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Increased gene transfer into human hematopoietic progenitor cells by
extended in vitro exposure to a pseudotyped retroviral vector
C von Kalle, HP Kiem, S Goehle, B Darovsky, S Heimfeld, B Torok-Storb, R Storb and FG Schuening
Fred Hutchinson Cancer Research Center, Seattle, WA 98104-2092.
Retroviral-mediated gene transfer is the most attractive modality for gene
transfer into hematopoietic stem cells. However, transduction efficiency
has been low using amphotropic Moloney murine leukemia virus (MoMLV)
vectors. In this study, we investigated modifications of gene transfer
using amphotropic MoMLV vectors in cell-free supernatant for their ability
to increase the currently low transduction of both committed hematopoietic
progenitors, granulocyte-macrophage colony- forming units (CFU-GMs), and
their precursors, long-term culture- initiating cells (LTC-IC). First,
based on the observation that bone marrow cells express more gibbon ape
leukemia virus (GALV) receptor (Glvr-1) than amphotropic receptor (Ram-1),
PG13/LN, which is a MoMLV vector pseudotyped with the GALV envelope, was
compared with the analogous amphotropic envelope vector (PA317/LN). Second,
progenitor cell transduction efficiency was compared between CD34 enriched
and nonenriched progenitor populations. Third, the duration of transduction
in vitro was extended to 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 to identical titers of pseudotyped PG13/LN vector
or PA317/LN vector in 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 vector supernatant was refed daily. Hematopoietic progenitor
cells as measured 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
antigen 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 (n = 4), 60% of CFU-GM and 100% of LTC-IC were resistant at 2
weeks (n = 2) and 74% of CFU-GM (n = 4) and 82% of LTC-IC (n = 2) were
resistant at three weeks.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 84,
Issue 9,
pp. 2890-2897,
11/01/1994
Copyright © 1994 by The American Society of Hematology
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