Optimization of the PiggyBac transposon system for the sustained genetic modification of human T lymphocytes

Nakazawa Y, Huye LE, Dotti G, Foster AE, Vera JF, Manuri PR, June CH, Rooney CM, Wilson MH.
Source: J Immunother
Publication Date: (2009)
Issue: 32(8): 826-36
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human peripheral blood unstim.
Species: human
Tissue Origin: blood
T cell, human stim.
Species: human
Tissue Origin: blood
Nucleofector® I/II/2b
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.
Optimal implementation of adoptive T-cell therapy for cancer will likely require multiple and maintained genetic modifications of the infused T cells and their progeny so that they home to tumor sites and recognize tumor cells, overcome tumor immune evasion strategies, and remain safe. Retroviral vectors readily transduce T cells and integrate into the host cell genome, but have a limited capacity for multigene insertion and cotransduction and are prohibitively expensive to produce at clinical grade. Genetic modification of T cells using transposons as integrating plasmids is an attractive alternative because of the increased simplicity and cost of production. Of available transposons, piggyBac has the higher transposase activity and larger cargo capacity, and we now evaluate piggyBac for potential adoptive therapies with primary T cells. PiggyBac transposons mediated stable gene expression in approximately 20% of primary T cells without selection. Treatment and maintenance of T cells with interleukin-15 increased stable transgene expression up to approximately 40% and expression was sustained through multiple logs of expansion for over 9 weeks in culture. We demonstrate simultaneous integration of 2 independent transposons in 20% of T cells, a frequency that could be increased to over 85% by selection of a transgenic surface marker (truncated CD19). PiggyBac could also deliver transposons of up to 13 kb with 10,000-fold expansion of transduced T cells in culture and finally we demonstrate delivery of a functional suicide gene (iCasp9). PiggyBac transposons may thus be used to express the multiple integrated transgenes that will likely be necessary for the broader success of T-cell therapy.