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Stromal support enhances cell-free retroviral vector transduction of human
bone marrow long-term culture-initiating cells
KA Moore, AB Deisseroth, CL Reading, DE Williams and JW Belmont
Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX
77030.
Gene transfer into hematopoietic stem cells by cell-free virions is a goal
for gene therapy of hematolymphoid disorders. Because the hematopoietic
microenvironment provided by the stroma is required for stem cell
maintenance both in vivo and in vitro, we reasoned that cell- free
transduction of bone marrow cells (BMC) may be aided by stromal support. We
used two high-titer replication-defective retroviral vectors to
differentially mark progenitor cells. The transducing vector was shown to
be a specific DNA fragment by polymerase chain reaction of colony-forming
cells derived from progenitors maintained in long-term culture (LTC). BMC
were infected separately by cell-free virions with or without
pre-established, irradiated, allogeneic stromal layers, and in the presence
or absence of exogenous growth factors (GF). The GF assessed were
interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor
(LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In
addition, we developed a competitive LTC system to directly assess the
effect of infection conditions on the transduction of clonogenic
progenitors as reflected by the presence of a predominate provirus after
maintenance in the same microenvironment. The results show gene transfer
into human LTC-initiating cells by cell-free retroviral vector and a
beneficial effect of stromal support allowing a transduction efficiency of
64.6% in contrast to 15.8% without a supporting stromal layer. A high
transduction rate was achieved independent of stimulation with exogenous
GF. We propose that autologous marrow stromal support during the
transduction period may have application in clinical gene therapy
protocols.
Volume 79,
Issue 6,
pp. 1393-1399,
03/15/1992
Copyright © 1992 by The American Society of Hematology
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