Single‐step, multiple retroviral transduction of human T cells

Abstract

Retroviral transduction of human peripheral blood T cells has considerable potential in the development of gene therapy strategies for immunological disorders. New vectors and experimental procedures have been developed for efficient transduction of several genes into human T cells. Bicistronic retroviral vectors encoding distinct cell markers were used for the simultaneous multiple transduction of a human T-cell line (MT-2), as well as of human peripheral blood T cells from normal donors. Transduction efficiencies were evaluated by flow cytometry and double- and triple-transduced cells were isolated by fluorescence cell sorting. Four new bicistronic retroviral vectors were developed that express different gene markers under the control of the internal ribosome entry site (IRES) of the encephalomyocarditis virus. These markers are, respectively, enhanced green fluorescent protein (EGFP), beta-galactosidase, and truncated versions of human nerve growth factor receptor (Delta NGFR) and human growth hormone receptor (Delta GHR). A single 1 h spinoculation infection, performed in the presence of polybrene and using transiently produced amphotropic retroviral particles, was sufficient to obtain transduction efficiencies consistently greater than 50% on human peripheral blood T lymphocytes which had been previously stimulated for 3 days with immobilized anti-CD3. The transient production of viral particles encoding EGFP, Delta NGFR, and Delta GHR markers in the same viral supernatant has allowed up to three different genes to be introduced simultaneously into human T cells. This study describes new experimental conditions for efficient single-step multiple transduction of human primary T lymphocytes. The procedure could be of interest for the development of gene therapy approaches.