EFFICIENT GENE-TRANSFER IN PRIMITIVE CD34(+) CD38(LO) HUMAN BONE-MARROW CELLS RESELECTED AFTER LONG-TERM EXPOSURE TO GALV-PSEUDOTYPED RETROVIRAL VECTOR/

Successful retroviral gene transfer into human hematopoietic stem cell s was demonstrated in preliminary clinical trials at low efficiency. W e have shown previously that gene transfer into committed hematopoieti c progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retrov iral vector. sere, we have conducted a systematic study of human hemat opoietic progenitor cells after extended transduction with a GALV-pseu dotyped retroviral vector. CD34(+)/CD38(lo) Cells were transduced for 5 days and reselected according to phenotype after culture and analyze d for cell cycle status, long-term culture-initiating cell (LTC-IC) ac tivity, and gene transfer. Reselection of rare, very primitive progeni tor cells was successful. Equal to fresh CD34(+)/CD38(lo) cells, >90% of reselected CD34(+)/CD38(lo) cells were in G(0)/G(1). CD34(+)/CD38(l o) reselection enriched for LTC-IC (10-fold), as compared to freshly i solated CD34(+)/CD38(lo) cells with excellent specificity (82.7% of to tal LTC-IC were recovered in the reselected CD34(+)/CD38(lo) populatio n) and recovery (62% of initial LTC-IC number in CD34(+)/CD38(lo) cell s were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418 -resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34(+)/CD38(lo) cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cel l cycle demonstrating retroviral gene transfer into rare, very primiti ve human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.