EX-VIVO EXPANSION AND SELECTION OF HUMAN CD34(-BLOOD PROGENITOR CELLSAFTER INTRODUCTION OF A MUTATED DIHYDROFOLATE-REDUCTASE CDNA VIA RETROVIRAL GENE-TRANSFER() PERIPHERAL)

Retroviral gene transfer into human myeloid precursor cells allows int roduction of marker genes as well as genes conferring resistance to ch emotherapeutic drugs. We transduced a human mutant dihydrofolate reduc tase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells fro m patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that per mitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive ce lls increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were prot ected completely against 2 x 10(-8) mol/L MTX, a concentration that si gnificantly reduced the CFU-GM detected in the control population. Gen e transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .00 1). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80 % of CFU GM and ex vivo expanded pre-CFU. We have demonstrated that hu man hematopoietic precursor calls can be expanded extensively after re troviral gene transfer. The same population of early progenitors can h e selected ex vivo with low dose MTX. As long-term expression of trans duced genes in human hematopoietic cells remains a problem in vivo, th ese results may have implications for future clinical trials, especial ly for the introduction of nonselectable genes. (C) 1995 by The Americ an Society of Hematology.