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

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.
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.
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.
Our study offers new experimental conditions for the transduction of human T cells, with obvious implications for the development of gene therapy protocols.
AuthorsM L Lamana, J C Segovia, G Guenechea, J A Bueren
JournalThe journal of gene medicine (J Gene Med) 2001 Jan-Feb Vol. 3 Issue 1 Pg. 32-41 ISSN: 1099-498X [Print] England
PMID11269334 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Phytohemagglutinins
  • Cell Line
  • Gene Expression
  • Gene Transfer Techniques
  • Humans
  • Phytohemagglutinins (pharmacology)
  • T-Lymphocytes (metabolism)
  • Transduction, Genetic
  • Transgenes

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