Low Temperature Electroporation Combined with Sleeping beauty Transposon System Enhances CAR-T Modification Efficiency
ZHAO Junhong, ZHENG Liqun, FU Yajuan, CAI Shaoli*
CAR-T cells (chimeric antigen receptor redirected T cells) have shown the beneficial effects in patients with B cell malignancies in clinical trials. The CAR-T cells were generated from patients’ blood using the PBMC and introduced in CAR constructs, then the genetically modified T cells will gain the anti-tumor ability and kill tumor cells. DNA electroporation could be more convenient and cost-effective, but this approach required to co-cultured CAR-T cells with artificial antigen-presenting cells for several rounds, which reduces the yield and efficiency. Thereby, a convenient, efficient and low-cost procedure for CAR-T cell production is urgently needed. In this study, we optimized a DNA electroporation procedure for making CAR-T cells by fresh PBMC, which includes using very high voltage to introduce the Sleeping beauty transposon/transposase system in the low temperature condition to directly express a CD19-specific CAR and produce modified CAR-T cells in the presence of specific cytokines. We examined the CAR gene integration and expression in the T cells derived from PBMC after proliferation. Results showed that the cells display cyto-activity and cytotoxicity against the cancer cells in the co-culturing system, and the lentivirus transduced CAR-T cells have a comparable efficiency. In conclusion, we developed a systemic approach for producing CAR-T cells based on the Sleeping beauty transposon/transposase system and electroporation, which essentially provides an alternative method for CAR-T-based therapies in clinical setting.
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