Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells.
Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes. We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene. Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33%. The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR). SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01). In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296. Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.
German Cancer Research Center, Heidelberg, Germany.
B
B
Schiedlmeier
K
K
Wermann
K
K
Kühlcke
H G
HG
Eckert
C
C
Baum
S
S
Fruehauf
W J
WJ
Zeller
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019450:Genes, MDR; D015316:Genetic Therapy; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
D017931:DNA Primers