Acute and cumulative myelosuppression is the dose limiting toxicity of BCNU and related chemotherapeutic agents. Resistance is conferred by 06-alkylguanine-DNA alkyltransferase (AGT) which is commonly present at high values in human tumors. Recently a strategy has been developed to overcome this resistance, using the AGT inhibitor BG. Unfortunately, in preclinical and early clinical trials, myelosuppression has again been dose-limiting. A mutant AGT (glycine to alanine at position 156 (G156A)) has been characterized which retains DNA repair activity but is resistant to inactivation by BG. In order to protect hematopoietic progenitors from BG and BCNU, the MFG based retroviral vector containing G156A mutant MGMT (deltaMGMT) was synthesized. deltaMGMT transduced human CD34+ cells and murine bone marrow cells were found to be significantly resistant to BG and BCNU in vitro. Furthermore mice transplanted with deltaMGMT transduced cells had a significant survival advantage over control animals when challenged by repetitive doses of BG and BCNU, along with enrichment of the transduced progenitors in vivo. The applicants now propose a clinical trial to test whether repopulating human hematopoietic progenitors can be retrovirally transduced by deltaMGMT, rendered resistant to BG and BCNU and enriched in vivo by chemotherapy in solid tumor patients. Transduced cells will be reinfused following BG and BCNU treatment at maximally tolerated doses (non-myeloablative) and engraftment of deltaMGMT transduced and BG & BCNU resistant progenitors will be analyzed before and after sequential doses of BG & BCNU. If successful this approach can improve the therapeutic efficacy of BCNU by both overcoming tumor resistance by BG pretreatment and simultaneously decreasing the dose limiting myelosuppression of this combination. In addition this approach may be used for the enrichment of the hematopoietic progenitors containing a therapeutic gene co-transduced with deltaMGMT.
