Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene

The genetic transfer of drug resistance to hematopoietic cells is an attrac tive approach to overcoming myelosuppression caused by high-dose chemothera py. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoi etic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a re troviral vector containing both a human cytosolic ALDH-1 cDNA and a human d oubly mutated DHFR cDNA (Phe22/Ser31; termed F/s in the description of cons tructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxy-cycloph osphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced hu man CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted w ith SFG-ALDH-IREs-F/S or mock-transduced bone marrow cells were treated wit h high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not tr ansduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity , whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marr ow were able to tolerate the same doses of CTX or CTX/MTX treatment posttra nsplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vi vo mouse model. This construct may be useful for protecting patients from h igh-dose CTX- and MTX-induced myelosuppression.