Limiting numbers of G156A O-6-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection

The limited efficacy of hematopoietic gene therapy can be improved by in vi vo selection for transduced long-term repopulating cells (LTRC), We selecte d for G156A MGMT (Delta MGMT) transduced LTRC present in 5 x 10(4) to 100 x 10(4) marrow cells infused into nonmyeloablated mice by the administration of O-6-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engra ftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, Delta MGMT was not detected in any hemato-poietic colony -forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, Delt a MGMT(+) CFU were frequently detected, and their proportions increased wit h each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% Delta MGMT(+) CFU for at least 6 mont hs, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell tox icity allows Delta MGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than t hat observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduce d hematopoietic cells, potentially decreasing cumulative drug-induced myelo suppression in patients with cancer, These data also suggest that Delta MGM T may be a potent, dominant, selectable marker for use in dual gene therapy . (C) 2000 by The American Society of Hematology.