A GENE-TRANSFER STRATEGY FOR MAKING BONE-MARROW CELLS RESISTANT TO TRIMETREXATE

Trimetrexate (TMTX) is an anticancer drug with potential advantages ov er the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors c ontaining mutant dihydrofolate reductase (DHFR) genes have been used t o protect bone marrow cells from MTX. suggesting a similar approach co uld be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A vari ant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) w as best, allowing a 100-fold increase in resistance over controls. Mur ine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mic e reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consiste nt protection from TMTX-induced neutropenia and reticulocytopenia at l evels that correlated with the proviral copy number in circulating leu kocytes. We conclude that the L22Y vector is highly effective in prote cting hematopoiesis from TMTX toxicity and may provide a means for inc reasing the therapeutic utility of TMTX in certain cancers. (C) 1996 b y The American Society of Hematology.