Prolonged high-level detection of retrovirally marked hematopoietic cells in nonhuman primates after transduction of CD34+ progenitors using clinically feasible methods.
Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell (HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells (STR), or the fibronectin fragment CH-296 (FN), and various cytokines such as flt3 ligand (FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and clinically feasible peripheral blood (PB) progenitor cell transduction methods. First, we compared transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor (SCF), and FLT (our best cytokine combination in prior studies) with a combination of megakaryocyte growth and development factor (MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10-15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.
Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
T
T
Wu
H J
HJ
Kim
S E
SE
Sellers
K E
KE
Meade
B A
BA
Agricola
M E
ME
Metzger
I
I
Kato
R E
RE
Donahue
C E
CE
Dunbar
J F
JF
Tisdale
D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics*; D015139:Blotting, Southern; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005353:Fibronectins / Q000378:metabolism; D018014:Gene Transfer Techniques; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008253:Macaca mulatta; D008565:Membrane Proteins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D019089:Stem Cell Factor / Q000494:pharmacology; D017154:Stromal Cells / Q000378:metabolism; D013926:Thrombopoietin / Q000494:pharmacology
D018952:Antigens, CD34; D005353:Fibronectins; D007377:Interleukin-3; D015850:Interleukin-6; D008565:Membrane Proteins; D019089:Stem Cell Factor; C084881:flt3 ligand protein; D016179:Granulocyte Colony-Stimulating Factor; D013926:Thrombopoietin