To overcome myelosuppression, a major dose limiting toxicity of many chemotherapeutic agents, this application proposes to use gene therapy to overexpress a drug resistance gene in hematopoietic progenitors. The overall goal is to reduce myelosuppression after repeated doses of conventional or high dose chemotherapy. The drugs targeted are alkylating agents which are associated with cumulative myelosuppression, persistent marrow damage and delayed secondary leukemias, most likely due to direct toxicity to early hematopoietic progenitors. This group of agents includes the chloroethylating agent,, BCNU, and the methylating agent, temozolomide, [TMZ] which exert their cytotoxic effect by attack of the O6 position of guanine. The applicant has shown that O6 -DNA alkyltransferase [AGT], encoded by MGMT, repairs O6 - alkylguanine lesions and is responsible for tumor resistance to these agents. The applicant has also shown that the AGT inhibitor, O6 - benzylguanine, [BG], markedly sensitizes tumors to BCNU although its use is associated with dose-limiting myelosuppression. To protect hematopoietic cells from the combination of BG & BCNU or BG & TMZ, the applicant proposes to use a mutant MGMT, glycine 156 to alanine, [delta-MGMT] which is resistant to BG inactivation. The investigators propose that expression of delta-MGMT from retroviral vectors in murine and human hematopoietic progenitors will selectively prevent myelosuppression after therapy with BG & BCNU and BG & TMZ. Evaluation of both BCNU and TMZ is important because their mechanism of action and potential therapeutic utility and toxicities are different. The following specific aims will be studied: 1. Define the ability of delta-MGMT to transduce human CD34 cells, generating resistance to the combination of BG and BCNU or TMZ; 2. Optimize delta-MGMT transduction into early hematopoietic progenitor long term culture initiating cells [LTC-IC] and determine whether these cells become resistant to the combination of BG & BCNU or TMZ; 3. Evaluate murine models of hematopoietic drug resistance following retroviral delta-MGMT transduction and transplantation; and 4. Determine the ability of transduced murine cells to competitively repopulate the marrow of mice by repeated drug exposure. These studies will optimize gene transfer strategies to protect hematopoietic cells from the acute and long term effects of chemotherapeutic agents.
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