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 retroviral vectors constitutes an attractive approach for the correction of a number of genetic diseases. In this study we have conducted a systematic analysis 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 improved 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 more efficient than spinoculation infections performed on FN-uncoated plates. Under optimized manipulation conditions (three infection cycles of anti-CD3i/28i-stimulated T cells in FN-coated plates) the total number of NGFR+ T cells harvested after 7 days of incubation represented, on average, twice 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 obtained 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 transduction of human T cells, with obvious implications for the development of gene therapy protocols.
Gene Therapy Programme, CIEMAT/Fundación, Marcelimobotín, Madrid, Spain.
M L
ML
Lamana
J C
JC
Segovia
G
G
Guenechea
J A
JA
Bueren
D002460:Cell Line; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D010835:Phytohemagglutinins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic; D019076:Transgenes
D010835:Phytohemagglutinins