INCREASED GENE-TRANSFER INTO HUMAN HEMATOPOIETIC PROGENITOR CELLS BY EXTENDED IN-VITRO EXPOSURE TO A PSEUDOTYPED RETROVIRAL VECTOR

Retroviral-mediated gene transfer is the most attractive modality for gene transfer into hematopoietic stem cells. However, transduction eff iciency has been low using amphotropic Moloney murine leukemia virus ( MoMLV) vectors. In this study, we investigated modifications of gene t ransfer using amphotropic MoMLV vectors in cell-free supernatant for t heir ability to increase the currently low transduction of both commit ted hematopoietic progenitors, granulocyte-macrophage colony-forming u nits (CFU-GMs), and their precursors, long-term culture-initiating cel ls (LTC-IC). First, based on the observation that bone marrow cells ex press more gibbon ape leukemia virus (GALV) receptor (Glvr-1) than amp hotropic receptor (Ram-1), PG13/LN, which is a MoMLV vector pseudotype d with the GALV envelope, was compared with the analogous amphotropic envelope vector (PA317/LN). Second, progenitor cell transduction effic iency was compared between CD34 enriched and nonenriched progenitor po pulations. Third, the duration of transduction in vitro was extended t o increase the proportion of progenitor cells that entered cell cycle and could thereby integrate vector cDNA. In 20 experiments, 1 x 10(6) marrow or peripheral blood mononuclear cells (PBMCs)/mL were exposed t o identical titers of pseudotyped PG13/LN vector or PA317/ LN vector i n the presence of recombinant human interleukin-1 (IL-1), IL-3, IL-6, and stem cell factor (SCF; c-kit ligand) for 5 days. 50% of fresh vect or supernatant was refed daily. Hematopoietic progenitor cells as meas ured by G418-resistant granulomonocytic colony (CFU-GM) formation were transduced more effectively with PG13/LN (19.35%) than with PA317/LN (11.5%, P = .012). In 11 further experiments, enrichment of CD34 antig en positive cells significantly improved gene transfer from 13.9% G418 -resistant CFU-GM in nonenriched to 24.9% in CD34-enriched progenitor cells (P < .01). To analyze gene transfer after extended growth factor -supported long-term culture, 1 x 10(6) marrow cells/mL were cultured with IL-1, IL-3, IL-6, and SCF (50 ng/mL each) for 1, 2, and 3 weeks. Fifty percent of PG13/LN supernatant with growth factors was refed on 5 days per week. Five percent of marrow CFU-GM and 67% of LTC-IC were G418 resistant at 1 week in = 4), 60% of CFU-GM and 100% of LTC-IC wer e resistant at 2 weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LT C-IC in = 2) were resistant at three weeks. These data suggest that th e efficiency of human hematopoietic progenitor cell transduction can b e significantly improved by using the GALV-pseudotyped PG13/LN vector, enriching for CD34(+) cells and extending the transduction culture in the presence of IL-1, IL-3, IL-6, SCF, and PG13/LN vector. (C) 1994 b y The American Society of Hematology.