INCREASED RESISTANCE TO METHOTREXATE IN HUMAN HEMATOPOIETIC-CELLS AFTER GENE-TRANSFER OF THE SER31 DHFR MUTANT

Retroviral gene transfer into hematopoietic cells has many experimenta l as well as clinical applications. Although transduction efficiency o f retroviral vectors is higher than with conventional methods, selecti on 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 meth otrexate (MTX) due to decreased binding of the drug. while its enzymat ic activity remains adequate for normal folate metabolism. Transductio n of this vector into murine hematopoietic cells has been recently des cribed end increase in MTX resistance could be observed (1,2). We inve stigated transduction of CD34-antigen positive subpopulations of hemat opoietic progenitor cells and CD34-postive/CD38-negative subpopulation s enriched for stem cells (3). We focused on two sources of primary he matopoietic 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 pluri potent progenitor cells and can be expanded extensively ex vivo (4-7). Potential clinical applications of gene therapy in such cell populati ons include correction of inborn enzymatic diseases and support of hig h-dose chemotherapy by transplanting ex vivo transduced progenitor cel ls rendered more resistant to cytotoxic drugs. The feasibility and eff iciency of retroviral transduction into UCB and PB has been reported r ecently (8,9).