Compositions and methods for combined therapy of disease

Abstract

A desired physiological state can be induced by altering the amount of gene products in target cells of a subject. The target cells are treated with at least one compound designed to reduce expression of at least one first gene by RNAi, and with at least one compound designed to increase expression from at least one second gene. The reduced expression of the first gene and the increased expression from the second gene in the target cells induces the desired physiological state in the subject. By altering target cell gene expression in this way, conditions such as angiogenesis or tumor growth and metastasis can be inhibited.

1 . A method of inducing a desired physiological state in a subject, comprising:
 (1) treating target cells of the subject with an effective amount of at least one RNAi compound to reduce expression of the at least one first gene by RNAi; and   (2) treating the target cells with an effective amount of at least one compound that increases the expression of at least one second gene,   wherein the reduced expression of the at least one first gene and the increased expression from the at least one second gene in the target cells induces the desired physiological state in the subject.   
     
     
         2 . The method of  claim 1 , wherein the desired physiological state is inhibition of angiogenesis in the subject. 
     
     
         3 . The method of  claim 1 , wherein the at least one first gene is a pro-angiogenic gene. 
     
     
         4 . The method of  claim 1 , wherein the at least one second gene is an anti-angiogenic gene. 
     
     
         5 . The method of  claim 3 , wherein the pro-angiogenic gene is selected from the group consisting of VEGF, Flt-1, Flk-1/KDR, HIF-1 alpha, ICAM-1, Ang1, Ang2 and Tie-2 genes. 
     
     
         6 . The method of  claim 1  wherein the at least one first gene is VEGF and HIF-1 alpha; VEGF and Ang2; VEGF and ICAM-1; or VEGF, HIF-1 alpha and Ang2. 
     
     
         7 . The method of  claim 4 , wherein the anti-angiogenic gene is selected from the group consisting of Ang2, angiostatin, antiThrombin-3, ATF, calreticulin, endostatin, IP-10, K1-5, kringle-5 domain, maspin, Mig/CXCL9, PEDF, PEX, CXCL4, PRP, TIMP-1, TIMP-2, TIMP-3, TIMP-4, troponin 1-2, T2-TrpRs, TSP-1 and tumstatin genes. 
     
     
         8 . The method of  claim 1 , wherein the at least one first gene is VEGF, and the at least one second gene is PEDF, endostatin or angiostatin. 
     
     
         9 . The method of  claim 1 , wherein the at least one first gene is HIF-1 alpha, and the at least one second gene is PEDF, endostatin or angiostatin. 
     
     
         10 . The method of  claim 1 , wherein the target cells are retinal pigment epithelial cells. 
     
     
         11 . The method of  claim 1 , wherein the desired physiological effect is induction of apoptosis in the target cells. 
     
     
         12 . The method of  claim 1 , wherein the at least one first gene is an anti-apoptotic gene. 
     
     
         13 . The method of  claim 1 , wherein the at least one second gene is a pro-apoptotic gene. 
     
     
         14 . The method of  claim 12 , wherein the anti-apoptotic gene is selected from the group consisting of Bcl-2 and Bcl-x L  genes. 
     
     
         15 . The method of  claim 13 , wherein the pro-apoptotic gene is selected from the group consisting of Bad, Bak, Bax, BclX S , Bik and Casp-3 genes. 
     
     
         16 . The method of  claim 1 , wherein the target cells are cancer cells. 
     
     
         17 . The method of  claim 1 , wherein the at least one RNAi compound comprises an isolated siRNA. 
     
     
         18 . The method of  claim 1 , wherein the at least one RNAi compound comprises an expression vector encoding at least one siRNA. 
     
     
         19 . The method of  claim 1 , wherein the at least one compound of step (2) comprises an isolated protein produced from the at least one second gene. 
     
     
         20 . The method of  claim 1 , wherein the at least one compound of step (2) comprises an expression vector encoding the at least one second gene. 
     
     
         21 . The method of  claim 1 , wherein the at least one RNAi compound and the at least one compound of step (2) together comprise a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to the at least one first gene; and   (2) nucleic acid sequences for expressing the at least one second gene.   
     
     
         22 . A method of inhibiting angiogenesis in a subject, comprising:
 (1) treating target cells of the subject with an effective amount of at least one RNAi compound to reduce expression of at least one pro-angiogenic gene by inducing RNAi of the pro-angiogenic gene; and   (2) treating the target cells with an effective amount of at least one anti-angiogenic compound to increase the level of anti-angiogenic factor from at least one anti-angiogenic gene,   wherein the reduced expression of the at least one pro-angiogenic gene and the increased level of anti-angiogenic factor from the at least one anti-angiogenic gene in the target cells inhibits angiogenesis in the subject.   
     
     
         23 . The method of  claim 22 , wherein the pro-angiogenic gene is selected from the group consisting of VEGF, Flt-1, Flk-1/KDR, HT-1 alpha, ICAM-1, Ang1, Ang2 and Tie-2 genes. 
     
     
         24 . The method of  claim 22 , wherein the at least one pro-angiogenic gene is VEGF and HIF-1 alpha; VEGF and Ang2; VEGF and ICAM-1; or VEGF, HIF-1 alpha and Ang2. 
     
     
         25 . The method of  claim 22 , wherein the anti-angiogenic gene is selected from the group consisting of Ang2, angiostatin, antiThrombin-3, ATF, calreticulin, endostatin, IP-10, K1-5, kringle-5 domain, maspin, Mig/CXCL9, PEDF, PEX, CXCL4, PRP, TIMP-1, TIMP-2, TIMP-3, TIMP-4, troponin 1-2, T2-TrpRs, TSP-1 and tumstatin genes. 
     
     
         26 . The method of  claim 22 , wherein the at least one pro-angiogenic gene is VEGF, and the at least one anti-angiogenic gene is PEDF, endostatin or angiostatin. 
     
     
         27 . The method of  claim 22 , wherein the at least one pro-angiogenic gene is HIF-1 alpha, and the at least one anti-angiogenic gene is PEDF, endostatin or angiostatin. 
     
     
         28 . The method of  claim 22 , wherein the target cells are retinal pigment epithelial cells. 
     
     
         29 . The method of  claim 22 , wherein the angiogenesis is pathogenic. 
     
     
         30 . The method of  claim 22 , wherein the angiogenesis is non-pathogenic. 
     
     
         31 . The method of  claim 22 , wherein the at least one RNAi compound comprises at least one isolated siRNA. 
     
     
         32 . The method of  claim 22 , wherein the at least one RNAi compound comprises an expression vector encoding at least one siRNA. 
     
     
         33 . The method of  claim 22 , wherein the at least one anti-angiogenic compound comprises an anti-angiogenic factor produced from the at least one anti-angiogenic gene. 
     
     
         34 . The method of  claim 22 , wherein the at least one anti-angiogenic compound comprises an expression vector encoding the at least one anti-angiogenic gene. 
     
     
         35 . The method of  claim 22 , wherein the at least one RNAi compound and the at least one anti-angiogenic compound together comprise a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to the at least one pro-angiogenic gene; and   (2) nucleic acid sequences for expressing the at least one anti-angiogenic gene.   
     
     
         36 . A method of treating an angiogenic disease in a subject, comprising:
 (1) treating target cells of the subject with an effective amount of at least one RNAi compound to reduce expression of at least one pro-angiogenic gene by RNAi; and   (2) treating the target cells with an effective amount of at least one anti-angiogenic compound to increase the level of anti-angiogenic factor from at least one anti-angiogenic gene,   wherein the reduced expression of the at least one pro-angiogenic gene and the increased level of anti-angiogenic factor from the at least one anti-angiogenic gene in the target cells inhibits angiogenesis in the subject.   
     
     
         37 . The method of  claim 36 , wherein the at least one RNAi compound comprises at least one isolated siRNA. 
     
     
         38 . The method of  claim 36 , wherein the at least one RNAi compound and the at least one anti-angiogenic compound together comprise a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to the at least one pro-angiogenic gene; and   (2) nucleic acid sequences for expressing the at least one anti-angiogenic gene.   
     
     
         39 . The method of  claim 36 , wherein the angiogenic disease is selected from the group consisting of cancer, AMD, diabetic retinopathy, psoriasis, rheumatoid arthritis and inflammatory diseases. 
     
     
         40 . A method of inducing apoptosis in target cells of a subject, comprising:
 (1) treating the target cells with an effective amount of at least one RNAi compound to reduce expression of at least one anti-apoptotic gene by RNAi; and   (2) treating the target cells with an effective amount of at least one pro-apoptotic compound to increase the level of pro-apoptotic factor from at least one pro-apoptotic gene,   wherein the reduced expression of the at least one anti-apoptotic gene and the increased level of pro-apoptotic factor from the at least one pro-apoptotic gene in the target cells induces apoptosis in target cells of the subject.   
     
     
         41 . The method of  claim 40 , wherein the anti-apoptotic gene is selected from the group consisting of Bcl-2 and Bcl-x L  genes. 
     
     
         42 . The method of  claim 40 , wherein the pro-apoptotic gene is selected from the group consisting of Bad, Bak, Bax, BclX S , Bik and Casp-3 genes. 
     
     
         43 . The method of  claim 40 , wherein the target cells are cancer cells. 
     
     
         44 . The method of  claim 40 , wherein the at least one RNAi compound comprises at least one isolated siRNA. 
     
     
         45 . The method of  claim 40 , wherein the at least one RNAi compound comprises an expression vector encoding at least one siRNA. 
     
     
         46 . The method of  claim 40 , wherein the at least one pro-apoptotic compound comprises an isolated pro-apoptotic factor produced from the at least one pro-apoptotic gene. 
     
     
         47 . The method of  claim 40 , wherein the at least one pro-apoptotic compound comprises an expression vector encoding the at least one pro-apoptotic gene. 
     
     
         48 . The method of  claim 40 , wherein the at least one RNAi compound and the at least one pro-apoptotic compound together comprise a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to the at least one anti-apoptotic gene; and   (2) nucleic acid sequences for expressing the at least one pro-apoptotic gene.   
     
     
         49 . A method of treating cancer in a subject, comprising:
 (1) treating cancer cells of the subject with an effective amount of at least one RNAi compound to reduce expression of at least one anti-apoptotic gene by RNAi; and   (2) treating the cancer cells with an effective amount of at least one pro-apoptotic compound to increase the level of pro-apoptotic factor from at least one pro-apoptotic gene,   wherein the reduced expression of the at least one anti-apoptotic gene and the increased level of pro-apoptotic factor from the at least one pro-apoptotic gene in the cancer cells inhibits tumor growth the subject.   
     
     
         50 . The method of  claim 49 , wherein the cancer to be treated is selected from the group consisting of breast cancer, lung cancer, head and neck cancer, brain cancer, abdominal cancer, colon cancer, colorectal cancer, esophagus cancer, gastrointestinal cancer, glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer, lymphomas and blood cancer. 
     
     
         51 . The method of  claim 49 , wherein the anti-apoptotic gene is selected from the group consisting of Bcl-2 and Bcl-x L  genes. 
     
     
         52 . The method of  claim 49 , wherein the pro-apoptotic gene is selected from the group consisting of Bad, Bak, Bax, BclX S , Bik and Casp-3 genes. 
     
     
         53 . The method of  claim 49 , wherein the at least one RNAi compound comprises at least one isolated siRNA. 
     
     
         54 . The method of  claim 49 , wherein the at least one RNAi compound comprises an expression vector encoding at least one siRNA. 
     
     
         55 . The method of  claim 49 , wherein the at least one RNAi compound and the at least one pro-apoptotic compound together comprise a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to the at least one anti-apoptotic gene; and   (2) nucleic acid sequences for expressing the at least one pro-apoptotic gene.   
     
     
         56 . A pharmaceutical composition comprising a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to at least one first gene; and   (2) nucleic acid sequences for expressing at least one second gene.   
     
     
         57 . A pharmaceutical composition comprising a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to at least one pro-angiogenic gene; and   (2) nucleic acid sequences for expressing at least one anti-angiogenic gene.   
     
     
         58 . A pharmaceutical composition comprising a single expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to at least one anti-apoptotic gene; and   (2) nucleic acid sequences for expressing at least one pro-apoptotic gene.   
     
     
         59 . An expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to at least one pro-angiogenic gene; and   (2) nucleic acid sequences for expressing at least one anti-angiogenic gene.   
     
     
         60 . The expression vector of  claim 59 , wherein the at least one pro-angiogenic gene is VEGF or HIF-1 alpha. 
     
     
         61 . The expression vector of  claim 59 , wherein the at least one anti-angiogenic gene is PEDF or angiostatin. 
     
     
         62 . An expression vector comprising:
 (1) nucleic acid sequences for expressing an siRNA targeted to at least one anti-apoptotic gene; and   (2) nucleic acid sequences for expressing at least one pro-apoptotic gene.