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Increased efficiency of retroviral-mediated gene transfer and expression in
primate bone marrow progenitors after 5-fluorouracil- induced hematopoietic
suppression and recovery
R Wieder, K Cornetta, SW Kessler and WF Anderson
Molecular Hematology Branch, NHLBI, National Institutes of Health,
Bethesda, MD 20892.
To define conditions for improved efficiency of retroviral-mediated gene
transfer and expression in primate progenitor cells, four rhesus monkeys
were treated with a 200 mg/kg intravenous bolus of 5- fluorouracil (5-FU).
The kinetics of hematopoietic suppression and recovery were assessed in
peripheral blood, bone marrow mononuclear cells, and bone marrow cells
fractionated in an albumin density gradient. Bone marrow mononuclear cells
were transduced with N2, a retroviral vector carrying the bacterial
neomycin phosphotransferase gene (NPT), which confers resistance to the
otherwise toxic neomycin analogue, G418. Circulating colony-forming
units-granulocyte-macrophage (CFU-GM) disappeared at 2 days. CFU-GM,
transducible CFU-GM, CD34+ cells, and the percent of cells in cycle
decreased at 3 days in unfractionated bone marrow cells and in a light
density population known to be enriched for these progenitors and for stem
cells. NPT activity in the light-density fraction, marginally detectable
before treatment, disappeared at 3 days as well. At day 7 the CFU-GM
plating efficiency, the CD34+ cell content, and the percentage of cells in
cell cycle began to increase in the light-density fraction. The NPT assay
became faintly positive again but the CFU-GM were not yet transducible,
implying that it was an earlier progenitor population that was dividing and
differentiating. By day 15, there was a marked rebound in all of the
progenitors measured, and transduction efficiency assessed by G418R CFU-GM
and NPT assay rebounded to several times pretreatment levels. The data
suggest that CFU-GM are optimally transduced at 15 days but that earlier
progenitors are more likely cycling and transducible before 5 days, a time
when a gene transfer experiment would probably have the best chance to
succeed.
Volume 77,
Issue 3,
pp. 448-455,
02/01/1991
Copyright © 1991 by The American Society of Hematology
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