PiggyBac transposon and transposase delivered in trans , in cis , and as mRNA for engineering CAR-T cells: a multi-objective optimization
Year: 2026
Type: preprint
Abstract: 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. Significance statement This study compared the efficiency, viability, and transgene copy number of CAR-T cells made using piggyBac transposon system delivered in three different formats: in trans as two plasmids (CONV), in cis as one plasmid (ONE), and transposase as an mRNA plus a transposon plasmid (MRNA). All three formats are being used in different clinical trials worldwide. While studies comparing ONE against CONV and MRNA against CONV exist and each demonstrates better results for their format against CONV, no three-way comparison study exists.
Language: English
Topic: CAR-T cell therapy research
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7222980896