Drug-resistant dihydrofolate reductases: generation, expression and therapeutic application

Abstract

Although methotrexate (MTX) and other antifolates are effective antitumor chemotherapeutic agents, their utility is limited by toxicity for normal hematopoietic and gastrointestinal tissues. Gene transfer and expression of variant dihydrofolate reductase (DHFR) which confers resistance to MTX could protect these tissues from MTX toxicity. Drug-resistant DHFR variants have been cloned from a number of sources and have also been generated by in vitro mutagenesis. A procedure is described which we used for saturation mutagenesis of the murine DHFR coding sequence at codon positions 22 and 31, resulting in a number of mutant DHFR genes encoding enzyme exhibiting a favorable combination of drug resistance with retention of catalytic activity. To evaluate the in vivo effectiveness of variant DHFR expression, we established several lines of FVB/N transgenic mice which expressed drug-resistant DHFR activity and which exhibited varying degrees of increased resistance to MTX administration. Transplantation of bone marrow from drug-resistant DHFR transgenic animals into normal, irradiated recipients conferred resistance to MTX at surprisingly high levels, indicating that drug-resistant DHFR expression in hematopoietic cells also protects gastrointestinal tissues from MTX toxicity. These studies have thus provided encouraging results with respect to the potential of drug-resistant DHFR gene transfer for improved use of MTX as an antitumor agent and for its use as an in vivo selective agent.