a-ras gene capable of expression, and wherein the cell is capab of proliferation; and introducing the oligonucleotide to the cell in some 25 embodiments, the oligonucleotide is between 15 and 30 bases in length. in some embodiments, the nlgonucleotide hybridizes to the promoter region of the e-Ha-ras gene at a position comprising between nucteotides 21220 of SEQ ID NO:66, 233,-860 of SEQ ID N:66, 1411-1530 of SEQ ID NO:66 or between nucleotides 163 t 1722 of SEQ ID NO:66. In some embodiments, the introducing results i the reduction ofproliferation of the 30 Ccl in certain embodiments, the introducing results in inhibition of expression of the C-Ha ras gene. In some embodimens, the cell is a cancer cell in some embodiniers the cancer is pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate, lymphoma, ovarian, or melanoma in other embodiments, the cell is in a host animal (e.g, a non-human mammal or a human), in some embodiments, the 113 ohigonucleotide is introduced to the host animal at a dosage of between o.01 pg to 100 g, and preferably between 1mg to 100 mg per kg of body weight. In some embodiments the oligonucleoide is introduced to the host animal one or more times per day. In other enbodiments. the ohgonucieotide is introduced to the host animal continuously (e.g. for a 5 period of between 2 hours and 2 weeks). In other embodiments, the cell is in cell culture, hi 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. In some embodiments, the method further provides a drug delivery system in some 10 embodiments, the drug delivery system comprises a liposome (e.g, a liposome comprising a neutral Hpd or a lipid like compound) in some embodiments, the drug delivery system comprises a cell targeting component (e., a ligand or 4igand 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 15 to 1:3/1 pg to 100 mg per kg body weight, In st furthr embodiments, the present invention provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a cIa-Hras gene. In some embodiments, at least one (. all) of the cytosine bases in the oligonucleotide are $-methylcytosine, In some embodiment, the 20 oligonucleotide is completely complementary to the promoter region of the c-Hairas gene, In other embodiments, the oligomielctide is partially complementary to the promoter region of the c-Ha-ras gene. For example, in certain embodiments, the oligonucleotide contains one mismatch to the promoter region of the C4a-ras gene. in sonm preferred embodiments, the olgonucleotide is complementary only to the promoter region of the c 25 Ha-ras gene and is not completely complememary to ohr regions of the human genome, In some embodiments, the oligonuclieotide is between 10 nucleotides and 60, and preferably between 15 and 35 nucleotides in length. The present invention further provides a composition comprising an oligonueleotide that hybridizes under physiological conditions to the promoter region of a c-Ha-ras gene 30 under conditions such that expression of the c-Ha-ras gene is inhibited. The present invention additionally provides a composition comprising an oligonucleotide that hybridires under physiological conditions to the promoter region of a c Ha-ras gene located on a chromosome of a cell under conditions such that proliferation of the cell is reduced. 14 The present invention also provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a c 4 aras gene, the oligonucleoride 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 5 oligonucleotide, the second oligonucleotide comprising at least on CG dinucleotide pair, w herein at least one of the cytosine bases in the CG dinucleotide pair comprises & rnethylcytosine In certain embodiments, the present invention provides a kit comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a c 10 Ha-ras gene, the oligonucleotide comprising at least on CO dinucleotide pair, wherein at least one of the cytosine bases in the CG dinueleotide pair comprises 5-methylcytosine and instructions for using the kit for reducing proliferation of a el comprising a c-Ha-ras gene on a chromosome of the cell or inhibiting gene expression. In some ebodirents, the composition in the kit are used for treating cancer in a subject and the instructions comprise 15 instructions for using the kit to treat cancer in the subject, ln some embodiments, the instructions are mstructions required by the U.S. Food and Drug Agency for labeling of pharmaceuticals, The present invention also provides a method, conprisng: providmg a biological sample from a subject diagnosed with a cancer; and reagents for detecting dhe present or 20 absence of expression of a oncogene in the sample; and detecting the presence or absence of expression ofan oncogene in die sample; administering an oligonucleotide that hybridizes inder 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-methyleytosine to 25 the subject. The present mention additionally provides a method of inhibiting the expression of a gene in a subject (eg. for the treatment of cancer or other hyperproiierative disorders) comprising providing an oligonucleotide that hybridizes under physiological conditions to the promioer region of a gene involved in cancer or a hyperproli ferative disorder expressed 30 in the biological sample, the oligonucleotide comprising a least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CO dinucleotide pair comprises 5 methylcytosine; and administering he oligonucleotide to the subject under conditions such that expression of the gene is inhibited. In some embodimens, the subject is a human, 15 n yet Ifther embodiments, the present invention provides a method of screening compounds comprising providing a cell comprising a suspected oncogene; and an ohgonucleotide that hybridizes to the promoter region of the gene; and administering the oligonuclcotide to the cell; and detemiing if proliferation of the cell is inhibied in the presence of the oligonucleotide relative to the absence of the oligonudeotide. 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, in some embodiments, the present invention provides a composition comprising an 10 oigonucleotide comprising SEQ i) NOs: 31, 32, 35, 36.37, or 38, in some embodiments, te oligonucleotide comprises at least one CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5i'nethylcytosine, In some embodiments, all of the cytosine bases in all of the CG dinucleotide pairs of the oligonucleotide are 5-methylcytosine, In certain embodiments, the oligonwleoide Is hybridizes under physiological conditions to the promoter region of a Her-2 gene, In some preferred embodiments, the hybridizaion of the oligonucleotide to the promoter region of a Her-2 gene inhibits expression of the et-2 gene. In some embodiments, the Her-2 gene is on a chromosome of a cell, and wherein the hybridization of the oligonucleotide to the promoter region of a Her-2 gene reduces proliferaton of the cel In some embodiments. 20 the composition further comprises a second oligonucieotide. in some embodimens, the second oligonucleotide comprising at least on CG dinueleotide pair, wherein at least one of the cytosine bases in the CC dinucleotide pair comprises 5-methyleytosine. In some embodiments, the second oligonucleotde comprises SEQ ID NOs: 31, 32, 35, 36, 37, or 38. In some embodiments, the second ohgonucleotide hybridizes to a promoter region of a 25 second gene, wherein the second gene is not Her-2. In some embodiments, the second gene is an oncogene (eg, c-ki-Ras, c-myc, bel-2, e4a-ras, or TGF-a), in other embodiments, the present invention provides a composition comprising an oligonueleotide that hybridizes to a promoter region of a e-myc gene at a position conprisingbetween nucleotides 205044 of SEQ ID NO:29 or between nucleotides 382-435 30 of SEQ ,D NO:29, in still further embodiments, the present invention provides a composition composing an oligonucleotide that hybridizes under physiological conditions to the promoter region of a Her-2 gene, 16 In yet other embodiments, the present invention provides a method, comprising pnoviding an oigonucleotide (e,g- SEQ ID NOs: 31, 32, 35, 3b, 37, or 38) and a cell comprising a Hkler-2 gene; and administering the oligonucleotide to the cell. In sone embodiments, the administering results in the reduction of proliferation of the celL. In certain embodimentst administration results in inhibitiono of expression of the Her-2 gene, In som embodiments, the cell is a cancer celt In other embodiments, the cell is in a host animal (e.g., a non-human mammal or a huran). In some embodiments, the oligonucleotide is administered to the host animal at a dosage of between 0,01 pg to 100 g, and preferably at a dosage of between 1mg to .100mg per kg of body weight In some 10 embodiments, the oligonucleotide is administered to the host animal one or more times per day. In other embodiments, the oligonucleotide is administered to the host animal continuously. in still further enbodimnents, the cell is im cl culture. In some embodiments the method further comprises the step of administering a test compound to the cell. In some embodiments, the test compound is a known chemotherapy agent, In some embodiments, I5 the cancer is pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate, lymphoma, ovarian, or melanoma. In some embodiments, the oligoaucleotide comprises at least one CG dinueleotide pair, wherein at least one, and preferably all, of the cytosine bases in the CG dinucleotde pair comprises 5 methylcytosne. 20 The present invention further provides a method, composing providing an oligonucleotide that hybridizcs to the promoter region of a H-er2 gene; and a cell comprising a Her-2 gene: and administering the oligonucleotide to the cell In still further embodiments, the present invention provides a composition comprising an oligonucleotide that hybrdizes under physiological conditions to the 23 promoter region of a Her-2 gene, the oligonucleotide comprising at least on CC dinucleotide pair, wherein at least one of the cytosine bases n the CG dinucleotide pawr comprises $-mothylytosine. ia soni embodiments, the oligonucleotide is completely complementary to the promoter region of the Her-2 gene. In other embodiments, the oligonucleotide is partially complementary to the promoter region of the Her-2 gene. For 30 example, in certain embodiments, the oligonucleotide contains one mismatch to the promoter region of the her-2 gene. hn some preferred embodiments, the oligonucleotide i complementsty only to the promoter region of the Her-2 gene arid is not completely complementary to other regions of the human genome. In some embodiments, the 17 oiigonucleotide is between (0 nueleotides and 60, and preferably between 15 and 35 nucleotides in length. The present invention further provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a Her- gene under 5 conditions such that expression of the Her-2 gene is ihibted, The present invention additionally provides a composition comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a ler-2 gene located on a chromosome o a cell under conditions such that proliferaion of the cell is reduced, 10 The present invention also provides a composition comprismg a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a Her-2 gene, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of th cytosine bases in the CG dinucteotide pair comprises $-methyleytosine; and a second oligonucleotide, the second oligonucleotide comprising at least on CG dinucleotide pair, 15 wherein at least one of the cytosine bases in the C dinucleotide pair comprises 5 miethyicytosine In certain embodiments, the present invention provides a kit comprising an oigonucleotide that hybrIdizes under physiological conditions to the promoter region of a Her-2 gene, the oligoruleotide composing at least on CG dinucleotide pair, wherein at 20 least one of the cytosine bases in the CG dinocleotide pair comprises 5-methylcytosine; and instructions for using the kit for reducing proliferation of a cell comprising a Her-2 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 25 instructions are instructions required by the U.S. Food and Drug Agency for labeling of phanmaceuticals, 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 30 expression of an oncogene in the sample; administering an oligonucleotide that hybridizes Mder physiological conditions to the promoter region of an oncogene expressed in the biological sample, the oligonucleodtde comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the C( dinucleotidc pair comprises 5-methylcytosine to the subject, 18 The present invention additionally provides a method of inhibitmg the expression of a gene in a subject (eg. for the treatment of cancer or other hyperproliferative disorders) comprising providing an oligonucleotide that hybridizcs under physiological conditions to the promtoter region of a gene evolved 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 dinuceoidde pair comprises 5 methylcytosine; and adniisierxng the ohgonueceotide to the subject under conditions such that expression of the gene is inhibited. In some embodiments, the subject is a hunan. In yet further embodirnents, the present invention provides a method of screening 10 compounds comprising providing a cel comprising a suspected oncogene; and an oligonueleotde 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 oligonucleoRide relative to the absence of the ohgonucleotide. In some embodiments, the cell is in culture (e.g., a cancer cell line), in other embodments, the cell 15 is in a host animal (e.g, a non-human mammal). In som embodiments, the method is a high-throughput screening method, In some embodiments, the present invention provides a composition comprising an oligonucleotide comprising SEQ ID NOs: 134, 136, 139, 140, 141, 142, 143, or 144. In soc enbodimnents, the oligonucleotide comprises at least one CG dinucleotide pair, 20 wherein at least one of the cytosine bases in the CCG dinucleotide pair comprises 5 miethyleytosine. In some embodiments, all of the cytosine bases in all of the CG dinucleotide pairs of the oligonucleotide are 5-methylcytosine, In certain embodiments, the oligonucleotide hybridizes under physiological conditions to the promoter region of' a TGF a gene, in some embodiments, the hybridikation of the oligonucleotide to the promoter 25 region of a TGF-a gene inhibits expression of the TGF-a gene. in some embodimens, the TGF-& gene is on a chromosome of a cell, and wherein the hybridization of the oligonucleotide to the promoter region of a TGF-a gene reduces proliferation of the cell. In some embodiments, the composition further comprises a second oigoneleotide. In some embodimems, the second oligonucleotide comprismg at least on CG dinuc etide pair, 30 wherein at least one of the cytosine bases in the CG dinucleotide pair composes 5 methylcytosine. in some embodiments, the second oligonuceotide is selected from the group consistmg of SEQ ID NOs: 134, 136, 139, 140, 141, 142, 143. and 144, il other embodients, the second oligonuceotide hybridizes to a promoter region of a second gene, 19 wherein the second gene is not TGFa. in still further emboiments, the second gene is an oncogene (e.g, c-d-Ras, c-Ha-Ras, bel-2, Her-2, or c-myc). in other embodiments, he present invention provides a composition comprising an oligonMcotide that hybridizes to a promoter region of a c-mye gene at a position 5 comprising between nucleotides -90 of SEQ ID NO: between oigonucleotdes 175 219 of SEQ ID NO:31, between nucleoides 261-367 of SEQ IL) NO:131, between nucleoides 431-930 of SEQ ID NO: 131, or between nucleotides 964,237 of SEQ ID NO131. hi yet other embodiments, the present invention provides a composition comprsig 0 an oligonucletide that hybridizes inder physiological conditions to the promoter region of a TGF-a gene, In still further embodiments, the present invention provides a method, comprising providing an oligonucleotide (e.g. SEQ ID NOs: 134, 136, 139, 140, 141, 142, 143, or 144); and a cell comprising a TGFhn gene; and administering the oligonueleotide to %he cell 15 In some embodiments, the administering results in the reduction of proliferation of the celt in certain embodiments, the administration results in inhibition of expression of the TCF-a gene. in some embodiments, the cell is a cancer ell In other embodiments, die cell is in a host animal (eg, a nonhuman mammal or a human). In some embodiments, the oligonucleotide is administered to the host animal at a dosage of between 0.01 gg to 100 g, 20 and preferably at a dosage of between 1mg to 100 mg per kg of body weight. In some embodiments, the oiigonucleotide is administered to the host animal one or more times per day, 4n other embodiments, the oligonucleotide is adminisered to the host animal cotinuousiy. in still further embodiments, the cell is in ell culture, In some embodiments, the method fAther cormprises the step ot administering a test compound to the clL in somc 25 embodiments, the test compound is a known chemotherapy agent, In some enbodmnients, the cancer is pancreatic caner, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate, lymphoma, ovarian, or melmoma. In some embodiments, the oligonucleotide comprises at least one CG dmiCeleotide pair, wherein at least one, and preferably all, of the cytosine bases in the CG dinucleocide pair comprises 5 30 methylcytosine. The present invention father provides a method, comprising providing an oligonucleotide that hybridizes o the promoter region of a TGF-a gene, and a cell comprising a TG-Fa gene; and adminsterng the oligonucleotide to the cell 20 In still further embodiments, the present invention provides a composition comprSMg an oligonucleotide that hybridizes under physiological conditions to the promoter region of a TGF-a gene, the oligonucleotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dicrucleotide pawIr comprises 5-netyleytosine. 1n some embodimcnts, the oligorucleotde is completely complementary to the promoter region of the TGOa gene. In other embodimens, the oligonucleotide is partially complementary to the promoter region of the TGF-a gene. For example, in certain embodiments, the oligonucleptide contains one mismatch to the promoter region of the TGF-a gene, In some preferred embodiments, the oligonuclo tide is 10 complementary only to the promoter region of the TGF-a gene and is not completely complementary to other regions of the human genomne, In some embodiments, the oligonucleotide is between 10 nucleotides and 60, and preferably between 15 and 35 nucleotides in length, The present invention further provides a composition comprising an oligonueleotide 15 that hybridizes under physiological conditions to the promoter region of a TGE- gene under conditions such that expression o the TOG gene is inhibited. The present invention additionally provides a composition conprisi ng an oligonucleotide that hybridizes under physiological conditions to the promoter region of a TFr-a gene located on a chromosome of a cell under conditions such that proliferation of 20 the cell is reduced. The present invetton also provides a composition comprising a first oligonucleotide that hybridizes under physiological conditions to the promoter region of a TOE-a gene, the oligonuceotide comprising at least on CO dinucleotide pair, wherein at least one of the cytosine bases i the CO dinucleotide pair comprises 5-methyeytosine; and a second :2 oligonucleotide, the second oligonucleotide comprising at least on CG dmucleotide pair, wherein at least one of the cytosine bases in the CG dinucleotide pair comprises 5 methylcyvtosine, in certam embodiments, the present invention provides a kit comprising an oligonucleotide that hybridizes under physiological conditions to the promoter region of a TG' gene, the oligonucieotide comprising at least on CG dinucleotide pair, wherein at least one of the cytosine bases in the CG dinucekotide pair comprises 5-methylcytosine; and instructors for using the kit fr reducing proliferation of a cell comprising a TOP-a gene on a chromosome of the cell or nhibiting gene expression. In some embodiments, the composition in the kit are used for treating cancer in a subject and the instructions comprise 21 i1.structions for using the kit to trea cancer in the subject. In some embodiments, the instructions are instructions required by the U_. Food and Drug Agency for labeling of pharmaceuticals. The present invention also provides a method, comprising: providing a biological sample from a subject diagnosed with a cancer; and reagents for detectmg 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 oligonucleotdde that hybridizes under physiological conditions to the promoter region of an oncogene expressed in the biological sample, the oligoniucleotide comprising at least on CG dinucleotide pair, wherein 101 at least one of the cytosne bases in the CG dinucicotide pair comprises 5-methyleytosine to the subject. The present invention additionally provides a method of inhibiting the expression of a gene in a subject (ecg, for the tratment of cancer or other hyperproliferative disorders) comprising providing an oligonucleotide that hybridizes under physiological conditions to 15 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 dinueleotide pair, wherein at least one of the cvtosine bases in the CG dinucleotide pair comprises 5 methyicytosine; 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, 20 In yet further embodiments, the present invention provides a method of screening compounds comprising providing a cell comprising a suspected oncogene; and an oligon ucleotide that hybridizes to the promoter region of the gene; and administerig the oligonueleotide to the cell; and determining if proliferation of the cell is inhibited in the presence of the oligonucleonide relative to the absence of the oligonucleotide, In some .23 embodimsents, the cell is in culture (e.g, a cancer cCel line), lb other embodiments, the cell is in a host animal (e.g, a non-human mammal). to some embodiments, the method is a high-throughput screening method, in other embodiments, the present invention relates to methods and compositions for cancer therapy. in particular, the present invention provides liposome based cancer .30 therapeutics. Accordingly, in some embodiments, the present invention provides a pharmaceutical composition comprising (e., consistIg of) a cationic. neutral, or anioi liposone "and an oligonucleotide, In some preferred embodiments, the liposome is a cardiolipin based cationic liposome (eg, NEOPHECTIN) in some preferred embodimentsthe charge ration of NEOPHECTIN to ohigonuclootide is 6:1. in other embodiments, the liposome comprises N-l(2,3-Dioleoyoxy)propyl ]NN,N-trimethylammonium methyksulfate(DOTAP).. In some embodiments, the present invention provides a kit, comprising an oligonucleotide (eg. an oligonuuleotide that hybridizes to the promoter region of an S onocogene) and a Orst phannaceutical composition comprising (e.g., consisting of) a cationic, neutral, or amonic hposone complies an optional second phanmaceutical composition, wherein the second pharmaceutical composition comprises a known chemotherapy agent (e., TAXOTERE, TAXOL, or VINCR1STNE) andT wherein the known chemotherapy agent is fornulated separately fnrm the first pharmaceutical 10 composition. 1 some embodiments, the chemotherapy agent is present at less than one half the standard dose, more preferably less than one third, e ven more preferably less than one forth and sill more preferable less than one tenth, and yet more preferably less than one hundredth the standard dose, In yet other embodiments. the present nvenion provides a method, comprising 15 prviding a pharmaceutical composition consisting of a cationic, neutral, or anionic liposome and an oligonucleotide (e.g, an oligonuculeotide that hybridizes to the promoter region of an onocogene); and exposing the pharmaceutical composition to a cancer cell. In some preferred embodiments, the liposome is a cardiolipin based cationic liposome (e.g, NEOPHECTJN). In some preferred embodimems, the charge ration of NEOPHECTIN to 20 oligornucleotide is 6:1 in other embodiments, the liposome comprises N414(2,3 Dlioeoylox)propy]NN,Nrimnethylammaonium mnethyL-sulftate (DOTAP). I some embodiments, the cancer cell is a prostate cancer cell, an ovarian cancer cell, a breast cancer cell, a leukemia cell or lymphoma cell. In some embodiments, the cell is in a host animal (eg, a human). In some embodiments the pharnaceutical composition is introduced to the 25 host animal one or more times per day (e.g, continuously), In some embodiments, the method further comprises the step of administering a known chemotherapeutic agent to the subject (e.g, TAXOTERE, TAXOL, or VINCRISTINET), wherein the known chemotherapeutic agent is Ibrmulated separately from the cationic, neutral or anionic liposome. In preferred em bodiments, the known chemotherapeutic agent is administered 30 separately from the pharnacctical composition. In som embodiments, the chemotherapy agent is present at less than one half the standard dose, more preferably less than. one third, even more preferably less than one forth and sti more preferable less than one tenth, and yet more preferably less than one hundred the standard dose. 23 DESCRIPTION OF THE FIGURES F Igure shows the nucleic acid sequence of the bel-2 gene (SEQ iD NOA). Figure 2 shows the sequences of antigenes to bcl-2 used in some embodiments of the present invention, X refers to a methyhated C nucleotide, Figure 3 shows the nucleic acid sequence of the c-erbB-2 (Her- 2 ) gene (SEQ ID NO:29). Figure 4 shows the sequences of antigenes to c-erb$-2 used in some embodiments of the present invention, X refers to a methylated C nucleotie Figure 5 shows the nucleic acid sequence of the lcRas gene (SEQ ID NO.46) Figure 6 shows the sequences of antigenes to c-ki-Ras used in some embodiments of the present mention. X refers to a methylated C nueleotide, Figure 7 shows the nucleie acid sequence o the c-Ha-Ras gene (SEQ 1W NO:66), Figure 8 shows the sequences of antigens to c-H a-Ras used in some embodiments of the present invention. X refers to a nmetylated C nuclotide. 5 Figure 9 shows the nucleic acid sequence of the e-myc gene (SEQ ID NO:108), Figure 1 shows the sequences of antigenes to c-myc used in some embodiments of the present invention X refers to a methylated C nucleotide, Figure 1 shows the nucleit acid sequence of the TGF-c gene (SEQ ID NO:13l) Figure 12 shows the sequences of amigenes to TOF-a used in some embodimens ot the present ivention X refers in a merthylaed C nucleotide. Figure 13 shows the inhibition of expression of cell growth by antigenes to e-ki-Ras used in some eibodiments of the present invemion Figure 14 shows the inhibition of expression of cell growth by antigens to bcb2 used in some embodiments of the present mnvtion, 25 Figure 15 shows the inhibition of expression of cell growth by antigenes to c-crb-2 used in somi embodiments of the present invention, Figure 16 shows the inhibition of expression of cel growth by antigenes to v4Ha Ras used in some embodiments of the presem invention, Figure 17 shows the inhibition of expression of ceil growth by antigens to c-mye 30 used in some embodirmens of the present invention. Figure 18 shows the inhibition of expression of cell growth by antigenes to TGF-c ased in soic embodiments ofthe present invention. Figure 19 shows the dose response curve of inhibition of expression of cell growth by amtigenes to e-kiRas. 24 25 Figure 20 shows the dose response curve of inhibition of expression of cell growth of FSCCL cells (A) and MCF-7 cells (B) by antigens to bcl-2. Figure 21 shows the dose response curve of inhibition of expression of cell growth by antigens to c-erb-2. s Figure 22 shows the dose response curve of inhibition of expression of cell growth by antigens to c-Ha-Ras used. Figure 23 shows the dose response curve of inhibition of expression of cell growth by antigens to c-myc. Figure 24 shows the dose response curve of inhibition of expression of cell to growth of T47D cells (A) and MDA-MB-231 cells (B) by antigens to TGF-a. Figure 25 shows exemplary variants of antigens to c-ki-Ras. Figure 26 shows exemplary variants of antigens to bcl-2. Figure 27 shows exemplary variants of antigens to c-erb-2. Figure 28 shows exemplary variants of antigens to c-ha-ras. 15 Figure 29 shows exemplary variants of antigens to c-myc. Figure 30 shows exemplary variants of antigens to TGF-a. Figure 31 shows inhibition of lymphoma cells by non-methylated oligonucleotides targeted toward Bcl-2. Figure 32 shows mean tumor volume of tumors in the PC-3 GFP prostate 20 carcinoma subcutaneous model following treatment with compositions of the present invention. Figure 33 shows mean body weight of tumors in the PC-3 GFP prostate carcinoma subcutaneous model following treatment with compositions of the present invention. 25 Figure 34 shows mean tumor volume of tumors in the PC-3 GFP prostate carcinoma subcutaneous model following treatment with compositions of the present invention. Figure 35 shows mean final tumor volume of tumors in the PC-3 GFP prostate carcinoma subcutaneous model following treatment with compositions of the present 30 invention. DEFINITIONS To facilitate an understanding of the present invention, a number of terms and phrases are defined below: 591205_1 LNB As used herein, the tem "wherein said chemotherapy agent is present at less than one half the standard dose" refers to a dosage that is less than one half (eg, less than 50%, preferably less than 40%, even more preferably less than 10% and still more preferably less than 1%) of the minimum va ue of the standard dosage range used for dosing humans. In 5 some embodiments, dhe standard dosage range is the dosage range recommended by the manufacturer. In other embodiments, the standard dosage range is the range utlized by a medical doctor in the fid. in still other embodiments, the standard dosage range is the range considered the normal standard of care in the field. The particular dosage within the dosage range is determined, for example by the age, weight, and health of the subject as 10 well as the type of cancer being treated, As used herein, the term "under conditons such that expressio of said gene is inhibited" refers to conditions vhere an oligonucleotide of the present invention hybridizes to a gene (e.g , the promoter region of the gene) and inhibits transcription of the gene by at least 10%, preferably at least 25%, even more preferably at least 50%, and still rnore 15 preferably at. least 90% relative to the level of transcription in the absence of the oligonucleotide. The present invention is not limited to the inhibition of expression of a particular gene, Exemplary genes include, but are not lin'ted to, c-kiRas, c-Ha-ras, c-ny her-2, TCF-c; and bW2. As used herein, the term "under conditions such that growth of said cell is reduced' 20 refers to conditions where an oligonuceotide of the present invention, when administered to a cell (e.g, a cancer) reduces the rate of growth of the cell by at least 10%, preferably at least 25%, even more preferably at least 50%, and still more preferably at least 90%h's relative to the rate of growth of the cell in the absence of the oligonncleotide, The term "epitope" as used here refers to that portion of an antigen that makes 25 contact with a particular antibody, When a protein or fragment of a protein is used to immunize a host ammal, numerous regions of the protein may induce the production of antibodies which bind specificly to a given region or three -dimensional structure on the protein; these regions or structures are referred to as "antigenic determnants". An antigenic determinant may 30 compete with the intact antigen (ie, the "*immunogen" used to elicit the immune response) for biding to an antibody. As used herein, the term "subject" refes to any animal (e.g, a mammal), including. but not limited to, humans, non-human primates 4 modents, and the like, which is to be the 26 recipient of a particular treatment. Typic-ally, tho teterms "subject" and patient " are used interchangeably herein in reference o a human subject. As used herein, the temns "computer memory" and "computer memory device" refer to any storage media readable by a computer processor. Examples of computer memory 5 include, but are not limited to, RAM.I, ROM, computer chips, digital video disc (DVDs). compact discs (CDs), hard disk drives (HDD), and magnetic tape, As used herein,t term "com tputer readable medium" refers to any device or system for storing and providing information 9 data and instructions) to a computer processor. Examples of computer readable media include, but are not limited to, DVDs, CDs, hard disk 10 drivesniagntic -tape and servers lbt streaming media over networks, As used herein, the terms "processor" and "central processing unit" or "CPU" are used interchangeably and refer to a device that is able to read a pro gram from a computer memory (e g. ROM or other computer memory) and perfonn a set of steps according to the program. 15 As used herein, the term "non-human animals" refers to all nonhman animals including, but are not limited to, vertebrates such as rodents, no-human primates, ovines, bovines, ruminants, lagomocphs, porcines, caprines, equines, canines feline. aves, etc. and and non-vebrate animals such as drosophila and nematode. In some embodiments, "non human animals" further refers to prokaryotes and viruses such as bacterial pathogens, vital 0 pathogens. As used herein, the term nucleicc acid molecule" refers to any nucleic acid containing molecule, including but not limited to, DNA or RNA. The term encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8Shydroxy-N6methyladenosine, aairidinylcytosine, 2$ pseutd&isoeytosine, 5-(carboxyhydroxylmethyl) uracit 54 luorouracil, S-bromouracil. $~ carboxymethylaminomethyL2thtouracil 5-carboxymsethyiaminomthyuracil, dihydrouraciI, inOSine, N6-isopentenyladenine, I -ethyladenine, 1nmethylpseudouracil, S-mnethylguanine, I -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyguanine, 3-muethylcytosine, Smethydeytosine, N6methyladenine, 30 7-mnethylguane, 5-nethylarnmomethyluracil, 5-methoxyaminomethyl4-thiouracii, beta-D-marnosylqueosine, 5'tmethox ycarbonynmethylurac~il, 5-mrethox yuracil, 2-methythio-N6sopentenyiadenine, uraci-5-oxyacetic acid methylester, uracil5-ox yacetic acid, oxybutoxosine, pseudouracil, queosine 2,thiocytosine, 5-methyl 2-ihiouracd, 2-thiouracil, 4-tiouracii, 5-methyluracil, N-uraci1-5-oxyacetic acid 27 methylester, uraei--oxyacetc acid, pseudouraci, queosine, 2~thiocytosine, and 2,6-diarninopurine. The term "gene" refers to a nucleic acl (eg. DNA) sequence that comprises coding sequences necessary tor the production of a poly'peptide, precursor, or RNA (eg. rRNA, 5 tRNAY The polypeptide can be encoded by a full length oding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e g enzymatic activity, ligand bMding, signal transduction immunogenicity, etc.) of the full length or fragment are retained. The tem also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' 10 ends for a distance of about I kh or more on either end such that the gene corresponds to the length of the ful-dength mRNA. Sequences located 5' of the codmg region and present on the mRNA are referred to as 5'nontransiated sequences. Sequences located V or downstream of the coding region and present on the mRNA are referred to as 3' non translated sequences 'The term "genC" encompasses both cDONA and genomnic forms of a iS gene, A genomic form Or clone of a gene contains the coding region interrupted with non coding sequences termed "introns" or "intervening regions" or "intervening sequences," Introns are segments of a gene that are transcribed nto nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA 20 (mRNA) transcript The mRNA functions during translation to specify the se quence or order of amino acids in a nascent polypeptide. As used herein, the tern "heterologous gene" refers to a gene that is not in it natural environment, For example, a heterologous gene includes a gent from one species introduced into another species, A heterologous gene also includes a gene native to an 25 organism that has been altered in sone way (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc) Heterologous genes are distinguished front endogenous genes in that the heterologous gene sequences are typically joined to DNA sequences that are not found naturally associated with the gene sequences in the chromosome or are associated with portions of the chromosome not found in nature e..g 30 genes expressed in loci where the gne is not normally expressed). As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (etg., nRNA, rRNA, tRNA, or snRNA) through "transcrption" of the gene (Me.s via the enzymatic action of an RNA polymerase). and for protein encoding genes, into protein through "translation" of mRNA Gene 28 expression can be regulated at many stages in the process. "Up~regulation" or "activation" refers to regulation that increases the production of gene expression products (ia, RNA or protein), while "down-regulation" r "repression" refers to regulation that decrease production. Molecules (eg, transcription factors) that are involved in up-regulation or 5 down-regulation are often called "activators' and "repressors," respectively, in addition to containing introns, genomic forms of a gone may also include sequences located on both the 5' and 3' end of the sequences that are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located ' or ' to the non-translated sequences present on the mRNA 10 transcnpth The 5 flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination oftrnscription, post-transcriptional cleavage and polyadenylation. The term "wildtype" refers to a gene or gene product isolated fromn a naturally 1S occurring source, A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutan" refers to a gene or gene product that displays modifications in sequence and or functional properties 0Lei, altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants 20 can be isolated; these are identifed by the fact that they have altered characteristics (including alered nucleic acid sequenets) when compared to the wild-type gene or gene product. As used herein, the term nucleicc acid molecule encoding," "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleoides 25 along a strand of deoxyribonucleic acid, The order of these deoxytibonucleotides determines the order of amino acids along the polypeptide (protein) chain The DNA sequence thus codes for the amino acid sequence. As used herein, the ieros "an oligonucleotide having a nucleotide sequence encoding a gene" and "polynucleotide having a nucleotide sequence encoding a gene," 30 means a nucleiL acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence that encodes a gene product. Th e coding region may be present in a eDNA, genomic DNA or RNA fnm. When present in a DNA form, the oligonucleotide or polynucleotide may be single-stranded (ie, the sense strand) or double-stranded. Suitable control elements such as enhancers/promoters, splice jtmctions, polyadenylation signals, etc. 29n nay ble placed in close proximity to the coding region of the gene if needed to permit proper initiaion of transcription andOr correct processing of the primary RNA transcrit Altematively, the coding region utilized in. the expression vectors of the present invention rmay contain endogenotus enhaneers/promuoters, splice junctions, intervening sequences 1 $ polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements, As used hereIn, the term "oligonucieotidei "efers to a short length of single-stranded poiynucleotide chain. Oligonucleotides are typically less than 200 residues long (-g, between 8 and 100), however, as used herein, the terrn is also intended to encompass longer 10 polynucleotide chams (eng,, as large as 5000 residues), Oligonucleotides are olRen rcerred to by their length. For example a 24 residue oligonucleotide is referred to as a 24-ner" Oligonucleotides can form secondary and tertiary structures by selfhybridizing or by hybridizing to other polynucleotides, Such structures can include, but are not limited to. duplexes, hairpins, eruciforms, bends, and triplexes, 15 in some embodiments, oligonucleotides are "anigenes , As used herein 1 the term "antgene" refers to an oligonucleotide that hybridizes to the promoter region of a gene. in some embodiments, the hybridization of the antigene to the promoter inhibits expression of the gene. As used herein, the terms "complementary" or "complemnem nty" are used in 20 reference to polynuclcotides (e, a sequence of nucleotides) related by the basepairing rules, For example, for the sequence "A-G~T," is complementary to the sequence "T-CA Complementarity may be "partia" in which only some of the nucleic acids* bases are matched according to the base pairing rles, Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between 25 nucleic acid strands has significant effects on the efficiency and strength of hybrdization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids, As used herein, the term "completely complementary," for example when used in reference to an olgonucleoide of the present invention refers o an oligonucleotide where 30 all of the nucleotides are complementary to a target sequence (eg, a gene). As used herein, the term "partially complementaryy' for example when used in referenee to an oIgonucleotide of the present invention, refers to an oligonucleotide where at least one nucleotide is not complementary to the target sequence. Preferred partially complementary oligonucleotides are those that can stil hybridize to the target sequente 30 under physiological conditions, The term "partially complementary" refers to oligonucleotides that have regions of one or more non-complenentary nucleotides both internal to the oligonucleotide or a either end, Oligonucleotides wkh mismatches at the ends may still hybridize to the target sequence. 5 The term "horology" refers to a degree of complementarity. There may be partal homology or complete homology (e identity), A partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous) The inhibition of hybridization of the completely complementary sequence to the target 10 sequence may be examined using a hybridization assay (Southem or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e the hybrdization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific 15 binding is perm itted; low stringency conditions require that the binding of two sequences to one another be a specific (i.et selective) interaction. The absence of non-specitic binding may be tested by the use of a second target that is substantially non-complementary (eg, less than about 30% identity); in the absence oflnon-specifi binding the probe will not hybridize to the second non-conpementary target. 20 When used in reference to a double-stranded rucleic acid sequence such as a eDNA or genonic clone, the term "substantially homologous" refers to any probe that can hybridize to either or both strands of the double-stranded nuclei acid sequence ander conditions of low stringency as described 'above. A gene may produce multiple RNA species that are generated by differential 25 splicing of the primary RNA transcript. DNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both eDNAs) and regions of complete non' identity (for examples representing the presence of cxoni "A" on cDNA I wherein DNA 2 contains exon "B" instead), Because the two cDNAs contain regions of sequence identity 30 they wil both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both DNAs; the two splice variants are therefore substantially homologous to such a probe and to each other, When used in reference to a single-stranded nucleic acid sequence, the tern "substantially homologous" refers to any probe that can hybridize (ie, it is the complement 31 of) the single-stranded nucleic acid sequence under conditions of low stringency as described above. As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids, Hybridization and the strength of hybridization (i e, the 5 strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids stringency of the conditions involved, the Tm of the formed hybnd, and the C:C ratio within the nucleic acids. A sIngle molecule that contains pairing of complementary nucleic acids within its streture is said to be "self hybridized.* 1t As used herein, the term "Tm" is used in reference to the meltingg temperature" The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands, The equation for calculating the Tin of nucleic acids is well known in the art As midicated by standard references, a simple estimate of the Th value may be calculated by the equation; Ti 81.5 + XA1{% CG 15 + C), when a nucleic acid is in aqueous solution at 1 M NCI (See t,g, Anderson and Young, Quantitative Filer Hybridization, in Nucleic Acid Hybridization [1985]) Other references include more sophisticated computaions that take structural as well as sequence characteristics into account for the calculation of Tm, As used herein the term "stringency" is used in reference to the conditions of 20 temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted, Under low stringency conditions" a nucleic acid sequence of interest will hybndize to its exact complement, sequences with single base mismatches, closely related sequences (e.g. sequences with 91% or greater honmology) and sequences having only partial homology e.g. sequences with 50-90% 25 bonology). Under "medium stringency conditions," a nucleic acid sequel nce of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely relation sequences (e. 90% or greater homology), Under "high stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, and (depending on conditions such a temperature) sequences with single base mismatches, in 30 other words, under conditions of high stringency the temperature can be raised so as to exclude hybridization to sequences with single base mismatches. "High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42C in a solution consisting 32 of 5X SSPE (418 g/l Na., 6,9 g/l Nal{ 2

PO

4 HI and i 85 g/l EDTA, pH adjusted to 74 with NaOH), 0.5% SDS 5X Denhardt's reagent and 100 pg/mi denatured salmon sperm DNA followed by washing in a solution comprising 0t IX SSPF, I,0% SDS at 42C when a probe of about 500 nucleotides in length is employed. 5 "Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42"C al a solution consisting of X SSPE (43 S g/l NaCL, 6.9 g/ NaH 2 4 H20 and 15 g! EDTA, pH adjusted to 7,4 with NaOH), 0,5% SDS, 5X Denhardrs reagent and 100 pg/mi denatured salmon sperm DNA followed by washing in a solution comprising 1 .0X SSPE, 10% SDS at 10 42"C when a probe of about 500 nuclotides in length is employed, "Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42C in a solution consisting of 5X SSPE (418 g/l Na, 6,9 g/l NaH 2 PO4

H

2 0 and 185 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reapent [50X Denhardi's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), S g BSA 15 (Fraction V; Sigma)] and 100 pg/mI denatured salmon sperm. DNA followed by washing in a solution comprising 5X SSPE, 41% SDS at 42 0 C when a probe of about 500 nucleotides in length is employed. The present inventon is not limited to the hybridization of probeof f about 500 nucieotides in length, The present inventIon contemplates the use of probes between 20 approximately S nucleotides up to several thousand (e g, at least 5000) nucleotides in length. One skilled in the relevant understands that stringency conditions may he altered for probes of other sizes (Sec eg. Anderson and Young, Quantitative Filter Hybridwzation, in Nucleic Acid Hybriduatin [ 1985] and Sambrook et al. Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, NY ['1989)) 25 The art knows well that numerous equivalent conditions may be employed to comprise low stringency conditons; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g, the presence or absence of formamide, dextran sulfte, polyethylene glycol) are 30 considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivaent to the above listed conditions. in addition, the art knows conditions that promote hybridization under conditions of high 33 stringency (e., mnrrasg gte temiperaurc of the hybridization and/or wash steps, the use of fonnamide in the hybridization solution, etci) (see definition 'above for "stringency"). As used herein, the term "physiological conditions" refers to specific stringency conditions that approximate or are conditions inside an anmal (eg, a human), Exenplary 5 physiological conditions fOr use in vitro include, but are not limited to, 374C, 95% air, 5% CO., commercial medium for culmre of mammalian cells (e.g., DMEM media available from Gibco, MD) 5-10% Aserum (e.g, calf serum C horse serum), additional buffers, and optionally hormone (mtg, insulin and epidermal growth factor), The term isolated" when used in relation to a nucleic acid, as m "an isolated 10 oligonucleotide" or "isolated polynucleotide" refers to a nucleic acid sequence that is identi fled and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a fom or sting that is different from that in which it is found in nature. In contrast, non-isotated nucleic acids as nucleic acids such as DNA and RNA found mu the state they exist in nature,. 15 For example, a given DNA sequence fe.g,, a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific rnRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRRNAs that encode a multitude of proteins. However, isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the 20 given protein where the nucleic acid is in a chromosomad location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature, The isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form. When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotidc 25 will contain at a minimum the sense or coding strand (i., the oligonucleotide or polynucotide n-ay be single-stranded), but may contain both the sense and ant-sense strands i.e the oligonucleotid or pol ynneotide may be double-stranded). As used here, the term "purified" or "to purify" refers to the removal of components (e.g, contaminants) from a sample. For example, antibodies are purifed by 30 removal of contaminating non-inmmunogiobuin proteins: they are also purified by the removal of immunoglobulin that does not bind to the target molecule, The removal of non nnniunoglobulin proteins and/or the removal of imnmunoglobulins that do not bind to the target molecule results in an increase in the percent of target-reactive immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host 34 cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample. "Amino acid sequence" and tens such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated 5 with the recited protein molecule. The term "native protein" as used herein to indicate that a protein does not contain amino acid residues encoded by vector sequences; that is, the native protein comains only those amino acids found in the protein as it occurs in nature. A native protein may be produced by recombinant moans or may be isolated from a naturally occurring source, 10 As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refrrs to fragments of that proteint The fragments may range in siz from four amino acid residues to the entire amino acid sequence minus one amino acid, The term "Southern blot" refers to the analysis of DNA on agarose or aerylatide get to fractionate the DNA according to size followed by transfer of the DNA from the gel 15 to a solid support such as nitrocelulose or a nylon membrane, The immobilize DNA is then probed with a labeled probe to detect DNA species complementary to the probe used, The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists (. 20 Sambrook e at, Molecular Cionig: A Laboratory Manua, Cold Spring Harbor Press, NY, pp 931 -9 $8 [1989T The term "Norhem blot as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support such as nitrocellulose or a nylon 25 membrane. The immobilized R(NA is then probed with a labeled probe to detect RNA species complementary to the probe used. Northem blots are a standard tool of molecular biologists ( Sambmok, et aL. supra, pp 7,39T52 [1 989) The term "Western blot" refers to the analysis of proteins) (or polypeptides) immobilized onto a support such as nitrocellulose or a membrane, The proteins are rn on 30 acrylamide getls to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane, The immobilized proteins are then exposed to antibodies with reactivity against an antigen of interest The binding of the antibodies may be detected by various methods, including the use of radiolabeled antibodies. 35 As used herein, the term "Kel culture" refers to any in varo culture of cells, included within this tern are continuous cell lines (ag,, with an immortal Phenotype) primary cel cultures, transfonned cell lines, ite cell lines (eag , non-transformed cells), and any other cell populanon maintained in Vitro. 5 As used, the term "eukaryote" refrs to organisms distinguishable from "prokaryotes" It is intended that the term encompass al. organisms with cells that exhibit the usual characteristics of eukaryotes, such as the presence of a true nucleus bounded by nuclear membrane, within which lie the chromosoas, the presence ofmembrane-hound organelles, and other characteristics commonly observed in eukaryotic organisms. Thus, 10 the term includes. but is not limited to such organisms as fungi, protozoa, and animals (e., humans). As used heren, the enn "in vir" refers to an artificial environment and to processes or reactions that occur within an artiicial environment, In vitro environments can consist of, but are not limited to, test tubes and cell cure. The tenn "in wvo" refers to the 15 natural environment (Cg, an animal or a cell) and to processes or reaction that occur within a natural environment, The terms "test compound' and "candidate compound" refer to any chemical entity, pharmaceuticat, drug, and the like that is a candidate for use to treat or prevent a disease, ilness, sickness, or disorder of bodily futnction (eg., cancer) Test compounds comprise 20 both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invenion in some embodiments of the present invention, test compounds include antisense compounds, As used herein, the term "known chemotherapeutic agents refers to compounds known to be useful in the treatment of disease (eg, cancer), Exemplary chemnotherapcuic 2 agents affective against cancer include, but are not limited to, daunorubicin. dactinomycin, doxorubicin, bleornycin, mitomych, nitrogen mustard, chlorambucil, ienphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), $-fluorouracil (5FU), floxuridine (5-FUdR methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin and diethylstilbestrol(DES), 30 As used herein, the term "sample" is used in its broadest sense, In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environnemal samples. Biological samples may be obtaied from animals (including humans) and encompass fluids, solids, tissues, and gases, Biological samples include blood products, such as plasma, serum and the like. Environmental samples include 36 environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the preset invention, DETAILED DESCRIPTION OF THE INVENTION The present invention relates to methods and compositions for the treatment of cancers, In particular, the present invention provides oligonucleotidebased therapeutics for the exhibition of oncogenes involved in a vanety of cancers. The present invention is not limited to the treatment of a particular cancer. Any cancer can be targeted, including, but 10 not limited to, breast cancers The present invention is also not limited to the targeting of cancers or oncogenes. The methods and compositions of the present invention are suitable for use with any gene that it is desirable to inhibit the expression of(e.g for therapeutic or research uses). 1$ L Oncogene Targets In some embodiments, the present invention provides antigen inhibitors of oneogenes, The present invention is not limited to the inhibition of a particular oncogene, indeedthe present invention encompasses antigene inhibitors to any number of oncogenes including, but not limited to, those disclosed herein, 20 A. Ras One gene which has captured the attention of many scientists is the human proto oncogene, e-Ha-ras. The nucleic acid sequence of the promoter region of c-I-ras is shown in Figure 7. This gene acts as a central dispatcher, relaying chemical signals into cells and 25 controllug cell division, Ras gene aheration may cause the gene to stay in the "on" position, The ras oncogene is believed to nderlie up to 30% of cancer, including colon cancer, hg cancer, bladder and mammary carcinoma (Bos, Cancer Res. 49:4682-4689 [1989'), The ras oncogene has therefore become a target for therapeutic drugs, There are several reports showing that oligonucleotides complementary to various 30 sites of ras RnKNA can inhibit synthesis of ras protein (p21). which decreases the cell proliferation rate in cell culture (US Pat. No. 5,576.208; U. Pat, NoS, ,582,986; Daska ea at Oncogene Res 5:267-275 [1990]; Brown eatl Oncogene Res. 4:243-2521 9891 Saison-Behnmoaras i nf EMBO J. 10:1111-1116 991 Oligonucleotides complementary to the 5' flanking region of the c-Ha-ras RNA transcript have shown to 37 inhibit tumor growth in nude mice for up to 14 days (Gray et an, Cancer Res 53:577-580 [19933). It was recently reported that an antrisense oligonucleotide directed to a point mutation (G>C) in codon 12 of the c-Ha-ras mRNA inhibited ecll proliferation as well as tumor growth in nude mice when it was ejected subcutaneously (U.S. Pat No. 5,576,208; U.S, Pat. No, 5,582,986; Schwab et at Proc. Nat. Acad, Sci. USA 91:10460-10464 j1994]; each of which is herein incorporated by reference), Researchers have also reported that antisense drugs shrank ovarian tumors in small cynical trials (Roush er at) Science 276:1192-1194 [1997). 10) B- Her-2 The IER-2 (also known as neu oncogene or erhB-2) oncogene encodes a receptor like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (1Hynes and Sten, iocehim, et Biophy. Acta 1 98:165-184 [1994); Dougall et, Oncogene 9:2109-2123 [1994]) and in mammnalian development (Lee 5S er at Nature 378-394- 39$ [1995]). The nucleic acid sequence of the promoter region of Her-2 is shown in Figure 3. The sequ fence of the "HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA frompiacena (Coussens ce at Science 230:1132-1139 [1985]) and from a gastric carcinoma cl line (Yaamoto e at, Nature 319:230-234 [1986)). The ER-2 nRNA was 20 shown to be about 4,5 kh (Coussens et at, Science 230:1132-1139 [1985]; Yamamoto e at. Nature 319:230-234 [1986]) and encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (pl 8HER-2) (Hiynes and Steen, Biochim, et Biophys Acta 1198:165-184 [1994); Dougal en aL, Oncogene 9:2109-2123 [1994]). Overexpression of HER-2 causes phenotypic transformation of cultured cells (DiFiore et at Scince 25 237 M7-82 [1987) Hudziak to at Proc NaP. cad, Sci. USA 84:7159-7163 [1987]) and has been associated wit aggressive clinical progression of breast and ovarian cancer (Slamon et at Science 235:177-182 [19873 Slamon er at Science 244:707-712 [19891). HER-2 is one of the most frequently altered genes i cancer It encodes a transmenbrane receptor (also known as p185) with tyrosine kinase. activity and is a member 30 of the epidermal growth factor (EF) family, and thu iis related to the epidermal growth factor receptor (EGFR or HER-). Aberrant HER-2 gene expression is present in a wide variety of cancers and are most common in breast) ovarian and gastrc cancers. HER-2 is overexpressed in 25-30% of al human breast and ovarian cancers. Levels of HER-2 overexpression correlate wel with clinical stage of breast cancer, prognosis and metastatic 38 potential. Overexpression of iER-2 is associated with lower survival rates. increased relapse rates and increased metastatic potential. Tan et aL. (Cancer Res., 57:1199 [1997]) have shown that overexpression of the HER-2 gene increases the metastatic potential of breast cancer cells without increasing their transformation ability, 5 Aberrant expression of HER-2 includes both increased expression of normal HER-2 and expression of mutant HER-2. Activation of the HER-2 proto-oncogene can occur by any of three mecbauisms-point mutation, gene amplification and overexpression. Gene amplification is the most conon mechanism. Unlike the other EGF family members for whom ligand acttvation is necessary for pronoting transformation, overexpression of HEW 10 2 alone is suffcient for transformation (Cohen, e at, 1 Bot Chem. 271:30897 []996J), Several therapeutic approaches have been used to reduce levels of the HER-2 gene product. The adenovirus type 5 gene product E i A has been studied as a potential therapeutic using a breast cancer model in nude mice. This gene product can repress HER 2/neu overexpression by repressing HER-2/neu promoter activity, and suppress the 15 tumorigenic potential of 1ER-2/neu-overexpressing ovarian cancer cels. In mice bearing HER-2/neu-overexpressing breast cancer xenografis, El A delivered either by adenovirus or liposone signficantly inhited tumor growth and prolonged mouse survival compared with the controls Chang a t Oncogene 14:561 [1997]) Clinical trials have been conducted to evaluate a bispecific antibody which targets 20 the extracellular domains of both the HER-2/neu protein product and Fc gamma R11 (CD16), the Fe gamma receptor expressed by human natural kiler cells, neutrophits, and differentiated mononuclear phagocyes (Weiner tat, . Heniatotherapy, 4:471 [1995]). Overexpression of HER-2 has also been found to be associated with increased resistance to chemotherapy. Thus, patients with elevated levels of HER-2 respond poorly to 25 many drugs. Methods used to inhibit HER-2 expression have been combined with connonly used chemotherapeutic agents (Uleno pt as.. Oncogone 15:953 [1997]), Combining the adenovirus type 5 gene product, ElA, with taxol showed a synergistic effect in human breast cancer cells. Zhang e at, (Oncogene, 12:571 [1996]) demonstrated that emodin, a tyrosine-specitic inhibitor, sensitized non-small cel lung cancer (NSCLC) cells 30 to a variety of chemothcrapeutic drugs, including cisplatin, doxorubicin and etoposide, A HER-2 antibody was found to increase the cflicacy of tamoxifen in human breast cancer ceHs (Witters el t Breast Cancer Res. and Treatment, 42:1 [1997]). Oligonucleotides have also been used to study the function of HER-2, triplex forming oligonucleotide targeted to the HER-2 promoter, 42 to 69 nucleotides upstream of .39 the miRNA transcription start site was found to inlibit HER-2 expression in virro (Ebbinghaus et at J. Cin vest, 92:2433 [1993)). Porunb e; at (Cancer Rea, 56:5 15 [1996]) also used a triplexorming oigonucleoide targeted to the same HER~2 promoter region. Decreases in HER-2 mRNA and protein levels were seen in cultured cells. Juhl et 5 at (j. BioL hem, 272:29482 [1997)) used anti-HER-2 bozymes targeted to a central region of the H ER-2 RNA just downstream of the transmembrane region of the protein to demonstrate a reduction in HER-2 mRNA. and protein levels in human ovarian cancer cells, A reduction in tumor growth in nude mice was also seer. An antisense approach has been used as a potential therapeutic for HER-2 10 overexpressi cancers. Pegues el at (Cancer Lett, 117 73 [1997)) cloned a S kb ftagment of HE.R-2 in an antisense orientation into an expression vector; tratnsfecting of this construct into ovarian cancer cells resulted in a reduction of anchorageindependent growth. Casalini et al (In j. Cancer 72:631 [1997]) used several human HER-2 antisense vector constructs, containing HER-2 fragments from 151 bp to 415 bp in length, to demonstrate 15 reduction in HER-2 protein levels and anchorage-independent growth in lung adenocarcinoma cells, Coloner er at ( r . Cancer, 7:819 [1994p showed that phosphodiester antisense oligonucleotides targeted at or immediately downstream of, the translation initiation codon inhibited proliferation of human breast caner cells by up to 60%. Wiechen et at (Int, J Cancer 63:604 1995]) demonstrated that an I8nucleotide 20 phosphorothioae oligonucleotide targeted to the coding region, 33 nucleotides downstream of the translation initiation codon, of HER -2 reduced anchorage-independent rowth of ovarian cancer ceNs. Bertram et a. (Biochem. Biophys, Res Comnun, 200:661 {1994}) used antisense phosphorothioate oligonucleotides targeted to the translation initiation region and a sequerce at the 3' par of the translated region of the mURNA which has high 25 homology to a tyrosine kinase consensus sequence, and demonstrated a 75% reduction in HER-2 protein levels in human breast cancer cells. ULi et a (Antisense and Nucleic Acid Drug Develop, 6:9 [1996) used antisense phosphorothioate oligonucleotides targeted to the 5' cap site and coding region. The Most effective oligonucleotide, targeted to the 5' cap site, reduced HER-2 protein expression by 90%. Cell proliferation was also reduced by a 30 comparable amount. Vaughn et al (Nuc. Acids. Res., 24:455 { 1996]) used phosphorothioate, phosphorodithioate and chimeric antisense oligonucleotides targeted at or adjacent to (either side) the translation initiation region of HER-2, An alternating dithioatefdiester oligonucleotide targeted to the translation initiation region worked slightly better than an all phosphorothioate oligonucleotide. Brysch et at (Cancer Gene Then, 1: 99 40 [ 1994]) used chemically moditled antisense oligonucleotides targeted lo the translationr initiation codon of HER-2 to reduce protein levels and cause growth arest of human breast cancer cell line. 5 C C-Mve The co-ye gene product is encoded by an imunediate early response gene, the expression of which can be induced by various mnitogens, The nucleic acid sequence of the promoter region of the enyc gene is shown in Figure 9. C-myc expression is involved in the signal transducton pathways leading to celi division, Studies have demonstrated that 10 proliferating cells have higher levels of e-mye mRNA and e-my c protein than do quiescem cells, Atibodies directed against the human canyc protein are known to inhibit DNA synthesis in nuclei isolated from human cells. Conversely, constitutive expression of c-ny produced by gene transfer inhibits induced differentiation of several cell ines. Constitutive expression of c-myc predisposes transgenic mice to the development of tumors, 15 Some studies have suggested that the c-nyc gene product may play a proliferative role in SMCs, Balloon de-endotheialization and injury of rat aortas is known to increase c myc mRNA expression of vascular SMC pior to their subsequent proliferation and migration, Also, SMCs in culture proliferate when exposed to several mitogens, including PDGF, FGF, EGF, JGF- and to serum. Each of these mitogens has been found to be 20 capable of increasing the expression n other cell lines of either c-myc protein, C-myc mRNA, or both. Additionally, blood serum has been found to increase csnyc mRNA levels in SMCs. Hare-Bellan et at (it bmun, 140; 2431-2435 (1988)) demonstrated that antisense oiigonueleotides complementary to c-my mRNA effectively inhibited the translation 25 thereof in human T cells, These T cells were prevented from entering the S phase of cell division. c-ny proto-oncogene sequences are described in Marcu et at, Ann Rev. Biochetm, 61:89-860 (19924 Watt et at, Nature, 30 3:725-728 [1983)4; Batey e at, Cell. 34:779-77 (1983); and Epstein et al NTIS publication PB93-100576 A30 1 Bell in many types ofhuman tumors, including lymphomas and leukemias, ine human hel-2 gene is overexpressed, and may be associated with tunogenicity (Tsujimoto et at, Science 2281440-1443 [1985]) The nucleic acid sequence of the promoter region of bd-2 is shown in Figure 1. High levels of expression of the human bcl-2 gene have been found in 41 all lynphomas with t (14; 18) chromosomal translocations including most follicular B cell lymphonmas and many largc cell non-Hodgkins lymphomas, High levels of expression of the bc2 gene have also been found in certain leukemias that do not have a 1(14; 18) chromosomal translation, including most cases of chronic lymphocytic leukemia acute 5 many lymphocytic leukemias of the pre-B cell type, neuroblastmnas, nasophryngeal carcinomas, and many adenocarcinomas of the prostate, breast, and colon, (Reed e at, CancerRes. 51:6529 [1991]; Yunis et a., New EnglandL Med. 320:1047; Campos eut .a Blood 81:30913096 [1993]; McDonnell at at, Cancer Res. 52:694046944 [1992); Lu et at, Int. j Cancer 53:29-35 [1993}; Bonnert at, Lab Invest, 68:43A [1993]), 10 E. TCF-a Transforming Growth Factor Alpha (TGF-a) is a polypeptide of SO amino acids. The nucleie acid sequence of the TGW-a promoter is shown in Figure 1 Y h was first isolated from a retrovirus-ransformed mouse cell line and subsequently was identified in 15 human tumor cells, in early rat embryo cells and in cel' cultures from the human pituitary gland, TGF-a is closely related to Epidermal Growth Factor (EGF), both structurally and functionally, and both bind tohe same receptor, .e, Epidernal Growth Factor Receptor

(EGFR)

The sequence and three dimensional structure of both GF and TGF-a have been 20 determined (Campbell et at, Prog, Growth Factor Res, 1: 13 [1989]). TGF-a is a 50 amino acid polypeptide having about 40% homology of residues with EGF, Both peptidesae characterized by three well defined loops (denoted A, B and C) and have three intramolecular disuiphide bonds. Several growth factors, including TGF-a and EGF, are believed to exert their 25 biological effects via interaction with the Epidermal Growth Factor Receptor (OGF Receptor). The EGF Receptor is a Type I receptor tyrosine kinase. The EGF Receptor and its ligands arc of interest for their roles in normal physiological processes as well as in hyperproliferative and neopiastic diseases. The in uvo precursor of TGF-a is a 160 amino acid residue menmbrane-bound 30 protein (pro-TOF alpha.) that is cleaved to yield a soluble compound (Massague, . Biol Chei, 265:21393-21396 [1990]) This cleavage removes an extracellular portion comprised of 50 amino acids with a roolecular weight of 6 Kd and is considered to be an important regulatory event (.Pandiela et at,.Proc. NaI. Acad. Sci. USA, 88:1726-1730 42 119901) that can be stimulated by phorbol esters acting via protein kinase C (Pandiella el al. I, Biol Chem 266:5769-5773 [1991) Cultured human prostatic tumor lnes contain elevated levels of TGF-a mRNA and proliferate in response to TGF-a (XWilding et at, The Prostate, 15:1-12 [ 19S9]), TGF-a 5 appears to have both autoec and paracrine function, stimulating physiologie activities such as cell division and angiogenesis, When induced in transgenic mic, TCP-a produced epithelial hyperplasia arid focal dysplastic changes that resembled carcinoma in situ (Sandgren et at, Cell, 61:1121- 135 f1 990]), 10 F e-ki-RAS The c-Ki-RAS (KRAS) oncogene is expressed ubiquousiy, KRAS, with a length of more than 30 kb, is much larger than HRAS or NRAS. The sequence of the promoter roeiIo ofciha is shown in Figure 5. Ahhugh the 3 rs genes, BRAS, KRAS, and NRAS, have different genetic structures, al code for proteins of 189 amino acid residues, 15 genricaly designated p 2 L These genes acquire malignant properties by single point mutations that affect the corporation of the 12th or 61st amino acid residue of their respective p 2 l KRAS is involved in malignancy much more often than is BRAS, In a study of 96 human tumors or tumor cell lines in the NIH 3T3 transforming system, (Pulciani et aL Nature 300: 539 (1982) found a mutated HRAS locus only in 724 bladder cancer 20 cells, whereas transfonring KRAS genes were identified in 8 different carcinomas and sarcomas. in a serous cystadenocarcinoma of the ovary, Feig et at (Scienwe 223: 698 (1981)) showed the presence of an activated KRAS oncogene not actWated in normal cells of the same patient. The transforming gene produce displayed an electophoretic mobility in SDS 25 polyacrylamide gels that differed fhm the mobility of KR AS transforming proteins in other tumors, Thus, a previously undescribed mutation was responsible for activation of KRAS in this ovarian carcinoma. To study the role of oncogenes in lung cancer, Rodethis et at (New Eng, 2. Med, 317: 929 (1987)) used an assay based on oligonucleotide hybridization following an in vito amplification step. Genomic DNA was examined from 39 tumor 30 specimens obtained at thoracotomy. The KRAS gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not netastasized. No HRAS, KRAS, or NRAS mutations were observed in 15 squamous cell carcinomas, 10 large cell carcinomas, I carcinoid. 2 metasratic adenocarcinomas From primary tumors outside the lung, and l small cell carcinoma, An 43 approximately 20-4old amplification of the uranutated KR.AS gene was observed in a tumor that proved to be a solitary ung metastasis of a rectal carnmoma, Yanez et at (Oncogene 1:315 (1987)) found mutations in cdon 12 of the KRAS gene in 4 of 16 colon cancers, 2 of 27 hng canes, and I of 8 breast cancers; no mutations were found t position 61. Of the 6 5 possible amino acid replacements in codon 12, all but onc were represented in the 7 muittations identid.( G, Other Oncogene Targets The present invenion is not limited to the oncogenes described above, The ewhods 10 of the present inventon are suitable fOr use with any oncogene with a known promoter region. Exemplary oncogenes included, but are not limited to, BCR/ABL, ABL1/BCR, ABL, BCL, CD24, CDK4, EFPR/ERBB-, ISTF, IINT1/WNTI, INT2, MDM2, MET, MYB, MYCjMYCN, MYCLI, RAPE, NRAS, REL, AKT2. APC, BCL2~ALPHA. BCL2~ BETA, BCL3, BCR. BRCA, BRCA2, CBL, CCND1, CDKN1A, CDKNiC, CDKN2A, 15 CDKN2B CRK, CRK~IT, CSFR/FMS, DBL, DDOST, DCC, DPC4/SMAD4, ECAD, E2F/RBAPE LK , E LK3, EPH, EPTAL FYI, EPHA, ERG, ETS L ETS2, FER, FGR, FLII/ERGB2, EMS, FPS/FES, FRA FRA2, FYN, HCK. HEK, UER3/ERBB-2, ERBB-3, HER4/ERBB-4, HST2, [NK4A, INK4B, JUN, JUNB3 JUND, KIP2, KIT, KRAS2A, KRAS2E, LCK, LYN, MAS, MX, MCC, MLH, MOS, MSH2, MYBA, MYBB, NF L 20 NF2, P53, PDGFB, PIM], PTC, RB13 RET, ROSY, SKI, SR.C, T AL , TGFBR2, THR, THIRB, TIAM1, T RK, VAV, VHL, W AF 1, WNT2, WTI, YES , ALK/NPM 1 AMI 4 l AXL, FMS GP, GLI, GSP, HOX 1I, HIST, IL3 INT2, KS3, K-SAM, LBC, LMOI1, LM 2, L-MYC, LYL I, LYT-10, MDM-2, MLH, MLL, MLM. N-MYC, OST, PAX5, PMS-l PMS-2, PRAD- t, RAF, RHOM- 1, RHM12, SIS, TAL2, TAN1, TIAM I TSC2, TRIKt 25 TSC1, STKI 1, PTCH, MENI, MEN2, P371UP2, PTEN, HPCl, ATM, XPA/XPG, BCL6, DEK, AKAPi3, CDHI. BLM, EWSRI/FLII, FFS, FGFS, FGF4, FGF6, FANCA, FLII!ERGB2, FOSLL, FOSL2, GL LERAS], HRX/MLLT, HRX/MLLT2, KRAS2 MADh4, MASI, MCF2, MLLT I/[MILL MLLT2/TH-RX, MTG&RUNXI, MYCLKI, MYHII l/CBFB, NFKB, NOTCH, NPMI/ALK, NRG/RFL, NTRK1, PBXI/TCF3, 30 PMURARA, PRCA RUNXI RUNX1/CBF A2T, SET, TCF3/PBX I, TFBI, TLX I P53. WNTI WNT2, WT. &v-o33, PKCa, TNFa. Clusterin, Snrvivmg, TGF7, c-fos. c SRC, and INT- L 44 1L, Non-Oncogene Targets The present mvnion is not limited to the targeting of oncogenes, The methods and compositions of the present invention find use in the targeting of any gene that it is desirable to down regulate the expression of, For example, i some embodiments, the genes to be targeted include, but are not limited to, an immunoglobulin or antibody gene, a clotting factor gene, a protease, a pituitary homnon, a protease inhibitor, a growth factor, a somaromedian, a gonadatmphin, a chemotactin, a chemokin a a plasma protein, a plasma protease inhibitor, an interleukin, an interferon, a cytokine, a transcription factor, or a 10 pathogen target (eg, a viral gene, a bacterial gene, a microbial gene, a ffmgal gene. Examples of specific genes include, but are not limited to, ADANTS4, ADAMTS5. APOA1. APOE, APP, 112M, COX2, CRP, DX25, DMCA, FKBP8, GH1, GHR, IAPP, TFNAI, fFNG, IL1, 1110, ILI2, 1L13, 1L2, ML4, I 1 7, IS, LPW, MAPKI4, Moil. MMPt3, MYD88, NDN, PACE4, PRNP, PSEN1, PSEN2, RAD5 L RAD5 C, SAP, SNRPN T LR4, 15 TLR9, TTR, UBE3A. XVLAA4, and PTP-B, c-RAF, n-TOR, LDL, VLDL, ApoB-100, HLDL. VEGF, rhPDGF-BB, NADs, ICAM-i MUC!, 2-dG, CTL, PSGL-1, E2F, NF-kB, HIP, and GiCPRs in other embodiments and gene from a pathogen is targeted. Exemplary pathogens include, bt are not limited to, Human 1umunodeficiency virus, Hepatitis B virus, hepatitis 20 C virus, hepatitis A virus, respiratory syncytiai virus, pathogens involved in severe acute respiratory syndrome, west nile virus, and food home pathogens (e.g., E, coli). M. DNA Methylation In some embodiments, the present invention provides oligonucleotide therapeutics 25 that are methylated at specific sites. The present invention is not limited to a particular mechanism, indeed, an understanding of the mechanism is not necessary to practice the present invention Nonetheless, it is contemplated that one mechanism for the regulation of gene activity is methylation of cytosine residues in DNA, 5-methylcvtosine (5-MeC) is the only naturally occurring modid base detected i DNA (Ehrlkck , at Science 212: 1350 30 1357(1981)). Although not all genes are regulated by methylation, hypometbyiadon at specific sites or in specific regions in a number of genes is correlated with active transcription (Doerfier, Annu, Rev. Biochem, 5293424 [1984; Christman, Curi, Top. Mitrobiol hnmunol 10849-78 [1988} Cedar, Cell 34:5503-5513 (198S]). DNA methylation &? wtro can prevent efficient transcripton of genes in a celhfree system or 45 iTasient expression of transfected genes. Methyation of C residues in some specific cis regulatory regions can also block or enhance binding of transcriptional factors or repressors (Doerfler, snpra; Christman, supra: Cedar, Celi 34:5503-5513 (1988); Tate r at, Curr. Opt. Genet. De. 3:225-231 [1993]; Christnmn ea al, Virus Strategies, eds, Doerfier, W, & MBohm. P. (VICK Weinheim, AY.) pp 319-333 [1993 1. Disruption of noral patterns of DNA iethylation has been linked to the development of cancer (Christcman et, Proc Natl. Acad, Sei USA 92:7347-7351 [993i}) The 5-MeC content of DNA from tumors and tmnor derived cell lines is generally lower than nornal tissues (Jones a a, AMv. Cancer Res 40:1-30 [1983)). Hypomethylation of 10 specific oncogenes such as i-my, c-i-ras and c "a-ras has been detected im a variety of human and animal tumors (Nambu er at, jpu. J, Cancer (Gann) 78:696-704 [1987]; Femberg e at, Biochem. Biophys. Res, Conmun, 111:47-54 [1983); Cheah et at. fNCI73:1057-1063 [1984; Bhave et a l Carcinogenesis (Lond) 9:343-348 [1988, In one of the beSt studied examples of human tumor progression, it has been shown that 15 hypomethylation of DNA is an early event in development of colon cancer (Goetm et at Science 228:187-290 [1985]). Interference with metylation in vivo can lead to tumor formation, Feeding of methyiatn ihibitors such as L-nethionine or 5-azacytodine or severe deficiency of 5-adenosine ethionmne through feeding of a diet depleted of lipotrupes has been reported to induce formation of liver tumors in rats (Wainfin w at Cancer Res, 20 52:2071s-2077s [1992)), Studies show that extreme lipotrope defcient diets can cause loss of methyl groups at speci fic sites in genes such as c-mye, ras and c-fos (Dizik et at, Cateinogenesis 12:1307-1312 [991& Hypomehylation occurs despite the presence of elevated evels of DNA MTase activity (Waintfn et at Cancer Rcs. 49:4094-4097 [1989). Gene required for sustained actve proliferation become inactive as rethylated during 35 differentiation and tissue specific genes become hypomethylated and are active, Hypomethylation can then shift the balance between the two states, In some embodiment, the present invention thus takes advantage of this naturally occurring phenomena, to provide compositions and methods for site specific methylation of specific gene promoters, thereby preventing transcription and hence translation of certain genes. In other embodiments, the 30 present invention provides methods and compositions for upreguiating the expression of a gene of interest (e.g, a tumor suppressor gene) by altering the gene's iehylation patterns, The present invention is not limited to the use of methylated oligonucleotides. Indeed, the use of non-methylated oligonucleotides for the inhibition of gent expression is specifcally contemplated by the present invention. Experiments conducted dunng the 46 course of development of the present invention (See e.g, Example 8) demonstrated that an unmethylated oligonucleotide targeted toward Bc2 inhibited the growth of lymphoma cells to a level that was comparable to that of a iethylated oligonucleotde, 5 P OligonIeVeotides in some embodiments, the present invention provides antigene oligonucleotides for inhibiting the expression of oncogenes. Exemplary design and production strategies for antigens are described below, The below description is not intended to limit the scope of antigene compounds suitable for use in the present invention One skilled in the relevant 10 recognizes that additional antigenes are within the scope of the present invenion, A. Oigonucleotide Design in some embodiments, oligonucleotides are designed based on preferred design criteria, Such oiigonucleotides can then be tested fbr efficacy using the methods disclosed 15 herein. For example, in some embodiments, the olgonucleotides are methylated at least one, preferably at least two, and even more preferably, all of the CpG islands. In other embodiments, the oligonuclectides contain no methylation, The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechanism is not necessary to practice the present invention. Nonetheless, it is contemplated that preferred 20 oligonucleotides are those that have at least a 50% GC content and at least 2 GC dinucleotides, It is preferred that oigonucleotides do not self hybridiz. in some embodiments, oligonucleotides are designed with at least I A or T to minimize self hybridization. In some embodiments, commercially available computer programs are used to survey oligonucleotides for the ability to self hybridize. Preferred oligonucleotides are at 25 least 10, and preferably at least 15 nucleotides and no more than 100 nucleotides in length. Particularly preferred oligonucleotides are 1824 nucleotides in length, In some embodimnents, otigonucleotides comprise the universal protein binding sequences COCCC and CGC or the complements thereof It is also preferred tat the oligonucleotde hybridie to a promoter region of a gene 30 upstream from the TATA box of the promoter It is also preferred that oligonucleotide compounds are not completely homologous to other regions of the human genon. The homology f the ogonucleoide compounds of the present invention to other regions of the genome can be determned using available search tools (e.g, BLAST, available at the Internet site of NCI, 4.7 in some embodiments, oligonucleoides are designed to hybridize to regions of the promoter region of an oncogene known to be bound by proteins (e"gr transcription factors). Exemplary oigonuclentide compounds of the present invention ar shown in Figures 2, 4, 6, 8, 10, and 12 The present invention is not limited to the oligonuleotides described h herein. th sitab Igonuceoides may be identified (g, u si nghe criteia descried above). Exemplary oligonueleotide variants of the disclosed oligonucleotides are shown in Figures 25-30, Candidate oligonucleotides may be tested for efficacy using any suitable method, including, but not limited to, those described in the illustrative examples below. Using the in wtro asay described in Examples 1 and 2 below, candidate igonucleotides 10 can be evauated for their ability to prevent cell proliferation as a variety of concentrations. Particularly preferred oligonucleotides are those that inhibit gene expression of cell proliferation as a low concentration (e'g- less that 20 pM, and preferably, less than t0 pM in the i vito assays disclosed herein). 15 W, Preferred Ohigonucleetide Zones in some embodimens, regions within the promoter region of an oncogene arc further defined as preferred regions for hybridization of oligonucleotides, In some embodiments, these preferred regions are referred to as hot zones. In son preferred embodiments, hot zones are defined based on oligonucleotide 20 compounds that are demonstrated to be effective (see above section on oligonucleotides) and those that are contemplated to be efieetive based or the preferred criteria for oligonucleotides described above, Preferred hot zones encompass 10 bp upstream and downstream of each compound included in each hot zone and have at least 1 CC or more within an increment of 40 hp further upstream or downstream of each compound, In 25 preferred embodimens, hot zones encompass a maximum of 100 bp upstream and downstream of each oligonucleotide compound included in the hot zone. In additional embodents, hot zones are defined at beginning regions of each promoter, These hot zores are defined either based on effective sequences) or contemplated sequences and have a preferred maximum length of 200 hp. Based on the above described critena, exemplary 30 hot zones were designed, These hot zones are shown in Table L, Numbering is based on the sequences described in the Figures of the present invention, 4:8 Table I Exemplary Hot Zones Gene. H-ot Zones B-2 -40 161-350 401-590 1002-1260 c-erbB-2 205-344 382-435 c-K-ras 1-289 432-658 1631-1722 c-mye 3-24 GF- 1-90 266-367 43V-930 C. Preparation and Formnulatiol of Oligonucleotides Any of the known methods of oligonueotide synthesis can be used to prepare the modifed oligonucleotides of the pre invention. In some embodiments uing metIhlated oigonucLieotis tide, C is replaced by 5-methyl-dC where appmpriate, as taught by the present invention. The modified or unmodifed igonuceotidesof te pre invetn ar most convenntly pg y the commaly avai e aomated nule acid synthesizer, They also be obtaind fTom commercial sources tht sntesie custom oligonucleetides pursuant to customer specications. 49 While oligonucleotides are a preferred form of compound, the present nventin comprehends other oligomeric oligonmcleotide compounds, including but not limited to oligonucleotide mimetics such as are described below. The oigonucleotide compounds in accordance with this invention preferably comprise from about 18 to about 30 nucleobases 5 (ie, from about IS to about 30 linked bases, although both longer and shorter sequences may find use with the present invention Specific examples of prefmed compounds usefIl with the present mention include oligonucleotides containing modified backbones or nom-natural intemucleoside linkages, As defined in this specification, oligonucleotides having modified backbones include those 10 that retain a phosphorus atm m the backbone and those that do not have a phosphorus atom in the backbone For the pwposes of this specification, modified otigonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. Preferred modified ohgonucleotide backbones include, for example, 15 phosphothioates, chiral phosphorotioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3Oalkylene phosphonates and Chiral phosphonates, phosphates, phosphoramidates including Yamino phosphoramid ate and ammnoalkylphosphoramridates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having 201 normal 35 linkages, 2&y linked analogs of these, and those having inverted polarity wherein the adjacent pars of nucleoside units are linked 35' to 5'3' or 2*5 to 5-2*. Varous salts, mixed salts and free acid forms are also included, Preferred modified oligonucleotide backbones that do not include a ph osphons atom therein have backbones that are formed by short chain alkyl or cycloalkyl 25 intenmucleoside linkages, mixed heteroatoin and alky! or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyce intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sude, sulfoxide and sulfone backbones; forimacetyl and thiofonnacetyl backbones; methylene formacetyl and thiofonnacetyl backbones; alkene 30 containing backbones; sulfamate backbones; methyleneinino and methylenehydrazino backbores; sulfonate and sulfonamide backbones; anide backbones: and others having mixed N, 0, S and CH 2 component parts. 50 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 nueic acid target compound, One such oligomneri compound, an oligonucleotide mimetic that has been shown to have S excellent hybridization properties, is referred to as a peptide nucleic acid (PNA), In PNA compounds, the suga-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethyiglycine 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, 10 but are nor limited to, US. Pat. Nosv 5,539,082; 5714,331; and 5,719,22, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen e al- Science 254:1497 ( 991). In some emtbodiments, ligonuclotides of the invent ion are oligonucleotides witl phosphorothioate backbones and oligonucleosides wit heteroatom backbones, and in S particular --CH 2 , -N -.- H 2 - -CH2-N(CH)0CH- (known a a methylknc (melhylimino) or MMT backbone, -CH--0-N(CH3}-CH2 -CH2-~N(CH 3

-N(CH

3 -C2^-, and -O-N(CH 3 -Ct-CH2 [wherein the natwe phosphodiester backbone is represented as -O-P-~2~~ of the above referenced U.S, Pat No. 5,489,677, and the arnide backbones of the above referenced US, Pat No. 0 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures (f the above-referened US. Pat No. 5,034,506. 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-dkenyl; O, S- or Nalkymy or 0-alkyl-O-alkyl, wherein the alkyl 25 alkenyl and aikyny. may be substituted or unsubstitauted C1 to C10 alkyl or C2 to Cg0 alkenyl and alikynyt Particularly preferred are O[CH2)nO mCH3, O(CH2)nOCH3, O(CH2 7

)

9

NH

2 , O(CH2')nCH3,~ O(CE 2 )nO)NH 2 , and O(CHM)nN[(CH2)nCH3)I2, where n and m are from I to about 10. Other preferred ohgonucleotides comprise one of the following at the 2' post in: C' to C" g lower alkyl, substituted lower alkyl, alikaryl aralkyt 3 0-aikaryi or C-aralkyt SH, SCH 3 OCN, Cl, Br, CN, CF3, OCF, S0CH 3 , S02CS 3 , 0N20, NO2, N 3 , NH 2 , hetercycloaikyl beterocycloalkaryl, aminoalkyiamino, polyaikylamino, substituted silyt an RNA cleavng group, a reporter group, an inercalator, 51 a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the phannacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2nmethoxycthoxy (2*-0--CH 2 C112003, also known as T-O-2-methoxyethyl) or 2MCE) (Martn et at S Hev, Chim. Acta 78:486 [1995}} mt an alkoxyaikoxy group, A farther preferred modification includes 2t-dimethylaminoxyethoxy {ts a O(CH2z)2ON(CH3)2 group) also known as 2DMAOE, and 2 t dimethyaminoethoxyethoxy (also known in the art as O0-dimnethy amninoethoxyethyi or 2>DMAEOE), ia, 2-O-CH 2 -- Q--CR 2

-N(CH

2 3 2 Other preferred modifcations include 2-methoxy(O-2-CH3) 20 X-amninopropoxy(2AXCH2CH2CH2NH2) and 2fluoro (2'-F). Similar modifications may also he made at otter positions on the oligonucleotidec particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2t3 linked oligonacleotides and the 5 position of 5* terminal& nucleotide. Oligonuclcotides may also have sugar mimics such as cyeiobutyI moienes im place of the pcntoftmosyl sugar, I5 Oligonucleotides may also include ncleobase (often referred to in the an simply as "base") modifications or substitutions. As used herein "unmodified" or "natural" nucleobases include the pune bases adenine (A) and guanine (C'j, and the pyrimidinc bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methyctosine (S-ne-C) 5-hydroxymethyt cytosine, 20 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-dhiouracii 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosme, 5-propynyl uracil and cytosne, 6-azo uracil, cytosme and thympine, 5-uracil (pseudouracil), 4-thiouracit 8-halo, S-amino, 8-thiol, 8-thialkyl, 8-hydroxyl and other 8-substituted adenines and guanines, S-halo 25 particularly 5-bromo, 5trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguaninc and 7-methyladenine., 8-azaguanine and 8-azaadenine, 7deazaguanine and 7-deazandenine and 3-deazaguanine and 3-deaadenine. Further nucleobases include those disclosed in U.. Pat, No, 3,687,808. Certain of these mactleobases are particularly useful for increasing the binding affinity of the oligomerie compounds of the invention. These 3 include 5~substituted pyrinidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2- arinopropy adenine, 5-propynyluracil and 5-propynylcytosine, 5-nthylcytosine substitutions have been show n to increase nucleic acid duplex stability by 52 0.6-1,2*C and are presently preferred chase substitutions, even more particularly when combined with 2-O-methoxyethyl sugar modifications Another modification of the oligonucleotides of the present invention involves chemical linking to the oligonucieotide one or more moieties or conjugates that enhance 5 the activity, cellular distribution or cellular uptake of the oligonueleoie. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid a thioether, g. bexyl-$-tritylthiol), a thiocholesterol, an aliphatic chain, (ag, dodecandiol or undecyl residues), a phospholipid(e~g, dibhexadecy-rac-glycerol or triethylammnonium 1;2-di-0-hexadecy-rac-glycero-3-l-phosphonate), a polyamine or a polyethylene glyco 10 chain or adamamane acetic acid, a palmityl moiety, or an octadecylamine or hexylamnino-carbonyl-oxycholesterol moiety, One skilled in the relevant art knows well how to generate oligonucleotides containing the above-described modifications, The present inveAion is not limited to the antisense otigonucleotides described above, Any suitable modification or substitution may i1S be utilized. It is not necessary for all positions in a given compound to be unifomily modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a smgle nucleoside within an oligonucleotide, The present invention also includes pharmaceutical compositons and formulations that include the 20 anisense compounds of the present mvention as described below. D, Cocktails in some embodiments, the present invention provides cocktails comprising two or more otigooucleotides directed towards promoter regions of genes (e.fg, oncogenes) In 25 some embodiments, the two oligonucleotides hybridize to different regions of the promoter of the same gene. In other embodiments, the two or more oligonucleotides hybridize to promoters of two different genes, The present invention is not limited to a particular mechanism. Indeed, an understanding of the mechansm is not necessary to practice the present invention. Nonetheless, it is contemplated that the combination of two or more 30 compounds of the present invention provides an inhibition of cancer cell growth that is greater than the additive inhibition of each of the compounds administered separately, 53 V. Research Uses The present invention is not limited to therapeutc applications, For example In sonc embodiments, the present i eynton provides compositions and methods for the use of chigonueleotides as a research toolt A, Kits For example, in some embodiments, the present mvention provides kits comprising oligonuleoides specihc for inhibition of a gene of interest, and optionally cell lines (eg, cancer eells tines) known to express the gene. Such kits find use, for example, in the t0 identification of metaboik pathways or the involvement of genes in disease (eg. cancer4 as well as in diagnostic applicanons. In some embodiments, the kits further comprise buffer and other necessary reagens, as well as instructions for using the kits, B. Target validation 15 In some embodiments, the present inversion provides methods and compositions for use in the vaidation of gene targets (eg, genes suspected of being involved in disease), For example, in some embodiments, the expression of genes identified n broad screening applications (egSy gene expression arrays) as being involved in disease is downregulated using the methods and compositions of the present invention. Thi methods and 20 compositions of' the present invention are suitable for use in vitro and in vivo (e.g, in a non human animal) for the purpose of target validation. In other embodiments, the compounds of the present invention find use in transplantation research (e.g HL A mihibition), C Drug Screening 25 In other embodiments, the methods and compositions of the present invention are used in drug screening applications. For example, in some enbodiments, oigonucleotides of the present invention are administered to a cell (e.g, in culture or in a no-human animal) in order to inhibit the expression of a gene of interest, in sonic embodiments, the inhibition of thec gene of interest minics a physiological or disease condition, In other embodiments, 30 an oncogene is Nhibited. Test compounds (e.g , small molecule drugs or oligonucleotide nimetics) are then administered to the test cell and the effect of the Lest compounds is assayed, The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including 54 biological libraries; peptoid libraries (libraries of molecules having the fmactionalties of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remam bioactive; see, e.g. Zuckennann er al, 'j, Med, Chem. 37: 2678-85 [1994]); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the'one-bead one-copound' library method; and synthetic library methods using afinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des 12:145). 10 Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et at Proc. Nat. Acad, Se. U.S A, 90:6909 [1993]; Er et at. Proc. Nad. Acad. SWi USA 91:11422 [1994]; Zuckermann et al., I Med. Chen. 37:2678 [1994]; Cho e at Science 261:1303 [1993]; Carel enat, Angew. Chem it Ed, Eng. 33.2059 [1994]: Carell er at, Angew. Chem. Int. Ed. Engh 33:2061 [1994]; and Gallop et at I 1 Med. Clhem 37:1233 [19)4], Libraries of compounds may be presented in solution (ag., Houghten, Biotechniques 13412-421 [1992}), or on heads (Lam Nature 354:82-84 11991 j) chips (Fodor, Nature 364:55-556 [1993]y bacteria or spores (U.S. Patent No. 5,223,409; herein incorporated by reference), phismids (Cull ea at Proc. Nad. Acad, Sci USA 89:1865 1869 [1992]) or on 20 phage (Scott and Smith, Sence 249:386~390 [1990]; Devln Science 249:404406 [1990]; Cwirla et at. Proc, Nati Acad. Sci. 87:6378-6382 [1990; Felici, , Mol Biot 222:301 [1991]), VL, Compositions and Delivery 25 I some embodiments, the oligonucleotide compounds of the present invention are formulated as pharmaceutical compositons for delivery to a subject as a pharmaceuhcal The novel antigen compounds of the present invention ind use in the treatment of a variety of disease states and conditions in which it is desirable to inhibit the expression of a gene or the growth of a celt In son preferred embodiments, the compounds are used to treat 30 disease states resulting from uncontrolled cell growth, for example including, but not limited to, cancer. The present invention is not limited to the treatment of a particular cancer. The oligonucleotide compounds of the present invention are suitable for the treatment of a vanety of cancers including. but not limited to, breast, colon, lung, stomach, S5 pancreatic, bladder, leukemia, and lymphoma. The below discussion provides exemplary, non- limiting examples of fbmulations and dosages. A., Pbarmaeutical Compositions The present invention further provides pharmaceutical compositions (e.g, comprising the oligonucleotide compounds described above'), 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 10 vaginal and rectal delivery) pulmonary (cg, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration eludes intravenous, intraarterial, subcutaneous, intraperItoneal or intramuscular infection or infusion; or intracrania], e-g, intrathecal or int'raventricul ar, administration, 15 Phamaceutical compositions and fomulations for topical administration may include transdermal patches, ointments, lotions, creams, gels 4 drops, suppositories, sprays, liquids and powders. Conventional pharmiacewtca rriers aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Compositions and fomulations for oral administration include powders or granules 20 suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsines, dispersing aids or binders may be desirable. Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents 25 and other suitable additives such as, bit not limited to, penetration enhances, carrier compounds and other phannaceutically acceptable carriers or excipiens. Pharmaceutical compositions of the present invention include, but are not limited to. solutions, emulsions, and liposome-containing fomulations. These compositions may be generated from a variety of components that include, but are not limited to, pretormed 30 liquids, self emulsifying solids and self-emulsifying semnisoids, The pharmaceutical formulations of the present invention, which may convemently 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 ingrdients with the pharmaceutical carriers) or excipient(s), In 56 general the fornulations are prepared by unifonnly 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 The iompositions of the present invention may be fomulated into any of nany 5 possle dosage forns such as, but not limited to, tablets capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present ivention may also he formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for examples sodium carboxymethylcellulose, sorbitol and/or dextran, The suspension may also 10 contain stabilizers. In one embodiment of the present invention the pharnaceutical compositions may be formulated and used as foams, Pharmaceutical foams include formulations such as, but not limited to, emulsions, microcrnUsions, creams, jelhes and liposones, While basically similar m nature these fornmulations vary in the components and the consistency of the final 15 product. Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and otter compositions of the present invention, For example, cationic lipids, such as lipofectin (U,.S Pat, No. 5 3 705,S8} cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 9730731 ), also enhance the cellular 20 uptake of oligonucleotides, The compositions of the present ivention may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus 3 for example, the compositions may contain additional, compatible, pharmaceuticaily-active materials such as, for example, antipruritics, astringents, local anesthetics or antiKnflanmmatory agents; or 25 may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, favoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of te components of the compositions of the present invetion. The fbnnulations can be sterilized and, if desired, 30 mixed with aiiary agents eg, lubr.ca, prservativs, stabilizers, wtng agent emulsifers, salts for influencmg osmotic pressurevtuffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. 57 Compositions and formulations for oral administration include powders or granules, micropartuates, nanopariculates, suspensions or solutions in water or non-aqueous media, capsules, ge capsules, sachets, tablets or minitables, Thickeners, flavoring agents, diluents, emulsifers, dispersing aids or binders may be desirable. Preferred oral 5 fornulations are those in which oligonucletides of the invention are administered in conjuncton 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 bite acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic 10 acid, glucholic acid, glycholic acid, glycodeoxychoie acid, taurocholic acid, tatwodeoxycholic aeid, sodium tauro-24,25dihydro-fusidate, sodium glycodihydrofusidate. Prefe'ed fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid. caprylic acid, capric acid, myristie acid, painitic acid, stearic acid, linoleic acid, linofenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl I -monucaprate, I 15 dodecylazacycloheptan-2-one, an avylcarnidine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (eg sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/sats in combination with bile acids/salts. A pati u aly preferred combination is the sodium sa of lauric acid, capric acid and UDCA. Further penetration enhances include polyoxyethylene-9auryl ether, 20 polyoxyethylene-20-cetyl ether, Gligonucleotides of the invention may be delivered orally in granular form including sprayed dried particles, or complexes to fon micro or nanoparticles. Oligonucleotide completing agents include poly-aino acids; polymines; polyacrylates; polyalkyiacryiates, polyoxethanes, poiyalkyicyanoacryiates; cationized gelatin, albunins, starches, acrylates, polyethyleneglycols (PEG) and starches; 25 polyalkyleyanoacrylates; DEAFEdenivatized polyimines, pollulans, cliuloses and starches. Particularly preferred complexng agents include chitosan, N-rimethylchitosan. poly-L lysine, polyhistidine, polyompithine, polyspermines. protamine, polyvinylpyidine, polythiodiethylammno-mnethylethyene P(TDAE), polyaminostyrene (e'g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylateY. poly(butylyanoacrylate), 30 poly(isobutylcyanoacryiate), poly(isohex ylcynaoacrylate)3, DEAE-mnethacrylate, DEAF hexylacrylate, DEAE-acrylamtide, DEAE-albumin and DEAE-dextras. polymethylaerylate, p lyhexylacrylate, poly(DLLactic acid), polIy(DL-lactic-co-glycolic acid (PLGA), alginate and poiyethylenegiycol (PEG). 58 Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more oligonucleotide compounds and (b) one or more other chemotherapeutic agents that function by a non-oligonucleotide mechanism. Examples of such chemotherapeutic agents include, but are not limited to, anticancer drugs such as 5 daunorubicin. dactinomycin, doxorubicin, bleonycin, nmitomycin, nitrogen mustard, chloramnbucii melph alan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA). 5-fluoruracil (5FU), floxuridine (5-FUdR), methotrexae (MTX), coichicine, vincristine, vinbiastine, etoposide, temposide, cisplatin and diethylstilbestrol (DES), Amti-iflanmatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs 10 and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarubine, acyclovir and ganciclovir, may also be combined in compositions of the invention, Other non-oligonucleotide chemotherapeutic agents are also within the scope of this invention Two or more combined compounds may be used together or sequentially, 15 B. Delivery The oligonucleotide compounds of the present invention may be delivered using any suitable method, In some embodiments, naked DNA is administered, in other embodiments, lipofection is utilized for the deNvery of nucleic acids to a subect In still further embodiments, oligonucleotides are modified with phosphothiolates for delivery (See 20 ,g. U S. Patent 6,169,177, herein incorporated by reference). In some embodiments, nucleic acids for delivery are compacted to aid in their uptake (See a g U.S, Patents 6,008,366, 6,383,811 herein incorporated by reference). in some embodiment, compacted nucleic acids are arrested to a particular cell type (g, cancer cell) via a rget cell binding moiety (See e.g. US, Patents 5,844,107, 6),77835, 25 each of which is herein incorporated by reference), 1n sonc embodiments oligonucleotides are conjugated to other compounds to aid in their delivery For example, in some embodiments, nucleic acids are conjugated to polyethylene glycol to aid in delivery (See eg, U. Patents 6,17,274, 6,2S7,5 1, 6,447,752, 6,447,753, and 6,440743, each of which is herein incorporated by reference). In 30 yet other embodiments, ohigonucieotides are conjugated to protected graft copolymers, which are chargeable" drug nano-carriers (Pharmailn), in still further emibodinents, the transport of oligonucleotides into cells is facilitated by conjugation to vitamins (Endocye, tc, Wes Lafayette, IN; See e.g., U.S. Patents 5108,921, 5,416,016, 5,635,382, 6291,673 and WO 02/085908; each of which is herein incorporated by reference), in other 59 embodiments, oligonucleotides are conjgatcd to nanoparticles (e.g. NanoMed Phannaceutic als; Kalamazoo. Ml). In preferred embodiments, oligonucleotides are enclosed in lipids (e.g., liposones or micelles) to aid in delivery (See eg,. U,S. Patents i, 458.,382, 6,29,>200; each of which is herein incorporated by reference). Preferred liposomes include, but are not limited to, cardiolipin based cationic liposomes (e.g , NEOPHECTIN available from NeoPharm, Forest Lake, IL). In some preferred embodiment, the charge raton of NEOPHECTIN to otigonuclcotide is 6:1, In still further embodiments. oigonucleutides are complexed with additional polymers to aid in delivery (See eg, U. Patents 6,379,966, 6,339,067, 10 5,744335; each of which is herein incorporate by reference and tntmdigm Conp., Rockvilie, MD). in sti1 further embodiments, the controlled high pressure delivery system developed by Miirus (Madison, WI) is utilized for delivery of oligonucleoides, 15 C Dosages 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 m the body of the patient The 20 administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oiigonuelcondes, and the delivery means, and can generally be esinated based on EC50s thund to be effective in in vitro and in vivo animal models or based on the examples described herein, la general, dosage is from 0.01 pig to 100 g per kg of body 25 weight, and may be given once or more daily, weekly, monthly or yearly. in some embodiments, dosage is continuous (e g, intravenously) for a penod of from several hours to several days or weeks In some embodiments, treatment is given continuously for a defined period followed by a treatment fee period. In some embodiments, the pattern of continuous dosing followed by a treatment free period is repeated several times (eg, until 30 the disease state is diminished). The treating physician can 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 subject undergo maintenance therapy 60 to prevent the recurrence of the disease stare, wherein the oligonucleotide is administered in maintenance doses, ranging from 0,01 pg to 110 g, preferably front 1mg to 50 mg, and even more preferably frotm 6 mg to 30 rug per kg of body weight, once or re daily, to once every 20 years. VI. Combination Therapy In some embodiments, the compositions of the present invention are provided in combination with existing therapies In other embodiments, two or nore compounds of the present invention are provided i combination. in some embodiments, the compounds of 10 the present invention are provided in combination with known cancer chemotherapy agents. The present invention is not limited to a particular chemotherapy agent. Various classes of antineoplastic (eg., anticancer) agents are contemplated for use in certain embodiments o the prese nt invention. Anticancer agents suitable For use with the present invention include, but are not limited to, agents that induce apoptosis, agents that 15 inhibit adenosine deamnase function, inhibit pyrimnidine biosynthesis, inhibit purine ring biosynthesis, inhibit nucleotido interconversions, inhibit ribonueleotide reductase, inhibit thymidine monophosphate (TMP) snhesis, inhibit dihydrofolate reduction, inhibit DNA synthesis, Form adducts wnh DNA, damage DNA, inhibit DNA repair, intercalate with DNA, deaminate asparagines, inhibit RNA synthesis, inibit protein synhesis or stability, 20 ilhibit microtubule synthesis or function, and the like. In some enbodinents, exemplary anticancer agents suitable for use in compositions and methods of the present invention include, but are not limited to: ) alkaloids, including microtubule inhibitors (eg, vineristine, vinblastine, and vindesine, etc ), nicrotubule stabiizers (e,g, paclitaxel (TAXOL), and doetaxel, ec. I and chromnain function .25 inhibitors, including topoisomerase inhibitors such as epipodophyllotoxins (etg, etoposide (VP-16), and teniposide (VM-26), etc.), and agents that target topoisomerase f (e.g, camptothecin and isirinotecan (CPT1 I, ec.); 2) covalent DNA-binding agents (alkylating agents), including nitrogen mustards (eg, nmechlorethamine, ehlorambucil, cyclophosphamide, ifospharmide, and busuifan (MYLER AN), etc), nitrosoureas (eg, 30 cannustine, lomustine, and semustne, etc.), and other akylating agents (e.g, dacarbazine, hydroxymethymelaine, thiotepa, and mitomycin, cIc); 3) noncovalent DNA.-binding agents (antitunor antibiotics), including nucleic acid inhibitors (e.g, dactinomycin (actinomycin 1)), ec), anthracyclines (e.g, daunorubicin (daunomycin, and cerubidin), doxoruhicin (adriam ycin and idarubicin (idamycin), etc.), anthracenediones (eg, 61 :3 anthracycline analogues, such as mitoxantrone, er.), bleomycins (BL ENOXAN etc, and plicamnycin (mithramnycinh etc, 4) antimetaboites, including antifolates (e~g.. methotrexate. FOLEX, and MEXA TE, etre), punne antimnetabolites (e.g,&6mercaptopurine (6-MP, PURINE THOL), 6-thioguanine (6-T2) azathioprine, acyclovir, ganciclovir, chlorodeoxyadenosine, 2-chlorodcoxyadenosme (XA), and 2-deoxycoformycin (pentostatin), etc.), pyrimidine antagonists (c~g. fluoropyrimidines (eg, 5-Thuorouracil (ADRUCIL), 5- fiuorodeoxyuridne (FdUrd) (floxuridine)) etc Yand cytosine arabinosides (e4g. CYTOSAR (ara-C) and fludarabine, etc); 5) enzymes, including L-asparaginase, and hydroxyurea, etc,; 6) hormones, including glucocorticoids, antiestrogens (e.g, tamoxifen, 10 etch nonsteroidal antiandrogens (e~g., flutamide, etc), and uromatase inhibitors (e.g anastrozole (ARIMIDEX), ec); 7) platinum compounds (eg., cisplatin and earboplatin, etc); 8) monoclonal antibodies conjugated with anticancer dmgs, toxins, and/or radionuclides, tc.; 9) biological response modifiers (e.g., interferons (e.g., FN-n etc.) and interleukins (egg TL-2, etc.), etc.); 10) adoptive immunotherapy; ii) hematopoietic growth 15 factors; 12) agents that induce tumor cell differentiation (e., al-trans-rctinoic acid, etc,); 13) gene therapy techniques; 14) antisense therapy techniques; 15) tumor vacces; 16} therapies directed against tumor metastases (e.g, batimastat, etc); 17) angiogenesis inhibitors; 18) proteosome mhibitors (e.g, VELCADE); 19) inhibitors of acetylation and/or methylation (e g. HDAC inhibitors); 20) modulators ofNF kappa B; 21) inhibitors oftceiI 20 cycle regulation (4g. CDK inhibitors); 22 modulators of p53 protein function; and 23) radiation, Any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention. For example, the U.S. Food and Drug Administration namtains a formulary of onculytic agents approved for use in the 25 United States, international counterpart agencies to the U. S.F.D.A maintain similar fonularies. Table 3 provides a list of exemplary antineoplastic agents approved for use in tihe U5. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved C1sapproved indications, dosing information, toxicity data, and the like, for the exemplary agents 30 Table3 Aldesleukin Prdeukin Chiron Corp,. (decs-alnyl- serine-125 human imedeuin2) Emreryvitte CA Aletmutusnsh Caavath Millennium and {Ig~Gl anti CDS2 antibady) ItEX Pa~rtners, LP, 62 Camtbridge, NA Pametin Lgand (9-cis-retinoic acid) Pharmalceuticals, Inc., San Diego CA )Aliopurinol Zyvtoprnm GiaxoSirntKhne, o ,$dbhydro,4 H pyrazoll3,4-dlpyrnuidun4one Research Triangle monsod~I am al) Par, NC Akretamine Hekxalcn US Biosience., West (NWNNNNTV texametthy1 1 )335raine-2, 4, Consbohockeni, PA jnane) ________ A n stine Ethyol US Bioscience (ethanthmi4 2- [3- nopryl~ fanfino), dibydrogen phosphate (ester)) ______ _________ Anastrozoke Armides AsaZeneca (1,JAlenzenediacetontrike a-, s, a', aVttnmrethyb Pharmauveuvicals, LP, 5-(tH44,tiao y flmthyr)) W i almington D Arsenic rioxide Trisenox Cell Terapeutic, Inet Seatte, WA A spat aginiie EIspar Merck & CoA The. (L-asparagmne anmidtohydrolase, type EC-2) Witehouse Station. NJ BCG Live T1CE SCG Organon Tektuka; (tyopbibzed prepartion of an attempted strain of Corp, Dulrhamn, NC Aiycohneteram bowis (Boeillus Cal4e4thGin [BCOI substnam Monrafl)_________________ bexarotene capsudes Targrttin 1gan (44 G(SA.7-trhydr2A3,Spentmety[2, Pharmaeucabs napthalenyl) ethenyQ benzoic acid) _________ bexaroten gel Targretin Lrcand Pharnmaeuticas Bleomycmn Beenaxar Bstal-Myers Squibb (cytotoxic glycopeptide antibionecs pmduced by Co.. NV, NY Strepromyces iedhus bleomycus At ea bicomycin B2t) _______ _________ Capecitabine Xeloda Roche (Sdeoxy-SluooN-(pntyloxyktibonyl'1 cytidine) ________ Carboplatia Parapiatin BristobMyers Sqmbb platinum , diamnnne (>l clcobuanedicaboaylato(2-)-0, Gt(SP-4-2)) ______ _________ Carmastiac BCNU. I$CNU Bnistol-Myers Squibb (.. .bis(2-chloroethyl>) itrosurea) Carmnustine with Pohfeprsan 20 implant Giiade4 Wafer CGuiefor Pharmnaceutticals, 1ne, Pa Baimore MD Ceex ib Cele brex Searle (as 445-(4-mnethyphenlN3 thucomethv1)- haraceuticals, W Hpytanoi h-yl] England enzenesulfonamidet) Chksrmbucii Leukezaa ClaxosmithKline 4-[bis(2chiorethyt~hmino bergenewbutanoic acid) __________ Cisplatin Platmoci BristokMyers Squibb (PtClai~INx)________ __________ Cladribine Leustaar, 2-CdA RAY lohnson (2-chloro~-txyb--adenosine) ______ Pharmaceutical 63 Reseach ispttuttem Rariian, N1 Cyclophosphamnide Cytoxan,194eosar BrisokMyers Squibb (2-[bis{2-ch oroethyar&muo trnhydro-2fl-13)2 oxcavaphosphorine 2-rxide m~onohydrate) Cytatabine Cytosar-U Pharmaacia & Upjohn (i-b-D-Arabinofuranlosykcytosme, CHi-N103k) Company eytsrabne tipoomafl~ DepoCyt Skye Pharmaceuicals, bac. TSan Diego, CA Dacarxbazmxu DiC-Dome Bayer AG, (5-(%3-imnethyi- brtent smidazole-4-~ teverkusen, carboxarnide (DT C)) Germany Dchnomycin, acnmycm D Cosmegen Merck (actinomnyci prodneed by Streptosmywes parvullus. Dabepoti .alf Aranesp Amjgen Tnhc. (recmbant pcpnde&) _Thousand Oaks, CA d4unonbicin liposmas DanoXcnme Nexst (($S-cs)8-ety1-(3imo-2)36-trideoxy-, Phamnaeaicais, In:. L-lyxo-hexopyranosyfloxy}7,8910-etrhydro- iBmdder) CO 1 N te cydroxy- I Tethoxy-5 , naphthaceedione hydrchioride) Dkaunorubicn iCL daunornyrn Cembtidine W yeth Ayert, (US 3 i3 S) N3-Actyl-I234,itUbahydro- Madeon N TS512-tnhydmnxy-10,mnethcnry,6,1-dhoso-i naphthacenyvl '-atnino-216-tuideoxdalpa)-L (oem -hexopyratioside hydrochoride) ______ Dminkm diflitox Omak Seragen, Inc irecombmparft peptide) H____ _ fopkmnton, MA Dexraaoxane Zinecard Pharmnacia & Upjohn (()44 -methyl-1;2,thnedybibis-2,6- Com~pay piperazinedione)__________ Docetaxel Taxotere Avents ((2R,3S)-N-arboxy-3-phenyhsosermec, N4-ters- Pharmaceuticas, Inck, buty1 eser, 13-ester wath 5b420epoxy- Bidgewater, N) 12a,4,7b, 0b13a-exahyxytax- I I-eni9one 4 acetate 2-benroate, trihydrate) Doxorabica H1(2 Adrtamyoir, Pharmacia & Uipjohn (8S 10S)-i0-[(3-amina236-ineoxy-a-Ldyxo- Rubes Company hexopyrannsyl)oxy]-A8-giycoy1-7 ,8,9,{ tetrahydro-S,$ 4~ trihydroxyi menThxy5 2 naphtaceedione hydrochdonrde) duxembhicin Adriamycin PFS Pharmiacia & Upjohn itrtavenous Company _____________________________________ injrection doxorubidialiposomal Dcxil Sequs Pharmactmicai. In.. Meno park, CA dromostanolone propicnate Dromoc:uanolone Ehi Lilly & Comnpany' ( I 7b-Hydroxyw2a-methy-5a-anxostan-3-ome Iudianapolis. IN propionate) dromostanolone propionate Masterone Sysex, Corp., Palo _____________________________inecton Aito, CA 64 Eliosts U Sokition Elliott's B Orphan Medicat hic SLuioxt SElence Pharacia & U pjdhn {{Scs-0(-mi26tdoya Comrtpany arabir, hexopyrayl)myp7&90 -tetabydro 6,8,1i~trihydroxyS- (hydroxyraetyDi-mehoxy 5,12-naphthancenedione hydrochloride) _________ Epotm aWa Epogen A mngente (recomlbinant peptide)_________ EstramustlineE y Phasinacia & Upjohn (estra-lAS(10)-ten -3 4 17dio(17(beta)), 3- Company 3b(2-chioroethy1)e rbamate} 174dihydrogen phosphate), disodrum salt, onohydrate or estradiol 3 Tbis(24-.hloroetby1)earbamnate 17 (dihydwoge.a phosphare), disodiumn salt, mionohydrate) Eto.poside pho~sphate& Eaopophos Boitol-Myers Squibb (t~emethylepipodophyilotoxmn 944,6,4R) ethyidene-(beta)-glucopyranod], 4' *(dihydxogen phosphate)) _________ e toposide, VP- 16 Vegesid Blristol -yers Squibb (4t-demethylepipodophy~lotom 944,6,04R) ethylidenba) -I dopyraoside Exemrestarne Aromsin ~ Pbanrmma & Upjoku (-rnethylenaandrosta-,4dene-3. I7-dione) Company Edgrastim Netpogen Amogen, na (r-mnetH AuG-CSF) Iloxuridine (intraarteriat) PU DR Roche (T-deoxy-3-ituorouridine) _________ Fldarabiie Fiudara IBerlex Laboratories, (ftormatned nucleotide analog of the ativ iral Incs. Cedar Knolls, agent vidarabine, 9-b -D-arahmofurnovladene NJ (ara-A)) __________________ Fluorouxacd, 5-U Adrued JCN Pharmaceutiakl, (5 -fluoro-2,4(H,3H )pyrmudmnedione) Ic, Hiumacac, Puerto Rico Fushestrant Faslodex flPR Ph-armaceutcats, (7-alpha4'9-(4At5 ,55-penua fluoropeotyhlstphinyl) Cuayama, Puerto nonyiestra3,5(10)- tene-31 7 -beta-dil 2 ,f ' Gemeitebine Gemaa Ett Lilly (2-dcoxy-2', 2-difluorocysidme mtonohtydrochlnde (b-somet )) Genuazmab Ozogamicm Myotnrg Wyeth A yers (an-CD33 hP.7 .6) Gosetelin acetate Zoladex implant AstraZeneca (actate sakt of [D-Ser(BluQ Azgl<}U_ HR; roi Pharmoaceutseals Glu-Hlis-Tp-Set-Tyr--Ser(3ut)4Leu-Arg-Pro Azgly-NH-2 acetate[jCs 5 ,N409+ (Cl2H______09____ Hydroxvurea Hvydrea Bantol-Myers Squibb Thrtnmomab Tiameans Zevaha Biogen JDEC, biic, (immnocnjuateresltogfro a hioresCambridge MA Ibritumomnab tad the linker-chelaror tinetan [N {2-bis(carboxcylet inmb-ob-(p isothioya natopheny1)- propylfMN-(2 brst carboxvnmethylnarino)-24-methylh ethylnglyaine) * darubicin Idamycin Pharmacia & Upjohni (3, 12-N apthacenetatng 9t-ary-74(3-atiao Company 2,36tridoox-(aiph)t lyxo hexopyranosyi)oxy)-7,Al0-detrahydro-6931 nohydroxyhydtochorde (?S- cis ))________ __________ lirsfamide .FBX Rrnstoi.-Myers Squibb (3-(2-chloroehy)2- 2 chloroethylParnxno~tt kayo2H-L 1 2.t oxazaphosphoutmre oxide) ________ imniib Mesilate Gleevec Novatis AC, Baset 3-i[4,(3-pyidinyl)-2-pyrimnidinyI~amn, phexiyl}benxamidc mtetanesifoniate) Interfetron ala-2a Roferon-A Uoffmnann-Ln Roche, (necombiaunt peptide) 1ue, Nudky, NJ interferon afafZb Iitton A -Schring AG, Berlm, (tecoliiam peptidek) (Lyophilized Germany Irinatecan fHCI Camiptosar Pharacia & Upjohn ((4 S-4,l-diethyi4-iydroxy-9~[4- pipen- Company dinopipetridmno)carbooyloxyj- I l-pyranc4': 6,7) indolizniu([1,2-b] qianohne-314(4H, 1 25) dine hydtochhiride trihydrate) Letrozole Femiara Novatrtis (4,4t(l1,2A -Trrazol- I yvmethylene) dibenzonitrile) __________________ Leucovotin Wellcvorin, hmnex, Crpx , (L-Glutamiic acid, N[4([(2amino-formtyb Leucovotin Seattle, WA I .4,5,8, hexahydro4exo6 ptenidinylhmethyamninoJhennoyl], calcium salt (I c)) Levamnisoke HCJ raamisoi Jassen Research ((4) S)-2,35, 64erahydro-6-phenyhmidato [2,1- Fontidacmn, b] thiazole snonohydrochloride Ca Hi &baS-HCI) Timtsville. NJ Lomnustinc Cee NU BristobMyers Squibb (1(2-chloro-ethyi}-3 -cyclohexyb itour) Mec torethanmne, nitrogen mustard Mustargen Merck (2-chioto-N-(2-oroethyl-N-tetylehanaine hydroehlortide) _________ Megestrol acetate Megace Bristoiktyers Squibb I 7a( acetyloxy)- 6& methylptegna- 4,6- diene 3,20- diMne Melphaian, L-PAM Alkersn Gla xoSmnithKlinie (4[bie(2.chloroethyA) amuinoL-phenykalanine) __________ Mercaptoputine, t 6-MP Purinethol GlaxoSmithlhne (E7-dihydro,6 H- -putine-64hione mnohydmte) ______ _________ MeTn Mesnex As a Medica (sodits 2-mnercaptoethane .sudfonatc) ________ __________ Medhotexate Methotrexat Lede;e Laboratories (N-4:R2A'-diarizso-6 pteridinylimehyimrhylamiinojbNszoyljpt glutamne acid) Methoxsalen Uvadex Therakos, Iac.. Way 66 (9-nethoxy-7-furso(3,2-ggf }bzopyanl-7-one) ________Exton, Pa Mitonyn C Mtrc Bristoi-Myers Sqibb rnikmycio C Mitozytrex Supeiflen< mac, Dzublin, CA Mimoane Lysodren Blristoh-Myera Squibb (1 &1dichlro2+(o-chloropheay1)-2-(p ch]orophenyI) eNanee Mitoxantrone Novantrone immrunex (14dhdoy58bs2 [(2- Corporation hydrcxyethy4)amitwlethyllammobwj930 anthracenedione diydrchlori de) ______ Nandlolone phenpropionate Daunaboli3-50 Organmn, Inc, West Orange, N NoftvAnnomab Vezrlumia Boelhringer Ingelheiun Phanma KG. Gemnnay Qmpaelek Neumega Generics Instintv, (IL-11) fUm, Alexandna, VA 0> alipatin Eoxatin Sanon Syntheinbo, (.is-[(IR,2R), ,2-eyclohe anediarine~NN'] Inc , NY, NY [ 'att(2)0,O* piattanm) PaclitaxYl TAXOL Bristol-Myars Squibb (5, 20-Epoxy 42a 4,70, 10, 13a hexahydroxytax-i1-en-9-onc 4<iO-diacetate 2 benzoate I Yester witlh (7R. 3 Sh- N-benzoylb phenylisoserine) _______ Pamidronate Areia Novarsl (phosphorsic acid (3-ainbydroxyproyienie) Adagen Eon ((monomtethonxypolyethylene glycol succinimidyl) (Pegadenmase Pharmaceuticals Inc., 11 -17 -adenosine deammiase) Bovine) Bridgewater, Ni Pegaspargase Oncaspar (.monomethoxypo1yethylene glyco succinimihdyi Laspara~pnase) Pegfllgrassin3 Neulasta Atngen. Tic (covalent conjugate ofreconminant mnethionyl human ()-CSF (?Ogrestim) and mnonomeihoxypnlyethy lene glycol) ________________ Pentosiatin Nipent Parke-Davis Pharmnceut&ca Co, Rockville, MD Pipobiroman Vercyte Abot Lstabatns, Abbott Park it Pliscaycin, Mihramnyc Miraracin Pat, inc., NY, NY (antibiotic produced by~ Sfrrposmoe pglicaras______________ Porfiner end iusn Photoftn QLT Phototherapeurti cs, Inc., ncouver, Canada Procarbazine Ma.ula..e Sigma .u (Nasopnopy[-p(2-methylbhydraztno)-p-troiuamide Phanraoeuticals, hic., manohydrocthioridec) _________Gisthetsburg, MD Quinaerine Atabane Abbot Lahs 67 (5-dhloro-9-( I -nethy-4-ityamine) buty'lamnino- 2-mthoxyacridine) ___________ Rasbunase Elitek Sannfi,Sythilabo% (recomrbmant peptide) Ritaximab Ritacan Ccnueeb, trac, (tecombinan. antiCD20 antibody) South San Franaso. CA SgmoimProkmew henexn Corp StetzenZanos r Pharmacma & Up3 ohn (strpteoci 2 -deoy -2 -Company (me'nthylmitmsnamfin~orbnyammiro3 , a(arnd b ) -D -gupynoead220 mg_ citc acid ainbydrous) TkSclerosol Blryan, Corp~, {MigaSi 6 O m (Q) 2 ) Wcbum, MA Tamoxien NolvadNx Amaneca ((2)2-[4-( 1,2-dipheiny bbutenyi) phenoxy}-N, NPamce&cl dirnehytethanrmme 2-4ydroy-2N3 propanetricarboxyate (1:1)) _________ Temnozoomide Temodar Schwrmng (MA-dihydrmanhy-oxemridanOj5,1- d)-as tetrazine,84arbnrTmidet) Teniposid, VM 4 Vumon BistokMyers Squibb (4-deinethylepipodophyllowoxn 9T ,4RF ihuay:Iidere-(be ta)-D glucopyxanmsde]) _________________ Testofattone Tesae Britoi-Myers Sqmbb (134hydroixyauxnod 3, I7Vecandmosa1 A-dien 17oK acid [dgr Ilactone) Tbivguanime,6-TGs Thioguanitie GiaxoSmnithKline (2SarinnI , .dihydro-6 H - perne&htone)___________ Thiowpa Thiople: 1mmunex (Azmdime ,) tphosphmotioyidynettis.,a Corp oration Tris (1Pazmrdmyl) phosphie saimdie4 ________________ Topoteuan HCI Hyanmtm GiaxoSmithKime ((S)A 0~[1dinrtthylamino) methryi}--thv4> dibydroxy-pyrano[3 4': 6J} iandoizino [1,2-ib gamalmie-3,1444H,1I2H)-dioae mionohydrocloride) Toemikne Pareston Roberts (.2-(p-[(2)-4-chioro ,2-dipey I-tbuenyikPamc-na phenoxy).N.Ndirmehylerhyla mine itrate (1) Corp., Earontown, NJ Tosiumomab , 1 131 Tosimmonab Bexxar Cosixa Corp, Seattle, (recombtina nt marine urnnunrothetapennc WA monoclonal Ii laubda ara-C)2 anybody [[ 131 is a radlioimnmunotherapeutic antibody)) _________ Trasturmab Haeep ti Genentech, Inc (reeomubinant ruonoeloal igGC kappa aruH ESR2 anuibody) Tretno, ATRA Vesanoid Roche (ralkrans retinoi acid) Ursac Mustard Uracl Mustard Roberts Labs Capsules Valrubicmin Ntn fluoroaceryhadriamnvcir 14- Valstar Antna -> Medeva 68 valrate ((2Si)-2-3,4,6,1,hebydma 12 trihydroxy47 methoxy-631 -dioxo-{[4 2,36, nioy [{tiuoroacey)ando+cLi y expyransyoxyl]-2aphthacenyt2-oxoethyl petiaoate)C v'inbLastine, Leuroemtine Vetan LiW Lifly (C- sK4NOgrIISOd ViciteOrnvnn Eti Litly (Ca 4 HaN4QjqHiSO4 __________________ Vuotebine Naveibine GaxoSithKiine (3' ,4'tddehydo44kox-C' norv inc afekctlste f,(R* R*)-23a dihydroxybutanedtoate (1:2)(sahon )o ) ) ______gee____In__ Zoledolate, 2otedronic acid Zoneta Novartis ((1-Hydroy2mundaz1h-yl-phosphonthyi) phosphonic acid inonchydrate) _______________ VMI CustOmenIIZd Patent Care in somne embodiments. the preset invention provides customized patient care 5 The compositions of the present invention are targeted to specific genes unique to a patients disease (eg. cancer). For example, in some embodiments, a sample of the patent's cancer or other affected tissue (eg, a biopsy) is first obtained The biopsy is analyzed for the presence of expressio of a particular gene (e.g, oncogene), In some preferred embodiments, the level of expression of an gene in a patient is analyzed, Expression may 10) be detected by monitoring for the presence ofRNA or DNA corresponding to a particular oncogene, Any suitable detection method may be utilized, including, but not limited to, those disclosed below. Following the characterization of the gene expression pattern of a patient's gene of interest, a customized therapy is generated for each patent In preferred embodiments, 1$ oligonucieotid compounds specific for genes that are aberrantly expressed in the patient (e.g. in a tumor) are combined in a treatment cocktail In some embodiments, the treatment cocktail further includes additional chemotherapeutic agents (et., those described above), The cocktail is then administered to the patient as described above, In some emnbodimients, the analysis of canter samples and the selection of 20 oligonucleotides for a treatment compound is automated. For example, in some embodiments, a software program that analyses the expression levels of a series of oncogernes to arrive at the optimum selection and concentration of oligonucleotides is utilzed. in some embodiments, the analysis is performed by the clinical laboratory 69 analyzing the patient sample and is transmitted to a second provider for fortmuation of the treatment cocktail In some embodiments, the information is transmitted over the intermet, thus allowing for the shortest possible time in between diagnosis and the beginning of treatment, A. Detection of RNA in some embodiments, detection of onogenes (eg, including but not limited to, those disclosed herein) is detected by measuring the expression of corresponding mRNA in a tssue sample (e g., cancer tissue). In other embodiments, expression of nRNA is 10 measured iin bodily fluids, including, but not limited to, blood, serum, mucus, and urine, In some preifrred enbodiments, the level of mRNA expression in measured quantitatively, RNA expression may be measured by any suitable method, including but not limited to, those disclosed below. in some embodiments, RNA is detected by Norther blot analysis. NortheN blot 15 analysis involves the separation of RNA and hybridization of a complementary labeled probe, In other embodiments, RNA expression is detected by enzymatic cleavage of specific structures (INVADER assay, Third Wave Technologies; See e.g, US, Patent Nos. 5,8467 17, 6,090,543; ,00P1,567; 5;985,5571 and 5,994,069; each of which is herein incorporated by reference), The INVADER assay detects specific nulec acid (eg., RNA) 20 sequences by using structure-specific enzynes to cleave a complex formed by the hybridization of overlapping oligonucleotide probes, in still further embodiment, RNA (or corresponding cDNA) is detected by hybridization to a oligonucleoidde probe). A variety of hybridization assays using a variety of technologies for hybridization and detection are available. For example, in some 25 embodiments, TaqMan assay (PE Biosystems, Foster City, CA; See e"g., U.S. Patent Nos, 5,962,233 and 5,538,848, each of which is herein incorporated by reference) is utilized, The assay is perfoennd during a PCR reaction, The Taq4an assay exploits the 5'-Y exonuclease activity of the AMPLTAQ GOLD DNA polytnerase. A probe consistng of an oligonucleotide with a 5-reporter dye (e.g, a fluorescent dye) and a 3Vquencher dye is 30 included in the PCR reaction. During PCR, if the probe is bound to its target, the SY nueleolytic activity of the AMPLITAQ GOLD polymerase cleaves the probe between the reporter and the quencher dye. The separation of the reporter dye from the quencher dye results in an increase of fluorescence, The signal accumulates with each cycle of PCR and can be monitored with a fluorimeter, 70 Tn vet other embodiments, reverse-ransenptasc PCR (RT-PCR) is used to detect the expression of RNA. In RT-PCR, RNA is enzymatically convened to complementary DNA or "cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a template for a PCR reaction. PCR products can be detected by any suitable method, including but not $ limited to, gel electrophoresis and staining with a DNA specific stain or hybridization to a labeled probe. In some embodiments, the quantitative reverse transcriptase PCR with standardized mixtures of competitive templates method described in US Patents 5439,606, 5,643,765, and 5,871978 (each of which is herein incorporated by reference) is utilized. B. Detectiou of Proteia In other embodiments, gene expression of oncogenes is detected by measuring the expression of the corresponding plotin or polypeptide. In some embodiments, protein expression is detected in a tissue sample. I other embodiments, protein expression is detected in bodily luids. ln some embodiments, the level of protein expression is 15 quantitated. Protein expression may be dtcted by any suitable method, in snome embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is welt known to those skilled in the art. Antibody binding is detected by techmques known in the art (e.g., radioinmmunoassay, EUSA (enzyme-linked immunosorbant assay) "sandwich" 20 immunoassays, inmunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in s111 immunoassays (e.g, using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, aggiutination assays (eg gel agglutination assays, hemaggiutination assays, etc4 complement fixation assays, immunoiluorescence assays, protein A assays, and immunoelectrophoresis assays, 25 et. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. hi another embodiment, the primary anybody is detected by detecting binding of a secondary antibody or reagent to the primary antibody, In a further enbodimnen the secondary antibody is labeled. Many methods are known in the an for deicnng binding in 30 an inmunoassay and are within the scope of the present invention, In some embodiments, an automated detection assay is utilized, Methods for the automation of immunoassays include those described in US, Patents 5,885,530, 4,981,75, 6,159,750. and 5,358,691, each ofwhich is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated, For example, in 7 ! some emibodiments, software that generates an expression profile based on the presence or absence of a series of proteins corresponding to oncogenes utilized. I other embodiments, the hmumoassay described in US. Patents 5,599,677 and 5,672480; each of which is herein incorporated by refrene. EX PERIMENTAL The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be constrmed as limiting the scope thereof 0 In thexpermen disclosure which olows, the followmg abbreviationis apply: N (normt); Mma;M (millimolar); pM (ntromolar); m (nmoles); mioli (miilmoles) pmol (msicromoles); nmol (nanomoles); pmnol (picomoles); g (grams) mg (milligrams; g (micrograms); n (nanograms); or L (liters); ml (milliliters); pl (microliters) cm (centimeters); mm (millimeters); pm (micrometers); nm (nanometers); and oC (degrees 15 CeOntigrad) Example I Materials and Methods This Example describes expenmental methods utilized in the below examples 20 A. Cell Lines Cell lines used in experiments of the present invention are described below, MA-mB-2T31 25 Timme dncrioa mmaygad rat pleurial effusion Pumorigenic: frsdeorcnnagade INl Receptors expressed: Ep'iderma Growth 'Factor (.EcGr, and rnthmn got facr T-alpha4 30 Oegn;wOt * and wem + 72 Sieibano ME, Barker PE, Cadlea Rt Mutualty exclsve gentic sig awtures of humain breast tumor ced! Wies wiM a common chromosomal marker Cancer Re&. 1979 MArJ(39019-22. C Kl~ ROlve 54, Cruciger Q4. Long-rer hun breast c.-arioma cel ie of in virc 197S? Nov,;14(l I 91 1- 5 Onsciger QV, Pathak S, Callean It. 1uanbratccio s:mkechmsms 0t invo.lvinlg 1q invencss CytoenetCell Ctiet 1976;j 1(4),,23 I -5. Satya-PrakashKL, P-athak S, 1%C T ve MA Caiteau PIt Cytogeneticaalysis on eight humnan breast tumior ell lins: high reuenie of lq, I qp and 1-ltalke are chromo1sole-s. C-'ancer, Ge-net Cytogenet !981 .n;3 I)6-73 15 MCF7 Tissue: adenocarmeincoa, amr gland, bhrea-st '-4' 1 20 Receenes2 25R ie Ce humn tumor Wes containing a stabilized wvild-type p53 protein, Cancer Rca., 57: -V. n 0 9 V36624568, 1997 Baccus SS et at Dierennaon of cultured hum cancr cells (AUnn6g and M42F71) as wcetd wAi 105f cell surface HER-Zfnou oligoimcle otide. MioP acii 35 -35 -362, 1990 MCEIOCAI MIH 0 cells -are derved fr benign breast tisue from a woan W fbrocyste disease 10 1CE1 0 lies consis of several lines, one in MC I OA, an immortalized normal human breastecllline. MC OilG was trans fod with TP24 H-a-ra to mak MVCPIO neoT] cells. MCFIOAT wi neopiastic porsinpotetial wsderivedi from xen~ograft asae MOP] ~ MC F!n& N4P1AT generates carcinoma in abot 25% of xenogmras- Fuly maligNanVI OC.AIlne were derived ftrom severl xeuogaft pa ssa ges of MCOAT '305 atr. IV injectin of the celos References: 20 sarntner Si ci t M ig aMCII O.l cell lnsdivdfro-m prmlgathumlan breast A MF OAT cl Bes e Resarch and treatment 63: 101-110, 23S A f e & C as mouse a mammary tor. The tumor was isolated an~d a small fres tissue is put into cume with a medium conditond by 1k. hshAm TJan at Karmanmos ace tiue This tumor cell Ine was established after 10 passa ge. 30 tissuem, M Iouse normal m nt-!aray ~ad p la Strain NAMRU, female Tunrrigenic: reduce bnign umor in rmce Cl'. 'ens RB-. Glnua epthlalcll ro ic:a ehod 6for seeJveciato. Nal I Cancr Inst. 52: 1375-1378, 1974 Owens RB et at Epiohelial cell cutures from nornal glandular tissue of mice. S. NatL. Cancer Int, 53: 261-269, 1974 Yin g JIM et al. Manmalian dwartiins are phosphorylated mn response to ransforming growth factor beta and are implicated in control f Wcel growth, Proc, Nat. Acad. Sci. USA 93: 89404944, 1996 1 0 is PC-3 Tissue: adenarcinomna, pancreas Cellular product: mucin, pancreatic cancer speci Mc antigen: CEA, carcinoma embryonic antigen, 15 Source: 61 year old female Tomorigenic: yes Oacogencs: c-Ki-ras References: 20 Tan MH eral Characterization of a new primary human pancreatic tumor line, Cancer invest 4: 15-23. 1986 Loor R ed at Use of pancreas-specific antigen in immunodiagnosis of pancreatic cancer, Clin. Lab Med 2: 567-578, 1982 25 Lan MS etal Polypeptide core of a human pancreatic tumornlucin antigen, Cancer Rs 50: 2997~001, 1990 Chambers A and Harris A. Expression of the cystic fibrosis gene and the major pancreatic mucin gne, MUC. in human ductal epithelal cells, j, Celi ScA, 105: 417-422, 1993 30 T-47D Tissuct ductal carcinoma manmary gland, breast, duct Metastade site: pleural effusion 7$ Sourtee plural eFlsion of a 54 years old female with infiltrating ducial carcinoma of te breast Receptor expression: estrogen, androgen calcitomn, progesterone, glucocorticoid and prolaclin positive, 5 Oncogees: wn3+ and wntlh+ This cell line is also know to ovrexpress e-erbW-2 References: Keydar I et at, Fstablishment and characterization of a ccli line of human breast carcinoma origin. Eur, 3 Cancer 15: 659-670, 1979 10 Judge SM and Chatterton RT Jr Progesterone-specic stimulation of triglycerde biosynthesis in a breast cancer cell line (T-47D). Cancer Res. 43: 4407-4412. 1983 Lamp SJ et a Calcitonin induction of a persistent activated stae of adenylate cyclase in human breast cancer cells (T-47D), J. BioL Chien 256: 12269-12274, 1981 15 Sher E e at Whole-cell uptake and nuclear localization of 25.dihydmxycholecalci ferol by breast cancer cells (T-47D) in culture, Biochent I 200: 315-320, 1981 Freak HC ex at 1,25-Dihydroxyvitamin D3 specifically binds to a human btast cancer cel, 20 line (T-47D) and stimuAles growth Biochem. Biophys Rcs Commun. 101: 1131-1138, 1981 Faust MB and Mocker IC Ap cation n expression of the 41 gene in human oid tumor CI lines. Cancer Rea 52: 2460-2463. 1992 RF335 1 Hugut Fe? a[ DfeeNi~al expr-ssion of human Wnit gnes 2, 3, 4, and 7Blnumi breast cell tins and normal and diseae stats ofrr.- humanbrast tiss.ue. Cancer Res. 54: 2615 2621, 1994 0 BT474 Tissue eta acnm atnr altt bras Source:, 60 year old female- Oncugene: caerbI-t Referencms Lasfargu EY Ft at isolation of two hmantoepihea clne fromsoi breast carnomas. J . an ifcr 11. 1YQ , Is 9 LasfarcesY ci at, A human breast tumor cell line (ST-474) thtsupports, mnouse mammary umr virs rep in I : 732901979 10 tewoodeEvas Al e at he osteociast-assoeiaied protease cathepniri K is express human breast carcinom ancr P es 6 30 9 WSE-FSCCL Human B c ne estab ihed 1993 1$ Saum: from peipheral blood of a malepatient ith l grad ar small cleaved cel lymphoma In Oncogenes: ,xhibmng chmosonal translocai for both cnye and bl2 Reference" 29 Mohammad RM, Mohamed AN, Smith MR, Jawadi NS, A L-Khatib A A unique EBV Negative Low Grade Lymphoma Line (WSU-FSCCL) Exhibing both t(14:8) and t(8:11), Cancer Genet Cyrogenet 70:62-67, 1993 BW Cell Culture 2$ Human breast cancer eL MCF7, MCF OCAa. MDA-IB 231, MDA-IB 435,eB. and human normal breast cells, M 10A were all obtained from Karmanos Cancer Institute. Afl cells were cultured in DMEM/F12 media (Gibco, MD) supplemented with 10 mM HEPES, 29 mM sodium bicarbonate, penicillin (100 units/mI) and streptomycin (100 gm) In addition, 10% cal fsrum 10 pg/ml insulin (Sigma Chemical, St Louis, MO), 30 and (15 nM estradiol was used in MCF7 media, 5% horse scrum and insulin (10 pg/mI) was used for MCF i OCala. and 10% fetal calf scrum was used for MDA-MB 231 and 435 tines, MACT IOA culture w as supplemented with 5% horse seum, insulin. (10 pg/mi}00 ng/ml 77 cholera enterotoxin (Calbiochem, CA), 0.5 pgim hydrocortisone (Sigma CmlaicaP) and 20 ng/ml epidermal growth factor (Sigma Chemical). All flasks and plates were incubated in a hunidified atmosphere of 95% ai and 5% CO2 at 374C. MYC-MT- cells were also cultured in DMEM/ 12 media containing 10 ng/ad EG (epithelial growth factor), I nM estradiol, 10 pg/ml insulin and 10% FfS (fetal bovine serum). BxPC-3 pancreatic carenoma cell line and BT-474, breast tumor cell line were culured in RPM! 1640 with 10% FBS, Breast tumor cell line, -4D was cultured in the same media as BT474 with the addition of 2,5 pg/mi insulin, NMuMG (nonnal nouse mammary gland cells) cell line was grown in DMEM media with 4.5 g/l glucose, 10 p.g/ml 10 insulin and 10% FBS, All the above cells were seeded at 2500 to 5,000 cells/well in 96 well plates, The cells were treated with oligonucleotide compounds in fresh media (100 pl total volume) 24 hours afler seeding. The media was replaced with fresh media without oligonucleotides 24 hours after treatment and every 48 hours for 6 to 7 days or until the control cells were 80 to 15 100% confluent, The inhibitory effect of oligomucleotide was evaluated using an MTT staining technique. Human follicular lymphoma celi line, WSU-FSCCL was used to evaluate the effect of antic-myc oligonucleotides as well as anti-Bcb2 oligonucleotides. PSCCL cells grow as a single cell suspension in tissue culture. The culture was maintained in RPMI 1640 20 supplemented with 10% fetal bovine senum, 1% L-glutamine, 100 units/mi penicillin and 1.00 pg/ni streptomycin, FSCCL cells were treated in 24 well plates (2x 10* cells/well/mI) with oligonucleotide compounds and incubated in a humidified atmosphere of 95% air and 5% C 0 2 at 37 0 C, The cells were counted every 24 hours using a hemocytometer, 25 C. Oigonucleotide Preparation All oligonucleotides were synthesized, gel purified anal lyophilized by BIOSYNTHESIS (Lcwisville, Texas) or Qiagen (Valencia, CA). Methylated oligonuclemtides were methylated at all CpG sites, Methylated Oigonucleotides were dissolved in pure sterile water (Gibco, Imvitrogen Corporation) and used to treat cells in 30 culture D, ipofectin Encapsulation 20 pg lpofectin (1nvitrogen) and 16 gg oligonucleotides were each incubated with 200 l OptMEM (invitrogen) media in separate sterile tubes at room temperature for 45 78 nn. They were then combined and incubated for an additional 15 min, 1 6 ml Opti-MEM media was then added to a final volume of 2 nl and a fmal concentration I aM Oligonucleotide. The concentration of lipofiCtin and oligonucleotides can be adjusted based on their molecular weight and desired concentration of compounds. There was no cvtoioic. 5 effect at this levels E. Cell Growth Inhibition Assay Cell growth inhibition was assessed using 3[4,-D imethyk-thiazol-2-ylI-2,diphenyltetrazolium bromide (MTT) purchased from 10 Sigma Chemical (St, Louis, MO), Cells were suspended n culure media at 5O,000) cells /in and 100 pl was distributed into each well of a 96well, flat bottomed plate (Costar Corning, NY, USA) and incubated for 24 hours, Media was changed to 100 pd fresh media containing the desired concentration of oligonucleotides and incubated for 24 hours, Controls had media with pure sterile water equal to the volume of oligonucleotide solution. 15 The media was changed withom further addition of oiigonucleotides every 24 hours until the control cultures were confluent (6 to 7 days). Thereafter the media was removed and plates were washed two times with phosphate-buffered saline (PBS) and 100 jtl of serum free media containing 0.5 mg/ml MTT dye was added into each well and incubated for i hour at 37"C. The media with dye was removed, washed with PBS and 100 pl of dimethyt 20 sulfoxide (DMSO) was added to solubilize the reactive dye, The absorbance values were read using an automatic multiwel I spectrophotometer (Bio-Tek Microplate Autoreader, Winooski, VT, USA), Each treatment was repeated at least 3 times with S independent wells each time. 25 F Protein Extraction and Western Bot Analysis The cells were seeded and cultured in T25 tissue cuhure flasks (Costar, Corning, NY, USA) at 200,000 cells/flask, The cells were allowed to attach for 24 hours. The media was replaced with fresh media containing 10 to 20 pM oligonucleotides and incubated for 24 hours. The media was changed every 48 hours without further addition of inhibitors and 30 cell cultures were continued until the control flasks were confluent (6-7 days). Cells were harvested using Ix trypsin:EDTA (TAvitrogen, Gibco, MD) and collected by centrifugation at 2000 rpm for 5 nin. Cells were resuspended in 125 mM Tris-HCL buffer (pH 6,8) soncated with 10-20% outut and lysed in an equal volume of 8% SDS for a final concentration of 4% SDS. Cells extracts were boiled for 10 rmi, chilled on ice and 79 centrifuged at 2.000 rpm for 5 min before collecting the supernatant. The protein was quantitated using BCA protein assay kit (Pierce, Roctford, IL). 50 to 100 pg of proteins were subjected to 10 to 1 5% ge (depending on molecular weight of each protein) electrophoresis and transferred to nitroceulose membrane (Schleicher & Schuell, Kence, 5 N) Each membrane was blocked with 10% dy mikW m TBSTc (Tris buffered saline. Tween 20) fo 2 hr, prior to incubation with primary antibodies in TBST ovenight. Antibodies to human c-my, c-ha-ras and erbB-2 were mice IgG (Pharmingen San Diego, CA, Membranes were washed 3 times, 15 rin each in TBST, then incubated with seondary antibodies conjugated with peroxidase for 1 hr, The membranes were washed 5 it) times. 10 mn each in TBST and incubated with 2 mi each of Lumino/Enhancer and Stable peroxide solution (P1ERCE) for 1 mi, The membranes were exposed to X-ray hilm for 2 nin (exposure time is adjusted from 10 seconds up to 24 hr if necessary), Exa mple 2 15 c-kiRAS This example describes the ability of oligonncleotde compounds targeted against the promoter of the c-ki-Ras gene to inhibit the growth of cancer cell lines, Experiments were performed as described in Example 1. Tc results are shown m Figures 13 and 19. The sequences of the oigonucleotides targeted against -ki-Ras as as the sequence of 20 c-ki-Ras gene are shown in Figures 5 and 6, Example 3 This example describes the ability ofoligontcleotide compounds targeted against 25 the promoter of the beb2 gene to inhibit the growth of cancer cell lines, Experiments were performed as described in Example 1. The results are shown in Figures 14 and 20. The sequences of the ohgonucieotides targeted against bct2 as well as the sequence of bc4 gene are shown in Figures 1 and 2. 30 Example 4 e-ha-&AS This example describes the ability of oligonucleotide compounds targeted against the promoter of the c-ha-Ras gene to inhibit the growth of cancer cell lines. Experiments were performed as described in Example L. The results are shown in Figures 16 and 22, 80 The sequences of the oligonucleotides targeted against c-ha-Ras as well as the sequence of c-ha-Ras gene are shown in Figures 7 and 8. Example 5 c -e rbl-2 This example describes the ability of oligonucleotide compounds targeted against the promoter of the c-erbB-2 gene to inhibit the growth of cancer cell hnes, Experiments were per formed as described in Example 1, The results are shown in Figures 15 and 21. The sequences of the oiigonucieotides targeted against c-erbB-2 as well as the sequence of 10 e-crbfl-2 gene are shown in Figures 3 and 4, Example 6 c-mye This example describes the ability of oiigonucleotide compounds targeted against 15 the promoter of the e-myc gene to inhibit the growth of cancer cell lines, Experiments were performed as described in Example 1. The results are shown in Figures 17 ant 23. The sequences of the oligonucleotides targeted against cnyc as well as the sequence of c-myc gene are shown in Figures 9 and 10, 20 Exanple 7 TG-a This example describes the ability of oligonucleotide compounds targeted against the promoter of the TGF-a gene to inhibit the growth of cancer cell lines, Experiments were performed as described in Example I The results are shown in Figures 18 and 24. 25 The sequences of the oligonucleotides targeted against TOF-a as wel as the sequence of TGF-a gene are shown in Figures I I and 12 Example $ Inhibition of eeH growth by Non-methytated oligonucleotides 30 This example describes the inhibiion of growth of lymphoma cell lines by non methylated oligonucleotides targeted towards Bel-2. VSU-FSCCL cells were plated in 24 well plates at 2x 10 cells /weli at t-24b. For each time point to he harvested, triplicate wels were treated at t=0 with the oligos at the concentrations indicated. Controls were plated in trplicate. Plates were incubated at 37C. All cultures were monitored through out 81 82 the experiment by cell count and viability every 24 hr for 4 days using trypan blue stain and hemacytometer. The MABL2 oligonucleotide is targeted to the promoter region of Bcl-2 [5' CAX GCA XGX GCA TCC CXG CCX GTG -3' (SEQ ID NO: 3)). Pho-Mabl-2 is an .5 unmethylated version of MABL-2 [5'- CAC GCA CGC GCA TCC CCG CCC GTG -3' (SEQ ID NO: 1438)]. WSU-FSCCL - derived from human B cell lymphoma (low-grade follicular small-cleaved cell lymphoma). The experimental protocol is shown in Table 2. Table 2 Target CFormulation Viability Harvest Group Gene Compound Cells Conc. Assay for Methyl I Bel-2 MABL2 FSCCL IOuM none n=3 @ 2 4 , n=3 @ 7 2 48 & 72 hr hr n=3@ 2 4 , n=3@ 7 2 2 Bcl-2 MABL2 FSCCL 3uM none 48 & 72 hr hr n=3 @ 24, n3 @72 3 Bcl-2 PhoMABL2 FSCCL IOuM none 48 & 72 hr hr n n=3 @ 24, n=3 @72 4 none none FSCCL n/a none 48 & 72 hr hr The results are shown in Figure 31. The results demonstrate that the unmethylated oligonucleotide directed against Bel-2 is as effective as the methylated 10 oligonucleotide in inhibiting cell growth. Example 9 In Vivo inhibition of Tumor Growth This example describes the inhibition of tumor growth by oligonucleotides of the present invention in a human prostate carcinoma model. is Animals: The human PC-3 GFP prostate carcinoma subcutaneous model was utilized (See e.g., Yang et al., Cancer Research 59, 781-786, [19991; Glinskii et al., Cancer Research 63, 4239-4243, [2003]; and Kalikin et al., Cancer Biology and Therapy 2:6, 17-21 [2003]). Male athymic NCr nude mice between 5 and 6 weeks of age were used. The animals were bred and maintained in a HEPA filtered environment with cages, 20 food and bedding sterilized by autoclaving. The breeding pairs were obtained from Taconic Quality Laboratory Animals and Services for Research (Germantown, NY). The animal diets (5010 1017n'; I I Nil autoclavabie rodent diet) were obtained front PMJ nutrition Latemational Inc. (Brertwood, MO), A total of 60 male arumals were used for the study, Study drugs: Nucleic acid based oligo compound PNTO0 and scrambled oligo comN PNT-C with cationic liposomal delivery (LDV formulation, 5 GFP expression vector: pLEiN was purchased from Clontech (Palo Alto. CA). The vector expresses enhanced GFP and the neomycin resistance gene on the same bicistronic message that contains an internal ribosome entry site, Cel culture, vector production, transetion, and subeloning: PT67, an NI13T3 derived packaging cell line, expressing the 10 Al viral envelopes, was purchased from 10 Clontech. PT67 cells were e hured in DMEM supplemented with 10% fetal bovine serum, For vector production, packaging cells (PT67), at 70% confluence, were incubated with a precipitated mixture of N- [ 1- (2 ,3 - dioleoyloxyl ) propyl-N, N, trimethylamnmomunethyl sulfate reagent and saurating amounts of pLEIN plasnid for 18 h. Fresh medium was replenished at this time, The cells were examined by fluorescence 15 micruscopy 48 h posuransfection. For selection, the cells were cultured in the presence of 200- 1000 pg/mi G418 for 7 days, CF gene trausduction of PCGFP cells: For OFP gene fransduction, 20% confluent PC-3 cells (ATCC, CRL 1435) were incubated with a I:1 precipitated mixture of 20 retroviral supematants of PT67 cells and Ham's F-12 K containing 7% fetal bovine serum for 72 h. Fresh medium was replenished at this time, P3 cells were harvested by trvpsin EDTA 72 h posttransduction and subeultured at a ratio of I 15 into selectve medium that contained 200 sgni 641& The level of 6418 was increased to 1000 Ng/m sepwise, The brightest P0-3 cell clones expressing GFP were selected, combined, and then amplified and 25 transferred by conventional culture methods. Subcutaneous Tumor Crowtht Tumor stocks were made by subcutaneously injecting PC-3-GFP cells at the concentration of 5 x 10' cells /200 y 1 into the flank of nude mice. The strong GFP expression of tumors grown in the subcutis of ince was certified before harvest. The tumor tissues harvested from subcutaneous growth in nude twice were 30 inspected, and any grossly necrotic or suspected necrotic or non GFP tumor tissues were removed. Tumor tissues were subsequently cut into small fragments of approximately 2 Subcutaneous tissue fragment kplantation: TuImor stock of the prostate cancer PC-3 GFP was established by subcutaneously inWectng P0- GFP cells to the flank of nude 83 mice The tumor was maintained in nude mice subcutaneously as tumor stock prior to use, Before implantation, strong GFP expression of the PC-3 GFP tunmcr tissue was confirmed by the fuorescent light On the day of mplanhtation, the tumor was harvested from the subcutaneous site md placed in RPMI-I 640 medium. Necrotic tissues were removed ard S viable tissues were cut into 2 mm 3 pieces. The tissue fragments were then implanted subcutaneously to right flank of the nude nice Whole body optical imaging of green fluorescent protein expressing tumors and tmetastases: A Leica stereo fluorescence microscope model LZ12 equipped with a mercury lamp power supply was used. Selective excitation of GFP was produced through a D425/60 10 band pass filer and 470 DCXR dichroic mirror. Emitted fluorescence was collected through a long pass fiter 0G475 (Chroma Technology, Brattleboro, VT) on a STh 33 Micromax High Speed TEA/CCD1 317K 1 thermoelectrically cooled camera (Princeton instruments, Tremon, NJ), with a i317x1035 pixel chip. Experiments were controlled and images were processed for contrast and brightness and analyzed with the help of Image Pro 15 Plus 3,1 soilwre (Media Cyberetics, Silver Spring, Maryland). High resolution images were captured directly on the computer or contnuously through video output. Study animals A total of 60 mice used for the study were divided into 6 groups 12 dlays after surgery. Grups for each of the cohort conditions were randomly chosen. Treatment Initiation: When primary tumors reach between 50-100 mm 3 estimated 20 volume, The study design is shown i Table 4 Table 4 Grop Subgroup Description Dose (mg/kg) Schedule Route N A PBS Control 200 I 'qd X5 sc 10 B PNT-C (5" 5 d X 5 sx. to NNNNNNNN NNNNNNNN NNNNNNINN -3* SEQ U) NO:I1439)+ .84 S Y 002PNT 5 10 (PhoMabl2; SEQ [D NO: 1438) + LDV D1 PNTI00 [5 qdX$5 s~c, IO I ET AXOTER5 10andS 5 Day 2and$ S v, 10 T AXOTERE 10 and 5+5 Dy2ad v S,1 +PNT.- +qdXS l00/LDV Data collection: Tumor sizes: Each animal was checked once a week for tumo~r growth by caliper 5 measurements until the end of the study, Measurements over a 40 days period were taken to calculate tumor volume response aver time. An approximate tumor volume was calculated using the fbmiula 1/2 (a x b), where bi is the smaller of two perpendicular diameters. Approximate tumor volume was calculated by the formula (Wx t ,) x 1 /2, GFP~ imaging: 12 days after implantation, whole body optical imaging of 10 GFPexpressintg tumors was taken once a week. Body weights: Blody weights for all animals was weighed once per week for the duration of the study, An electronic balance was used to measure the body weight. Termination: The final tumor weights were~ acquired after animals were sacrificed at the fortysixthi day of the study. Each tumor was weighed using an electronic balance. I 5 Statistical methods Qsed in efficacy evaluation: Tumor volumes and final tumor weights of all 6 groups were analyzed using the Student's 0 test with an =0105 (twosided). Results: The results are shown in Figures 32-35. Figure 32 shows mean tumor volume of tumors in the PC-3 G3FP prostate carcinoma subcutaneous model following 20 treaument with PNT-100 and/or TAXOTERE. Figure 33 shows mean body weight of tumors in the PC-3 GFP prostate carcinoma subcutaneous model following treatment with PNTI 00 and/or TA XOTERE. Figure 34 shows mean tumor volume of tumors in the PC->3 85 86 GFP prostate carcinoma subcutaneous model following treatment with PNT400 and/or TAXOTERE. Figure 35 shows mean final tumor volume of tumors in the PC-3 QFP prostate carcinoma subcutaneous model following treatment with PNT-100 and/or TAXOTERE. The results indicate that PNT-100 decreased tumor size. The effect was s increased in the presence of TAXOTERE. Example 10 In Vivo Inhibition of Tumor Growth This example describes the inhibition of tumor growth by in vivo delivery of oligonucleotides of the present invention in a human prostate carcinoma model. Io 1) PC-3 prostate Xenograft tumor response to PNTl 00 administered i.v. with randomized xenografts at different vascularization states was investigated. This experiment was performed with the two step Neophectin formulation with five daily doses of 1 mg/kg PNT100. All mice survived the dosing regiment without noticeable toxicity responses. 15 H) WSU-DLCL2 Xenograft i.v. PNTOO study; A second study was performed to establish i.v. delivery and efficacy of PNTI00-Neophectin in a non Rodgkin's-lymphoma model (NHL). The study was designed to administer five daily doses of 5 mg/kg PNT 100, and in certain cohorts, combination therapy with vincristine. After one dose of PNT100, noticeable weight loss in the animals injected with PNTIOO 20 and PNT-C (Scrambled control) was observed. The data shows substantial effect of combination therapy with PNT100 and PNT-C. Results showing tumor burden 20 days post WSU-DLCL2 transplantation indicate that PNTIOO, alone and in combination with vincristine, shrinks tumors in mice. All publications and patents mentioned in the above specifloation are herein 23 incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, 30 various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims (20)

1. A composition comprising an oligonucleotide that hybridizes under physiological conditions to a promoter region of a bcl-2 gene, wherein said first oligonucleotide is SEQ ID NO: 1418 and complements thereof.

2. The composition of claim 1, further comprising an additional oligonucleotide.

3. The composition of claim 2, wherein said additional oligonucleotide is selected from the group consisting of SEQ ID NOs: 2, 3, 7, and complements thereof.

4. The composition of claim 2, wherein said additional oligonucleotide hybridizes to a promoter region of a gene is selected from the group consisting of c-ki-as, c-fIa-Ras, c-myc, Her-2, and TGF-a.

5. A method, comprising a) providing i) a composition of any one of claims 1-4; and ii) a ce-l capable of expressing a bl-2 gene, wherein said cell is capable of proliferation; and b) introducing said oligonucleotide to said cell, wherein said administering results in a reduction of proliferation of said cell.

6. The method of claim 5, wherein said cell is a cancer cell selected from the group consisting of pancreatic cancer, colon cancer, breast cancer, bladder cancer, lung cancer, leukemia, prostate cancer, lymphoma, ovarian cancer, and melanoma.

7. The method of claim 5, wherein said cell is in an animal.

8. The method of claim 7, wherein said animal is a human. 88

9. The method of claim 5, further comprising the step of introducing a test compound to said cell.

10. The method of claim 9, wherein said test compound is a known chemotherapy agent.

I 1, The method of claim 5, further comprising providing a drug delivery system for introducing said oligonucleotide to said cell.

12. The method of claim H1, wherein said drug delivery system comprises a liposome.

13. The composition of claim 1 further comprising a known chemotherapy agent,

14. The composition of claim 13, wherein said chemotherapy agent is docetaxel ((2R,3S)-N carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5, 20-epoxy-1, 2, 30 4, 7, 10, 13 hexahydroxytax- I -en-9-one 4-acetate 2-benzoat trihydrate).

15. The method of claim 12, wherein said liposome is a cardiolipin based cationic liposome.

16. The method of claim 12, wherein said liposome comprises N-[1-(2,3 Dioleoyloxy)propy l]-N,N,N-trimethylammonium methyl-sulfate (DOTAP).

17, The method of claim 10, wherein said known chemotherapeutic agent is docetaxel ((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5, 20epoxy-1, 2, 4, 7, 10, 13-hexahydroxytax-I 1-en-9-one 4-acetate 2-benzoate, trihydrate).

18. A pharmaceutical composition comprising an oligonucleotide having the nucleic acid sequence of SEQ ID NO: 1438 and a liposome.

19. The composition of claim 18, wherein said liposome is a cardiolipin based cationic liposome. 89

20. The composition of claim 18, wherein said liposome comprises N-[1-(2,315 Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP). Pronai Therapeutics, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON