Abstract | 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.
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Authors | M L Lamana, J C Segovia, G Guenechea, J A Bueren |
Journal | The journal of gene medicine
(J Gene Med)
2001 Jan-Feb
Vol. 3
Issue 1
Pg. 32-41
ISSN: 1099-498X [Print] England |
PMID | 11269334
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
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Topics |
- Cell Line
- Gene Expression
- Gene Transfer Techniques
- Humans
- Phytohemagglutinins
(pharmacology)
- T-Lymphocytes
(metabolism)
- Transduction, Genetic
- Transgenes
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