DNA alkylating agents are an important part of most dose-intensified chemotherapy protocols. In spite of increased use of myeloid growth factor and stem cell support, myelosuppression continues to be a dose-limiting toxicity of many alkylating agents. This is particularly true in patients with relapsed disease previously treated with intensive chemotherapy during induction and consolidation phases of initial therapies. One approach to circumvent the dose-limiting myeloid toxicities of chemotherapy agents has been the use of gene transfer to introduce and express genes important in chemotherapy resistance in bone marrow-derived cells. Work in our laboratory has concentrated on retroviral vectors encoding DNA repair proteins as a mechanism to generate resistance to chloroethylnitrosourea (CENUs) and other alkylating agents. In our previous studies, we have developed a murine model of CENU-induced fatal bone marrow suppression. Reconstitution of murine bone marrow with hematopoietic stem cells expressing vector-derived MGMT protects mice from BCNU-induced bone marrow hypoplasia and peripheral blood pancytopenia. Bone marrow cells harvested from these mice are more resistant to CENU in vitro, and demonstrate a higher level of DNA repair activity compared to CENU-treated mock-infected control mice. In the studies outlined here, we propose to continue our work on CENU-resistance by developing a new series of retroviral vectors which encode DNA repair activity resistant to depletion by the free base 06-benzylguanine, which would allow differential depletion of tumor versus bone marrow repair activity. In addition, we propose to expand this approach to include proteins of the base excision repair (BER) pathway, which are important in the repair of DNA damage from CENUs and other alkylating agents. Finally, we wish to develop vectors which would potentially prevent DNA damage from alkylating agents by increasing the expression of detoxifying proteins in bone marrow cells. Overall, this project seeks to use gene transfer technology to increase resistance of normal bone marrow to the cytotoxic effects of alkylating agent damage.
The specific aims i nclude: 1) Develop retroviral vectors which increase the therapeutic window of chloroethylnitrosourea (CENU)-based tumor killing in vivo utilizing 6-benzylguanine resistant methylguanine DNA methyltransferases expressed in bone marrow cells; 2) Examine the effect of transgene expression of components of the base excision repair (BER) pathway on CENU and alkylating (bleomycin, cyclophosphamide and thiotepa) agent resistance; and, 3) Examine the role of increased expression of aldehyde dehydrogenase and glutathione-S- transferase in resistance to cyclophosphamide. These basic studies are likely to lead to innovative clinical studies utilizing gene transfer technology in the future.
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