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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2287-2292
The Use of Granulocyte Colony-Stimulating Factor During Retroviral
Transduction on Fibronectin Fragment CH-296 Enhances Gene Transfer
Into Hematopoietic Repopulating Cells in Dogs
Martin Goerner,
Benedetto Bruno,
Peter A. McSweeney,
Greg Buron,
Rainer Storb, and
Hans-Peter Kiem
From Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle, WA; and Department of Medicine, University of
Washington School of Medicine, Seattle, WA.
A competitive repopulation assay in the dog was used to develop
improved gene transfer protocols for hematopoietic stem cell gene
therapy. Using this assay, we previously showed improved gene transfer
into canine hematopoietic repopulating cells when CD34-enriched marrow
cells were cocultivated on gibbon ape leukemia virus (GALV)-based
retrovirus vector-producing cells. In the present study,
we have investigated the use of fibronectin fragment CH-296 and 2 growth factor combinations to further improve gene transfer efficiency.
CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were
placed into flasks coated with either CH-296 or bovine serum albumin
(BSA) and virus-containing medium supplemented with growth factors, and
protamine sulfate was replaced 4 times over a 48-hour period. One
fraction was cocultivated on irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 animals, cells of the different fractions were transduced in the presence of human FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakaryocyte growth
and development factor (MGDF), and in 2 other dogs, transduction was
performed in the presence of FLT3L, cSCF, and canine granulocyte-colony stimulating factor (cG-CSF). The vectors used contained small sequence
differences, allowing differentiation of cells genetically marked by
the different vectors. After transduction, nonadherent and adherent
cells from all 3 fractions were pooled and infused into lethally
irradiated dogs. Polymerase chain reaction and Southern blot analysis
were used to determine the persistence of the transferred vectors in
the peripheral blood and marrow cells after transplantation. The
highest levels of gene transfer were obtained when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (gene transfer levels of more than 10% for more than 8 months so far). Compared with
the 2 animals that received cells transduced with FLT3L, cSCF, and
MGDF, gene transfer levels were significantly higher when dogs received
cells that were transduced in the presence of cG-CSF. Transduction on
CH-296 resulted in gene transfer levels that were at least as high as
transduction by cocultivation. In summary, the overall levels of gene
transfer obtained with these conditions should be sufficiently high to
allow stem cell gene therapy studies aimed at correcting genetic
diseases in dogs as a model for human gene therapy.
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