Deedit, N.; Suppipat, K.; Tawinwung, S.; Nakazawa, Y.; Hirankarn, N.; Chinsuwan, T.

Chimeric antigen receptor (CAR)-T cell therapy has become a revolutionary strategy in cancer immunotherapy. However, conventional manufacturing strategies, which rely on viral vectors to deliver genetic material to host genomes, are hindered by many obstacles that have limited their widespread clinical application. This study aims to optimize the delivery of piggyBac transposon for simple and cost-effective manufacturing of CAR-T cells. We tested three different piggyBac delivery strategies: (i) conventional two-plasmid system (CONV), (ii) helper and donor one-plasmid system (ONE), and (iii) mRNA transposase in combination with a DNA donor plasmid (MRNA). Each method was evaluated for transfection efficiency, cell viability, and transgene copy number. The results demonstrated that, without enrichment, ONE approach generated CAR-T cells with the highest gene delivery efficiency at the cost of lower viability and higher transgene copy number, while transfection using MRNA offered a reduction in transgene copy numbers at the cost of lower efficiency. Interestingly, with enrichment by autologous feeder, the shortcoming in viability was completely overcome while retaining higher efficiency and higher transgene copy number. The result of this study lays the groundwork for a more efficient and safer piggyBac-engineered CAR T cell manufacturing process, and it should facilitate the decision in method selection for further optimization and clinical scale-up.