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Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells
Author(s) -
Lamana María L.,
Segovia José C.,
Guenechea Guillermo,
Bueren Juan A.
Publication year - 2001
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/1521-2254(2000)9999:9999<::aid-jgm153>3.0.co;2-r
Subject(s) - transduction (biophysics) , transgene , flow cytometry , biology , microbiology and biotechnology , gene , genetics , biochemistry
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. Copyright © 2000 John Wiley & Sons, Ltd.