Abstract
Objective To establish a non-viral gene transduction method of T cells, providing a gene modification strategy for CAR-T cell immunotherapy. Methods CD19CAR cassette (1 470 bp) was inserted into XbaⅠ and EcoRⅠ sits of PiggyBac transposon vector PB513B-1, recombinant vector was named as pb19CAR. Dual plasmids (pb19CAR and transposase) were transducted into primary T cells using electroporation (1×20 ms 830 V/840 V/850 V) and nucleofection (U014/T023). The transduction efficiency and cell viability were compared and optimized. T cells were activated by plates coated with human CD19 (5 μg/ml) protein or CD3/CD28 mAb (5 μg/ml), and then amplified by interleukin (IL)-2 (500 U/ml) or mixed cytokines IL-7 (10 ng/ml)/IL-15 (10 ng/ml)/IL-21 (20 ng/ml) CD19CAR expression and phenotype of T cells were compared after activation and amplification. Results PiggyBac transposon vector (pb19CAR) was confirmed by XbaⅠ and EcoRⅠ enzyme digestion and gene sequencing analysis. Based on the flow cytometry and cell vitality detection, the transduction efficiency and cell viability of primary T cells by electroporation were higher than that of nucleofaction (51.0±2.3)% vs. (36.5±1.8)%, (75.0±3.8)% vs. (60.0±3.0)% (P=0.000). After amplification of 12 days post-electroporation, CD19CAR expression on T cells in CD19 protein activation group was significantly higher than that in CD3/CD28 mAb activation group (70.0±3.5)% vs. (54.0±2.7)% (P=0.000). The cell phenotypic analysis showed that the proportion of Tscm/Tscm-like in mixed cytokine group was a higher than that in IL-2 group (25.00±1.25)% vs. (12.20±0.60)%, and so is the Tcm cells (10.20±0.51)% vs. (3.00±0.15)% (P=0.000). Conclusion The method of pb19CAR/transposase electroporation reveals higher transduction efficiency and cell viability. The activation by target antigen CD19 protein contributes to the preferential amplification of CAR-T cells. The mixed cytokine group (IL-7/IL-15/IL-21) supports the growth of CAR-T cells with central memory and stem memory phenotypes. Key words: PiggyBac transposon; Chimeric antigen receptor; Electroporation
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