Increased resistance to methotrexate in human hematopoietic cells after gene transfer of the Ser31 DHFR mutant.
Retroviral gene transfer into hematopoietic cells has many experimental as well as clinical applications. Although transduction efficiency of retroviral vectors is higher than with conventional methods, selection of successfully transduced cells may become mandatory for efficient in vivo transfer. We have been evaluating a retroviral construct that meets the criteria for a clinically acceptable selection system. The Ser31 dihydrofolate reductase (DHFR) mutant confers resistance to methotrexate (MTX) due to decreased binding of the drug. while its enzymatic activity remains adequate for normal folate metabolism. Transduction of this vector into murine hematopoietic cells has been recently described and increase in MTX resistance could be observed. We investigated transduction of CD34-antigen positive subpopulations of hematopoietic progenitor cells and CD34-positive/CD38-negative subpopulations enriched for stem cells. We focused on two sources of primary hematopoietic cells, umbilical cord blood (UCB) and peripheral blood (PB) harvested from patients after mobilization with chemotherapy and/or cytokines. Both contain a large number of lineage restricted and pluripotent progenitor cells and can be expanded extensively ex vivo. Potential clinical applications of gene therapy in such cell populations include correction of inborn enzymatic diseases and support of high-dose chemotherapy by transplanting ex vivo transduced progenitor cells rendered more resistant to cytotoxic drugs. The feasibility and efficiency of retroviral transduction into UCB and PB has been reported recently.
D018952:Antigens, CD34; D003114:Colony-Forming Units Assay; D004351:Drug Resistance; D005312:Fetal Blood; D015316:Genetic Therapy / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000473:pathology; D006801:Humans; D007231:Infant, Newborn; D007375:Interleukin-1 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D008727:Methotrexate / Q000633:toxicity*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012190:Retroviridae; D012694:Serine; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018952:Antigens, CD34; D007375:Interleukin-1; D007377:Interleukin-3; D011994:Recombinant Proteins; D012694:Serine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate