| Limitations in the therapeutic window of chemotherapy drugs can be addressed by transducing tumor cells with viral vectors expressing genes encoding for prodrug-activating enzymes. After transduction, treatment with a prodrug that is nontoxic in the absence of enzymatic activation causes the death of transduced tumor cells, as well as nearby nontransduced tumor cells by means of a "bystander effect." Despite initial success, chemosensitizing gene therapies remain hindered by several limitations, most notably low transduction frequencies. This thesis explores three approaches to improve the efficacy of chemosensitizing gene therapies. First, combination gene therapy using herpes simplex virus thymidine kinase (HSV-TK), which phosphorylates the nucleoside analogue ganciclovir (GCV), and E. coli cytosine deaminase (CD), which activates the prodrug 5-fluorocytosine (5-FC), was studied. Synergism was found in culture and in vivo using Chou-Talalay analysis, and evidence was offered that synergism stems from 5-FC's active metabolite affecting nucleotide pools in a manner that promotes GCV phosphorylation. Second, increasing the folylpolyglutamyl synthetase (FPGS) expression of glioma cells, a novel chemosensitizing gene therapy, was investigated. Enhanced antifolate sensitivity was demonstrated in transfected cells in culture and in subcutaneous tumors in vivo . A bystander effect was demonstrated, and shown to be due to the release of stored antifolate upon removal of extracellular drug. The presence of a bystander effect justified the construction of a vector expressing FPGS. Transduction of two glioma cell lines with this vector enhanced their antifolate sensitivity. Third, combination gene therapy using HSV-TK and the rat cytochrome P450 2B1 (CYP2B1) gene, which activates the prodrug cyclophosphamide (CPA), was studied. Synergism was shown in culture using Chou-Talalay analysis, and evidence was provided suggesting that the synergism results because GCV's active metabolite inhibits repair of DNA crosslinks caused by CPA's active metabolite. Synergism was also shown upon infection of glioma cells in culture with a replication-conditional herpes vector expressing both transgenes. Infection of subcutaneous tumors with this virus led to tumor regression only after a triple treatment combining virus and both prodrugs. The efficacies of these three strategies can now be thoroughly assessed in an intracranial model. These studies will hopefully contribute to the development of an improved strategy for chemosensitizion of brain tumors using gene therapy. |
