Efforts to utilize genetically-modified T lymphocytes for adoptive T cell therapy have been limited by the relatively inefficient transduction efficiency obtained using retroviral vectors. In preparation for utilizing the SIV/macaque model for in vivo studies of adoptive T cell therapy for AIDS, we have analyzed conditions affecting retroviral transduction of rhesus T cells. To more accurately assess transduction efficiency, we utilized retroviral vectors encoding the murine CD2 molecule, thereby allowing precise determination of transduced cells by flow cytometry. Transduction efficiency of rhesus lymphocytes using retroviral vector pseudotyped with the gibbon ape leukemia virus envelope was 5 to 10-fold greater as compared with vector containing the murine amphotropic envelope. The introduction of centrifugation and phosphate depletion steps to our standard transduction protocol further enhanced transduction efficiency in NIH-3T3 or COS cells. However, although we can consistently enhance transduction efficiency upon phosphate starvation of various fibroblast cell lines, this step alone was not always effective for rhesus lymphocytes. In fact, transduction efficiency of rhesus lymphocytes was significantly enhanced solely by centrifugation. Results from these experiments will be used to design a protocol for the large scale transduction and expansion of genetically-modified rhesus T cells for use in in vivo studies in macaques.
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