| Gene therapy is a field that may lead to new options for the treatment of inherited disorders and cancers. The goal of drug resistance gene therapy is to protect hematopoietic cells from the myelosuppressive side effects of chemotherapy by transfer of a cDNA that confers resistance to a class of chemotherapeutics. Protection of hematopoietic cells can not only decrease chemotherapy-induced side effects, but also can be used for the preferential expansion of gene-modified cells. In vivo selection may allow low numbers of transplanted gene-modified cells to repopulate an individual and express a protein encoded within the transfer vector. The work described in this dissertation characterizes an E. coli thymidylate synthase cDNA optimized for expression in mammalian cells (OPTecTS) and a human cDNA encoding cytosolic 5-nucleotidase I (cN-I) for potential use in drug resistance gene therapy. Retroviral constructs based on a murine stem cell virus (MSCV) backbone were engineered to express cDNA sequences specific for these enzymes from the 5' LTR. After transduction of OPTecTS into murine NIH3T3 cells, we observed high level resistance to TS-targeted antifolates, and could select GFP+ cells after co-transduction with OPTecTS and antifolate treatment. Furthermore, OPTecTS can protect murine hematopoietic progenitor cells from concentrations of antifolates that eliminate nontransduced cells. However, an attempt to develop a chemotherapy regimen for the in vivo selection of OPTecTS-expressing hematopoietic stem cells using the TS-targeted antifolate raltitrexed with concurrent thymidine phosphorylase treatment failed due to severe gastrointestinal toxicity at doses that were insufficient to cause stem cell toxicity. Transduction of MSCV-cNI conferred resistance to 2-CdA and 5-FU independently and in combination for murine fibroblasts and progenitor cells. We also developed a system to select cN-I-expressing hematopoietic stem cells by administering 5-FU to sensitize stem cells to 2-CdA. However, stem cell toxicity was only observed at doses of 2-CdA that were highly myelosuppressive. Therefore, while OPTecTS remains an excellent in vitro drug resistant marker, its use in vivo is limited by the lack of an available selection system. A potential selection system was developed for cN-I, however it causes severe myelosuppression and represents no clear benefit over currently available systems. |
