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Retrovirus-mediated gene transduction into canine peripheral blood
repopulating cells
HP Kiem, B Darovsky, C von Kalle, S Goehle, D Stewart, T Graham, R Hackman, FR Appelbaum, HJ Deeg and AD Miller
Fred Hutchinson Cancer Research Center, Seattle, WA 98104-2092.
Genetically marked peripheral blood progenitor cells were used to
investigate their contribution to long-term hematopoietic reconstitution
after autologous marrow and peripheral blood cell transplantation. After
autologous marrow harvest and cryopreservation, canine peripheral blood
progenitor cells were mobilized in three dogs by treatment with recombinant
canine stem cell factor for 8 days. Peripheral blood mononuclear cells were
collected and enriched for major histocompatibility complex (MHC) class II
antigen-positive cells by avidin-biotin immunoadsorption, thereby enriching
for repopulating cells. Subsequently, the cells were cocultivated for 24
hours on irradiated vector-producing packaging cells (PA317/LN), followed
by an 11-day incubation in a vector containing long-term marrow culture
system. On the day of transplantation, the animals were irradiated with 9.2
Gy total body irradiation (TBI), and transduced peripheral blood cells and
untransduced cryopreserved marrow cells were infused within 2 hours of TBI.
All three dogs engrafted. Two dogs are long-term survivors showing
intermittently G418-resistant marrow-derived colony- forming unit
granulocyte-macrophage colonies at a median of 1% and 2%, respectively
(range, 1% to 10%), for now up to 48 weeks after transplantation.
Neo-specific sequences were detected by polymerase chain reaction in
peripheral blood granulocytes for now up to 65 weeks and in peripheral
blood lymphocytes for up to 75 weeks after transplantation. Peripheral
blood samples of the dogs were free of helper virus and no side effects
from the transduction were observed. One of the three dogs died from
chronic canine distemper sclerosing encephalitis on day 84, whereas the
other two dogs are alive at 15 and 17 months. Our data show successful
retroviral transduction of canine peripheral blood repopulating cells.
Long-term persistence of marked myeloid and lymphoid cells after
transplantation suggests that peripheral blood contains repopulating cells
that contribute to long- term hematopoietic reconstitution after otherwise
lethal TBI.
Volume 83,
Issue 6,
pp. 1467-1473,
03/15/1994
Copyright © 1994 by The American Society of Hematology
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