This competing renewal application will investigate the capability of drug resistance gene expression in allowing improved anti-tumor chemotherapy and selective in vivo enrichment of cells expressing the drug resistance gene. In this study, methotrexate (MTX) will be used as an anti-tumor or selective agent in combination with variant forms of drug-resistant dihydrofolate reductase (DHFR). The proposed work is based on the applicant's successful demonstration of increased MTX dose tolerance of transgenic mice and subsequently of DHFR transgenic marrow transplant recipients. There are five Specific Aims.
In Aim 1, DHFR variants will be generated which contain two mutations which are known from the applicant's enzyme kinetic studies to contribute to either increased drug-resistance (arg22 or tyr22) or increased catalytic activity (ser31). The applicant will test the arg22/ser31 and tyr22/ser31 combinations for enzyme kinetic and MTX inhibition character which may contribute to increased drug-resistance of animals when expressed in vivo. The potential of drug-resistant DHFR gene expression for improved chemotherapy will be addressed in Aim 2 through pharmacokinetic studies to determine whether or not the increased MTX resistance conferred by DHFR transgenic marrow is associated with decreased MTX in blood and tissues, a condition which would not only protect MTX-sensitive normal tissues but tumor cells as well.
In Aim 3, experiments will be conducted to determine whether the drug resistance conferred by transplantation with MTX-resistant DHFR transgenic marrow is sufficient to allow improved survival of tumor-bearing animals by administration of higher doses of MTX. MTX-mediated selective engraftment of drug-resistant DHFR expressing marrow will be tested directly in Aim 4 through competitive repopulation experiments in which animals transplanted with mixtures of normal and DHFR transgenic marrow will be administered MTX and analyzed after secondary transplant for increased representation of marrow derived from drug-resistant stem cells.
In Aim 5, hematopoietic stem cells will be isolated from DHFR transgenic marrow in a highly purified state, testing for the capability of small numbers of stem cells to confer drug resistance in irradiated transplant recipients as a model for DHFR gene transfer into purified human hematopoietic stem cells. Results from these studies will address the feasibility of applying MTX resistance gene transfer for improved chemotherapy in humans as well as using MTX as an in vivo selective agent to deal with the low levels of gene transfer currently observed in humans and in large animal models.
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