Deficiency of oncoretrovirally transduced hematopoietic stem cells and correction through ex vivo expansion.
BACKGROUND
Extensive efforts to develop hematopoietic stem cell (HSC) based gene therapy have been hampered by low gene marking. Major emphasis has so far been directed at improving gene transfer efficiency, but low gene marking in transplanted recipients might equally well reflect compromised repopulating activity of transduced cells, competing for reconstitution with endogenous and unmanipulated stem cells.


METHODS
The autologous settings of clinical gene therapy protocols preclude evaluation of changes in repopulating ability following transduction; however, using a congenic mouse model, allowing for direct evaluation of gene marking of lympho-myeloid progeny, we show here that these issues can be accurately addressed.


RESULTS
We demonstrate that conditions supporting in vitro stem cell self-renewal efficiently promote oncoretroviral-mediated gene transfer to multipotent adult bone marrow stem cells, without prior in vivo conditioning. Despite using optimized culture conditions, transduction resulted in striking losses of repopulating activity, translating into low numbers of gene marked cells in competitively repopulated mice. Subjecting transduced HSCs to an ex vivo expansion protocol following the transduction procedure could partially reverse this loss.


CONCLUSIONS
These studies suggest that loss of repopulating ability of transduced HSCs rather than low gene transfer efficiency might be the main problem in clinical gene therapy protocols, and that a clinically feasible ex vivo expansion approach post-transduction can markedly improve reconstitution with gene marked stem cells.
Bryder
David
D
Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Hematopoietic Stem Cell Laboratory, Lund University, BMC B10, 221 84, Lund, Sweden.
Björgvinsdóttir
Helga
H
Sasaki
Yutaka
Y
Jacobsen
Sten Eirik W
SE
D000818:Animals; D049109:Cell Proliferation; D017931:DNA Primers; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D051379:Mice; D041681:NIH 3T3 Cells; D009369:Neoplasms / Q000235:genetics / Q000628:therapy*; D016133:Polymerase Chain Reaction; D012190:Retroviridae; D018709:Statistics, Nonparametric; D014161:Transduction, Genetic / Q000379:methods*
D017931:DNA Primers; D049452:Green Fluorescent Proteins