Clinical trials aimed at ameliorating the myelosuppressive side effects of chemotherapy by retroviral transduction of chemoprotecting gene have been established for the treatment of solid tumors. Thymidylate synthase (TS) is an excellent candidate for drug resistance gene therapy because 1) it plays a critical role in DNA biosynthesis by providing the only de novo source of thymidylate, 2) several novel inhibitors of TS have recently been developed and are in clinical trials, and 3) the dose limiting toxicity of many TS-directed inhibitors is myelosuppression. Included among these inhibitors are ZD1694, LY21514, ZD9331, 1843489 and AG337. In addition to attenuating side effects of chemotherapy, genes that confer drug resistance can also be used as dominant selectable markers. The applicant has recently shown the feasibility of using dihydrofolate reductase as a chemoprotecting agent for in vivo selection of genetically modified hematopoietic cells. The goal of this application is to establish the possibility of using gene transfer of thymidylate synthase to attenuate the toxic side effects of TS targeted inhibitors and determine if TS can be used as a dominant selectable marker.
The specific aims of this application can be summarized as: 1) Determine mutations of TS that result in decreased affinity for nucleotide and antifolate inhibitors, 2) Construct retroviral vectors that encode variants of TS and a previously characterized variant of DHFR. 3) Test if resistance to TS and DHFR inhibitors protect against chemotherapy induced myelosuppression and if in vivo selection of hematopoietic stem cells can be achieved in transplanted mice. 4) Determine if recombinant retroviruses that encode variants of TS and DHFR confer chemoprotection to transduced human bone marrow cells, human umbilical cord blood cells, and human mononuclear peripheral blood cells.
