Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.
The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.
Horn
Peter A
PA
Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA.
Keyser
Kirsten A
KA
Peterson
Laura J
LJ
Neff
Tobias
T
Thomasson
Bobbie M
BM
Thompson
Jesse
J
Kiem
Hans-Peter
HP
D000818:Animals; D018952:Antigens, CD34; D001773:Blood Cells; D001854:Bone Marrow Cells; D018929:Cell Culture Techniques / Q000379:methods; D019070:Cell Lineage; D004285:Dogs; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D019650:Hematopoietic Stem Cell Mobilization; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics; D023421:Models, Animal; D019089:Stem Cell Factor / Q000494:pharmacology; D014161:Transduction, Genetic / Q000379:methods* / Q000592:standards
D018952:Antigens, CD34; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor