Improved retroviral gene transfer into murine and Rhesus peripheral blood or bone marrow repopulating cells primed in vivo with stem cell factor and granulocyte colony-stimulating factor.
In previous studies we showed that 5 days of treatment with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) mobilized murine repopulating cells to the peripheral blood (PB) and that these cells could be efficiently transduced with retroviral vectors. We also found that, 7-14 days after cytokine treatment, the repopulating ability of murine bone marrow (BM) increased 10-fold. In this study we examined the efficiency of gene transfer into cytokine-primed murine BM cells and extended our observations to a nonhuman primate autologous transplantation model. G-CSF/SCF-primed murine BM cells collected 7-14 days after cytokine treatment were equivalent to post-5-fluorouracil BM or G-CSF/SCF-mobilized PB cells as targets for retroviral gene transfer. In nonhuman primates, CD34-enriched PB cells collected after 5 days of G-CSF/SCF treatment and CD34-enriched BM cells collected 14 days later were superior targets for retroviral gene transfer. When a clinically approved supernatant infection protocol with low-titer vector preparations was used, monkeys had up to 5% of circulating cells containing the vector for up to a year after transplantation. This relatively high level of gene transfer was confirmed by Southern blot analysis. Engraftment after transplantation using primed BM cells was more rapid than that using steady-state bone marrow, and the fraction of BM cells saving the most primitive CD34+/CD38- or CD34+/CD38dim phenotype increased 3-fold. We conclude that cytokine priming with G-CSF/SCF may allow collection of increased numbers of primitive cells from both the PB and BM that have improved susceptibility to retroviral transduction, with many potential applications in hematopoietic stem cell-directed gene therapy.
Dunbar
C E
CE
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. dunbarc@gwgate.nhlbi.nih.gov
Seidel
N E
NE
Doren
S
S
Sellers
S
S
Cline
A P
AP
Metzger
M E
ME
Agricola
B A
BA
Donahue
R E
RE
Bodine
D M
DM
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D001853:Bone Marrow; D002460:Cell Line; D002478:Cells, Cultured; D018432:Drug Resistance, Multiple / Q000235:genetics; D005260:Female; D005822:Genetic Vectors; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells; D006801:Humans; D019868:Kanamycin Kinase; D008253:Macaca mulatta; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D009699:N-Glycosyl Hydrolases / Q000032:analysis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D012190:Retroviridae; D019089:Stem Cell Factor / Q000494:pharmacology*; D013234:Stem Cells; D014162:Transfection / Q000379:methods*; D014182:Transplantation, Autologous
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase; D009699:N-Glycosyl Hydrolases; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; C498156:Cd38 protein, rat; D051997:ADP-ribosyl Cyclase 1