Methotrexate is a potent inhibitor of dihydrofolate reductase and a useful chemotherapeutic agent in the treatment of a variety of human malignancies. However, the antiproliferative effects of this drug result in considerable toxicity to normal hematopoietic and gastrointestinal tissues as well as to the tumor cells. This limits the drug dose tolerated by the host and, therefore, the potential of the drug in tumor elimination. It is proposed herein that insertion of a methotrexate-resistant dihydrofolate reductase gene (MTXr-DHFR) into the cells of donor bone marrow would allow administration of methotrexate to the bone marrow transplant recipient at high levels, increasing the likelihood of tumor elimination. The experimental approach can be considered in three phases. First, recombinant retroviral vectors will be generated and used to insert a MTXr-DHFR gene into hematopoietic stem cells of the mouse. Expression will be assessed both biochemically (transcriptional and/or enzymatic analysis) and physiologically (drug resistance). This work will include the isolation of a cDNA sequence encoding a more highly resistant form of DHFR. The second phase will be demonstrating that insertion of MTXr-DHFR gene confers substantial drug resistance in vivo and will allow regeneration of hematopoietic tissue during administration of methotrexate at doses which preclude regeneration of tissue not containing the gene insertion. The final phase will be to determine if the higher methotrexate doses tolerated by regenerating tissue can be used to enhance survival and tumor elimination in experimental animals (mouse L1210 leukemia). The extent to which drug-resistance gene insertion improves the anti- tumor potential of methotrexate administration in transplanted animals will be directly applicable to the use of MTXr-DHFR gene transfer in a more aggressive chemotherapeutic approach for patients suffering from a variety of neoplastic disease. The results will also be important in considering the potential for gene transfer in the treatment of human genetic diseases.
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