Optimization of retrovirus mediated gene transfer into primary human t lymphocytes

Infusion of suicide gene transduced donor T cells after allogeneic hematopoietic stem cell transplantation could be an efficient method to profit from their initial graft-versus-leukemia effects and still retain the possibility to abrogate graft-versus-host disease if it occurs by prodrug administration. Clinical application of suicide gene transduced T cells requires a high transduction efficiency and a rapid and efficient method to select the transduced T cells. To establish optimized transduction conditions for human T cells, we analysed the effects of different transduction procedures on transduction efficiencies. The use of the SFCMM retroviral vector that contains both the Herpes Simplex Virus thymidine kinase gene and the gene encoding a truncated version of the human low-affinity nerve growth factor receptor (LNGF-R) allows the accurate measurement of retroviral transduction efficiencies at the level of protein expression using anti-LNGF-R antibodies and FACS analysis. Culturing PHA-stimulatfil human T cells in phosphate-free medium one day prior to initiuiion of retroviral transduction (in order to upregulate retrovirusreceptor expression at the cell surface of the T cells) had no effect on transduction efficiency. Transduction efficiencies were significantly higher at a transduction temperature of 37 C than at 32 C. Transduction 1-3 days after mitogenic stimulation resulted in significantly higher transduction efficiencies than transduction 4-6 days after mitogenic stimulation despite the fact that T cell proliferation was maximal 3-6 days post-mitogenic stimulation. The percentage LNGF-R-positive T cells reached a plateau level 2 to 3 days after termination of the 48 hours transduction and remained stable at that level thereafter. Using the optimized co-cultivation-based transduction protocol, transduction efficiencies of a panel of cryopreserved human peripheral blood T cells ranged from 25 to 38%. At present, we are optimizing a supernatant-based transduction protocol of human T cells.