CRISPR/Cas9 介导的胞内分子 SHP-1 敲除增强靶向 CD133 的 CAR-T 细胞的体外肿瘤杀伤能力

CAR T cell therapy has been successfully used in the treatment of hematological malignancies, and the strategy that deletion of inhibitory receptor on the CAR T cell surface, such as PD-1, greatly enhance the antitumor effects.

Here, we describe a one-step electroporation for the co-transfection of Cas9:sgRNA and CAR plasmids on primary T cells to demonstrate the effect of SHP-1 deletion in CAR T cells. By using PiggyBac Transposase system, we can achieve more than 90% of T cells express CAR gene and nearly 60% SHP-1 knockout efficiency in T cells.

We show that knockout of SHP-1 in CD133 CAR T cells resulted in significantly improve the cytolysis effect on CD133 positive glioma cell lines.

We further demonstrate that the enhanced antitumor efficacy of SHP-1 deletion is due to the increased release of TNF- , IL-2 and IFN- in vitro.

Finally, we evaluated the biosafety of Cas9 genome editing and did not find any insertions of Cas9 and obvious editing in off-target sites in CAR T cells. These data provide an approach for achieving both intracellular inhibitory molecule, SHP-1 deletion and CD133 CAR gene over-expression in human T cells. And SHP-1 could be a new potential target for adoptive CAR T cells immunotherapy.