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Blood, 15 May 2004, Vol. 103, No. 10, pp. 3710-3716.
Prepublished online as a Blood First Edition Paper on January 22, 2004; DOI 10.1182/blood-2003-07-2414.
Submitted July 17, 2003
Accepted December 18, 2003
Efficient Lentiviral Gene Transfer To Canine Repopulating Cells Using An Overnight Transduction Protocol
Peter A Horn, Kirsten A Keyser, Laura J Peterson, Tobias Neff, Bobbie M Thomasson, Jesse Thompson, and Hans-Peter Kiem*
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
* Corresponding author; email: hkiem{at}fhcrc.org.
The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest due 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 SCF and G-CSF primed marrow or mobilized peripheral blood in a competitive repopulation assay in three dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B-cells, T-cells, granulocytes, red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels 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 where the maintenance of stem cells in culture is a major limitation.
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