Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical...

Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.