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Autologous transplantation of canine long-term marrow culture cells
genetically marked by retroviral vectors
RF Carter, AC Abrams-Ogg, JE Dick, SA Kruth, VE Valli, S Kamel-Reid and ID Dube
University of Toronto Hospitals' Cancer Cytogenetics Program, Toronto
General Hospital, Ontario, Canada.
Retroviral infection of bone marrow cells in long-term marrow cultures
(LTMCs) offers several theoretical advantages over other methods for gene
transfer into hematopoietic stem cells. To investigate the feasibility of
this approach in a large animal model system, we subjected LTMCs from nine
dogs to multiple infections with retrovirus containing the neomycin
phosphotransferase gene (neo) during 21 days of culture. Feeder layers,
cocultivation, polycations, and selection were not used. The in vitro gene
transfer efficiency was 70% as determined by polymerase chain reaction
amplification of neo sequences in colony- forming unit
granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC
cells were infused into autologous recipients with (four dogs) and without
(three dogs) marrow-ablative conditioning. At 3 months posttransplant, up
to 10% of marrow cells contained the neo gene. This percentage declined to
0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in
individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix
progenitors derived from marrow up to 21 months postinfusion and in
cultures of peripheral blood- derived T cells up to 19 months postinfusion.
There was no difference in the percentage of neo-marked cells present when
dogs that received marrow ablative conditioning were compared with dogs
receiving no conditioning. Detection of neo-marked marrow cells almost 2
years after autologous transplantation in a large mammalian species shows
that retroviral infection of marrow cells in LTMCs is a potentially
nontoxic and efficient protocol for gene transfer. Further, our results
suggest that marrow conditioning and in vivo selection pressure to retain
transplanted cells may not be absolute requirements for the retention of
genetically marked cells in vivo.
Volume 79,
Issue 2,
pp. 356-364,
01/15/1992
Copyright © 1992 by The American Society of Hematology
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