Retrovirus-mediated gene transfer into T cells:: 95% transduction efficiency without further in vitro selection

This study was designed to retrovirally transduce T cells by a protocol that would be simple, short, cost effective, applicable for clinical use, and efficient enough to avoid further selection of transduced T cells. Because retrovirally mediated infection is depending on the cell cycle, we first optimized the conditions for activating T cells in the presence of immobilized CD3 monoclonal antibodies and recombinant interleukin 2. Cell cycle analysis indicated that CD8(+) and total T cells reach a maximum of cycling within 4 days whereas CD4(+) T cells attain their maximum of cycling only by day 6. Taking into account these data, CD4(+), CD8(+) and total T cells mere preactivated for 5 and 3 days, respectively, and then infected for 24 hr with supernatant containing retrovirus pseudotyped with gibbon-ape leukemia virus envelope, using a cell centrifugation protocol. Results show that approximately 95% of CD4(+), CD8(+), and total T cells can be transduced, this transduction efficiency being significantly higher than that obtained,vith amphotropic retrovirus vectors. Furthermore, under permanent growth stimulation, transduced T cells can be expanded similar to 1000-fold in 4 weeks of culture with maintenance of transgene expression. Ho cr ever, Immunoscope analysis revealed alterations of T cell repertoire diversity after 2-3 weeks in culture that was not due to retroviral transduction per se. Overall, these data provide evidence that T cells can be transduced at levels that may alleviate the need for both further selection of transduced cells and in vitro expansion, thereby preserving the repertoire diversity of the transduced T cells to be reinfused.