1005. Expression of Porcine [alpha](1-3)galactosyltransferase in Non-Human Primate Hematopoietic Progenitors Following Simian Immunodeficiency Virus Mediated Gene Transfer

• Published in: Molecular therapy Vol. 13; no. S1; p. S387
• Main Authors: Benatuil, Lorenzo, Iacomini, John
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Limited 01.05.2006
• Subjects: Adenoviruses; Bone marrow; Cancer; Efficiency; Fibroblasts; Gene expression; Gene therapy; Genetic engineering; Genomes; Immune system; Infections; Kinases; Medical research; Monkeys & apes; Proteins; Viruses; United States > US; Chicago Illinois
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2006.08.1099

Background.Preformed anti-donor antibodies are believed to play a significant role in chronic allograft rejection. We have shown that genetic engineering of autologous bone marrow stem cells, can be used to establish B cell tolerance in mice. Here we examined the ability of simian immunodeficiency virus vector (SIV) to transduce hematopoietic CD34+ progenitor cells from non-human primates and achieve expression of αGT in these cells.Methods.CD34+ progenitors were purified from bone marrow (BM) of Rhesus macaques. After 36 h of stimulation with SCF, Flt3-L, and hTPO, cells were exposed to amphotropic SIV vector carrying the gene encoding the αGT enzyme (SIV-αGT), or enhanced green fluorescent protein (SIV-GFP) to achieve different multiplicities of infection (MOIs) for 24 h. After transduction, CD34+ cells were suspended in methylcellulose medium to perform Colony-forming unit (CFU) assays. Cell surface staining and flow cytometry were used to analyze αGal epitopes or GFP expression on the surface of transduced CD34+ cells as well as in CFUs.Results.CD34+ progenitors were readily transduced with amphotropic enveloped SIV vector carrying the gene encoding either αGT or GFP. Infection of CD34+ cells with SIV-αGT using MOI of 1, 3, 5 or 10 resulted in 35.6 ± 2.9%, 50.5 ± 5.8%, 55.6 ± 5.8% and 58.8 ± 5.4% of cells expressing αGal, respectively. Infection of CD34+ cells with SIV-GFP using an MOI of 1, 3, 5 or 10 resulted in 47.9 ± 2.4%, 55.4 ± 6.3%, 67.4 ± 2.4% and 76.0 ± 6.6% of cells expressing GFP, respectively. MOIs greater than 5 did not augment the transduction efficiency and MOIs greater than 10 decreased viability. Infection with SIV-αGT for more than 24 h did not improve the transduction efficiency. In all cases, mock-transduced cells remained negative for αGal or GFP expression. There was no significant difference in the numbers, frequency or size of CFUs observed following infection with both vector or mock-transduced cells. The transduction efficiency of CD34+ cells with SIV-αGT was similar to the frequency of CFUs expressing αGal.Conclusion.These data demonstrate that rhesus macaque CD34+ BM cells can be efficiently transduced with amphotropic enveloped SIV vector carrying the gene encoding αGT. The transduction efficiency was similar to that observed using viruses carrying the marker gene GFP. Using the transduction conditions described, the majority of transduced cells expressing αGal or GFP remained CD34+. Knowing that the SIV vectors represent a reliable system to evaluate gene transfer in a non-human primate, it may be possible to use SIV-αGT vector and genetic engineering of BM in primates in order to establish expression of and tolerance to αGal. This approach could be an important way to avoid preformed anti1-donor antibody response in allotransplantation.