Five million PBMCs were mixed with PB-transposon plasmids and/or PB-transposase plasmids and then transfected using the Nucleofector Device (program U-014) in combination with the Human T-cell Nucleofector Kit. After transfection, PBMCs were stimulated and after that restimulated weekly. To obtain large numbers of transduced cells with a high frequency of transgene expression, authors cotransfected IL-15-cultured PBMCs with 2 transposons expressing GFP and DCD19, respectively. On day 8, transduced cells were selected based on their expression of CD19. On day 22, over 80% of cells expressed both transgenes, with the remaining cells expressing only DCD19.
To overcome toxicity of nucleofection, authors optimized culture conditions to compensate for the toxicity. Stably transduced T cells could be expanded by 4 to 5 logs to over 10e10 transduced T cells in 4 to 5 weeks and to 10e12 in 6 to 7 weeks. These numbers would be more than adequate for any current T cell transfer clinical study.