Protection and in vivo selection of hematopoietic stem cells using temozolomide, O-6-benzylguanine, and an alkyltransferase-expressing retroviral vector

Transfer of drug resistance genes to hematopoietic stem cells offers the po tential to protect cancer patients from drug-induced myelosuppression and t o increase the number of gene-modified cells by in vivo selection. In this study, a retroviral vector expressing both a P140K variant of human O-6-met hylguanine-DNA methyltransferase (MCMT) and an EGFP reporter gene was evalu ated for stem cell protection in a murine transplant model. Mice transplant ed with vector-transduced cells showed significant resistance to the myelos uppressive effects of temozolomide (TMZ), an orally administered DNA-methyl ating drug, and O-6-benzylguanine (BC), a drug that depletes cells of wild- type MCMT activity. Following drug treatment, increases in EGFP(+) peripher al blood cells were seen in all peripheral blood lineages, and secondary tr ansplant experiments proved that selection had occurred at the stem cell le vel. In a second set of experiments in which transduced cells were diluted with unmarked cells, efficient stem cell selection was noted together with progressive marrow protection with repeated treatment courses. Altogether, these results show that P140K MCMT gene transfer can protect stem cells aga inst the toxic effects of TMZ and Be; and that this vector/drug system may be useful for clinical myeloprotection and for in vivo selection of transdu ced stem cells.