Induction of apoptic or cytotoxic gene expression by adenoviral mediated gene codelivery

Abstract

The present invention generally relates to viral vectors and their use as expression vectors for transforming human cells, both in vitro and in vivo. More particularly, the present invention relates to adenoviral vectors containing propapoptotic genes and their use in cancer therapy.

1 - 38 . (canceled)  
     
     
         39 . A method for treating a subject with cancer comprising the steps of: 
 (i) providing an adenoviral expression construct comprising a first nucleic acid encoding a proapoptotic member of the Bcl-2 gene family and a first promoter functional in eukaryotic cells wherein said nucleic acid is under transcriptional control of said first promoter; and    (ii) contacting said expression construct with cancer cells of said subject in a manner that allows the uptake of said expression construct by said cells,    wherein expression of said proapoptotic gene results in the treatment of said cancer.    
     
     
         40 . The method of  claim 39 , further comprising contacting said cancer cell with a further cancer therapeutic agent.  
     
     
         41 . The method of  claim 40 , wherein said cancer therapeutic agent is selected from the group consisting of tumor irradiation, chemotherapeutic agent, a second nucleic acid encoding a cancer therapeutic gene.  
     
     
         42 . The method of  claim 41 , wherein said chemotherapeutic agent is a DNA damaging agent selected from the group consisting of verapamil, podophyllotoxin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate.  
     
     
         43 . The method of  claim 41 , wherein said radiation is selected from the group consisting of X-ray radiation, UV-radiation, γ-radiation, or microwave radiation.  
     
     
         44 . The method of  claim 40 , wherein said cancer therapeutic agent comprises a second nucleic acid.  
     
     
         45 . The method of  claim 44 , wherein said second nucleic acid is a cDNA or genomic DNA.  
     
     
         46 . The method of  claim 44 , wherein said second nucleic acid is a second gene operatively linked to a promoter in said first expression construct.  
     
     
         47 . The method of  claim 44 , wherein said second nucleic acid is a second gene operatively linked to a promoter in a second expression construct.  
     
     
         48 . The method of  claim 47 , wherein said second expression construct is selected from the group consisting of an adenovirus, an adeno-associated virus, a vaccinia virus and a herpes virus.  
     
     
         49 . The method of  claim 39 , wherein said contacting is effected by regional delivery of the expression construct.  
     
     
         50 . The method of  claim 39 , wherein said contacting is effected by local delivery of the expression construct.  
     
     
         51 . The method of  claim 39 , wherein said contacting is effected by direct injection of a tumor with said expression construct.  
     
     
         52 . The method of  claim 39 , wherein said contacting comprise delivering said expression construct endoscopically, intratracheally, intralesionally, percutaneously, intravenously, subcutaneously or intratumorally.  
     
     
         53 . The method of  claim 39 , further comprising the step, prior to said contacting, of tumor resection.  
     
     
         54 . The method of  claim 39 , wherein said cancer is selected from the group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial, prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood.  
     
     
         55 . A method of inhibiting the growth of a cell comprising the steps of: 
 (i) providing an adenoviral expression construct comprising a first nucleic acid encoding a proapoptotic member of the Bcl-2 gene family and promoter functional in eukaryotic cells wherein said nucleic acid is under transcriptional control of said first promoter; and    (ii) contacting said expression construct with said cell in an amount effective to inhibit the growth of said cell;    wherein expression of said proapoptotic gene by said cell results in slower growth of said cell relative to the growth of said cell in the absence of said proapoptotic gene.    
     
     
         56 . The method of  claim 55 , wherein said cell is a cancer cell.  
     
     
         57 . The method of  claim 56 , wherein said inhibition of growth comprises killing of said cancer cell.  
     
     
         58 . The method of  claim 56 , wherein said cancer cell is selected from the group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial, prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node, small intestine, blood cells, colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood.  
     
     
         59 . The method of  claim 56 , wherein said cell is located within a mammal.  
     
     
         60 . The method of  claim 59 , wherein said inhibition of growth comprises an inhibition of metastatic growth of said cancer cell.  
     
     
         61 . A method of inducing apoptosis in a cell comprising the steps of: 
 (i) providing an adenoviral expression construct comprising a first nucleic acid encoding a proapoptotic member of the Bcl-2 gene family and promoter functional in eukaryotic cells wherein said nucleic acid is under transcriptional control of said first promoter; and    (ii) contacting said expression construct with said cell in an amount effective to kill said cell;    wherein expression of said proapoptotic gene by said cell results in an increase in the rate of death of said cell relative to the growth of said cell in the absence of said proapoptotic gene.    
     
     
         62 - 67 . (canceled)  
     
     
         68 . A method for expressing a polypeptide in a target cell comprising introducing into said target cell: 
 (a) a first vector comprising a coding region for said polypeptide under the control of a first promoter inducible by an inducer polypeptide not expressed in said target cell; and    (b) a second vector comprising a coding region for said inducer polypeptide under the control of a second promoter active in said target cell.    
     
     
         69 . The method of  claim 68 , wherein said first and said second vectors are viral vectors.  
     
     
         70 . The method of  claim 68 , wherein said first and said second vectors are non-viral vectors.  
     
     
         71 . The method of  claim 68 , wherein said first vector is a viral vector and said second vector is a non-viral vector, or said first vector is a non-viral vector and said second vector is a viral vector.  
     
     
         72 . The method of  claim 68 , wherein said second promoter is a constitutive promoter, an inducible promoter or a tissue specific promoter.  
     
     
         73 . The method of  claim 69 , wherein said viral vectors are the same or different and selected from the group consisting of an adenoviral vector, a herpesviral vector, a retroviral vector, an adeno-associated viral vector, a vaccinia viral vector or a polyoma viral vector.  
     
     
         74 . The method of  claim 68 , wherein said first vector and said second vector are introduced into said target cell at a ratio of 1:1, respectively.  
     
     
         75 . The method of  claim 68 , wherein said first vector and said second vector are introduced into said target cell at a ratio of 2:1, respectively.  
     
     
         76 . The method of  claim 68 , wherein said first vector is introduced at 900 MOI and said second vector at 1500 MOI into said target cell.  
     
     
         77 . The method of  claim 68 , wherein the first promoter is GAL4 and the inducer polypeptide is GAL4/VP16, respectively.  
     
     
         78 . The method of  claim 68 , wherein the target cell is a hyperproliferative cell.  
     
     
         79 . The method of  claim 78 , wherein said cell is a pre-malignant cell.  
     
     
         80 . The method of  claim 78 , wherein said cell is a malignant cell.  
     
     
         81 . The method of  claim 80 , where said cell is a lung cancer cell, a prostate cancer cell, a brain cancer cell, a liver cancer cell, a breast cancer cell, a skin cancer cell, an ovarian cancer cell, a testicular cancer cell, a stomach cancer cell, a pancreatic cancer cell, a colon cancer cell, an esophageal cancer cell, head and neck cancer cell.  
     
     
         82 . The method of  claim 68 , wherein said first and second vectors are introduced into said target cell at the same time.  
     
     
         83 . The method of  claim 68 , wherein said first vector is introduced into said target cell prior to said second vector.  
     
     
         84 . The method of  claim 83 , wherein said second vector is introduced into said target cell within 24 hours of said first vector.  
     
     
         85 . The method of  claim 83 , wherein said second vector is introduced into said target cell within 12 hours of said first vector.  
     
     
         86 . The method of  claim 83 , wherein said second vector is introduced into said target cell within 6 hours of said first vector.  
     
     
         87 . The method of  claim 83 , wherein said second vector is introduced into said target cell within 3 hours of said first vector.  
     
     
         88 . The method of  claim 83 , wherein said second vector is introduced into said target cell within 1 hour of said first vector.  
     
     
         89 . The method of  claim 68 , wherein said second vector is introduced into said target cell prior to said first vector.  
     
     
         90 . The method of  claim 89 , wherein said first vector is introduced into said target cell within 24 hours of said second vector.  
     
     
         91 . The method of  claim 89 , wherein said first vector is introduced into said target cell within 12 hours of said second vector.  
     
     
         92 . The method of  claim 89 , wherein said first vector is introduced into said target cell within 6 hours of said second vector.  
     
     
         93 . The method of  claim 89 , wherein said first vector is introduced into said target cell within 3 hours of said second vector.  
     
     
         94 . The method of  claim 89 , wherein said first vector is introduced into said target cell within 1 hour of said second vector.  
     
     
         95 . The method of  claim 78 , wherein said target cell is further contacted with a DNA damaging agent.  
     
     
         96 . The method of  claim 95 , wherein said DNA damaging agent is radiotherapy.  
     
     
         97 . The method of  claim 95 , wherein said DNA damaging agent is chemotherapy.  
     
     
         98 . The method of  claim 72 , wherein said promoter is an inducible promoter and the inducing factor is present in said target cell.  
     
     
         99 . The method of  claim 72 , wherein said promoter is an inducible promoter and the inducing factor is added to said target cell.  
     
     
         100 . The method of  claim 68 , wherein one or both of said vectors further comprise a polyadenylation signal.  
     
     
         101 . The method of  claim 68 , wherein said polypeptide expressed in said target cell is cytotoxic.  
     
     
         102 . The method of  claim 101 , wherein said cytotoxic polypeptide is selected from the group consisting of an inducer of apoptosis, a cytokine, a toxin, a single chain antibody, a protease and a antigen.  
     
     
         103 . The method of  claim 102 , wherein said inducer of apoptosis is selected from the group consisting of Bax, Bak, Bik, Bim, Bid, Bad and Harakiri.  
     
     
         104 . The method of  claim 103 , wherein said inducer of apoptosis is Bax.  
     
     
         105 . The method of  claim 102 , wherein said toxin is selected form the group consisting of ricin A-chain, diptheria toxin A-chain, pertussis toxin A subunit,  E. coli  enterotoxin A subunit, cholera toxin A subunit and pseudomonas toxin c-terminal.  
     
     
         106 . The method of  claim 105 , wherein said toxin is diptheria toxin A-chain.  
     
     
         107 . The method of  claim 102 , wherein said cytokine is selected form the group consisting of oncostatin M, TGF-β, TNF-α and TNF-β.  
     
     
         108 - 110 . (canceled)  
     
     
         111 . A method of treating a disease comprising introducing into cells of a subject having said disease: 
 (a) a first vector comprising a coding region for said therapeutic polypeptide under the control of a first promoter inducible by an inducer polypeptide not expressed in said target cell; and    (b) a second vector comprising a coding region for said inducer polypeptide under the control of a second promoter active in said target cell.    
     
     
         112 . The method of  claim 111 , wherein said disease is selected from the group consisting of lung cancer, prostate cancer, brain cancer, liver cancer, breast cancer, skin cancer, ovarian cancer, testicular cancer, stomach cancer, pancreatic cancer, colon cancer, esophageal cancer and head and neck cancer.  
     
     
         113 . The method of  claim 111 , wherein said therapeutic polypeptide is selected from the group consisting of Bax, Bak, Bik, Bim, Bid, Bad, Harakiri, ricin A-chain, diptheria toxin A-chain, pertussis toxin A subunit,  E. coli  enterotoxin A subunit, cholera toxin A subunit, pseudomonas toxin c-terminal, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.