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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1878-1886
RAPID COMMUNICATION
Improved Gene Transfer Into Baboon Marrow Repopulating Cells Using
Recombinant Human Fibronectin Fragment CH-296 in Combination With
Interleukin-6, Stem Cell Factor, FLT-3 Ligand, and Megakaryocyte
Growth and Development Factor
Hans-Peter Kiem,
Robert G. Andrews,
Julia Morris,
Laura Peterson,
Scott Heyward,
James M. Allen,
John E.J. Rasko,
Jennifer Potter, and
A. Dusty Miller
From the Clinical Research and Molecular Medicine Divisions, Fred
Hutchinson Cancer Research Center, Seattle, WA; the Departments of
Medicine, Pediatrics, and Pathology, University of Washington School of
Medicine, Seattle, WA; and the University of Washington Regional
Primate Research Center, Seattle, WA.
We have used a competitive repopulation assay in baboons to develop
improved methods for hematopoietic stem cell transduction and have
previously shown increased gene transfer into baboon marrow
repopulating cells using a gibbon ape leukemia virus (GALV)-pseudotype retroviral vector (Kiem et al, Blood 90:4638, 1997). In this
study using GALV-pseudotype vectors, we examined additional variables that have been reported to increase gene transfer into hematopoietic progenitor cells in culture for their ability to increase gene transfer
into baboon hematopoietic repopulating cells. Baboon marrow was
harvested after in vivo administration (priming) of stem cell factor
(SCF) and granulocyte colony-stimulating factor (G-CSF). CD34-enriched
marrow cells were divided into two equal fractions to directly compare
transduction efficiencies under different gene transfer conditions.
Transduction by either incubation with retroviral vectors on
CH-296-coated flasks or by cocultivation on vector-producing cells was
studied in five animals; in one animal, transduction on CH-296 was
compared with transduction on bovine serum albumin (BSA)-coated flasks.
The highest level of gene transfer was obtained after 24 hours of
prestimulation followed by 48 hours of incubation on CH-296 in
vector-containing medium in the presence of multiple hematopoietic
growth factors (interleukin-6, stem cell factor, FLT-3 ligand, and
megakaryocyte growth and development factor). Using these conditions,
up to 20% of peripheral blood and marrow cells contained vector
sequences for more than 20 weeks, as determined by both polymerase
chain reaction and Southern blot analysis. Gene transfer rates were higher for cells transduced on CH-296 as compared with BSA or cocultivation. In one animal, we have used a vector expressing a cell
surface protein (human placental alkaline phosphatase) and have
detected 10% and 5% of peripheral blood cells expressing the
transduced gene 2 and 4 weeks after transplantation as measured by flow
cytometry. In conclusion, the conditions described here have resulted
in gene transfer rates that will allow detection of transduced cells by
flow cytometry to facilitate the evaluation of gene expression. The
levels of gene transfer obtained with these conditions suggest the
potential for therapeutic efficacy in diseases affecting the
hematopoietic system.
© 1998 by The American Society of Hematology.
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