Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells

Background The transduction of human peripheral blood T cells with retrovir al vectors constitutes an attractive approach for the correction of a numbe r of genetic diseases. In this study we have conducted a systematic analysi s of the relevance of a large number of parameters currently considered to affect the transduction of, and transgene expression in, human T cells. Methods Retroviral vectors encoding the human nerve growth factor receptor (NGFR) were used for transducing human T cells from normal volunteers. The proportion of T cells that expressed the marker transgene was determined by flow cytometry using anti-NGFR antibodies. Results Spinoculation and static fibronectin (FN)-assisted infections impro ved to a similar extent the transduction efficiency of PHA/IL-2 stimulated T cells, when compared with samples subjected to standard static infections . When immobilized anti-CD3 (anti-CD3i) or anti-CD3i/28i-stimulated T cells were considered, static infections in FN-coated plates were reproducibly m ore efficient than spinoculation infections performed on FN-uncoated plates . Under optimized manipulation conditions (three infection cycles of anti-C D3i/28i-stimulated T cells in FN-coated plates) the total number of NGFR(+) T cells harvested after 7 days of incubation represented, on average, twic e the total number of T cells seeded at Day 0, and up to 95% of the human T cells efficiently expressed the marker transgene. Similar results were obt ained regardless of whether samples were manipulated in medium supplemented with fetal bovine serum or with heat-inactivated autologous serum. Conclusions Our study offers new experimental conditions for the transducti on of human T cells, with obvious implications for the development of gene therapy protocols. Copyright (C) 2000 John Wiley & Sons, Ltd.