An, J.^#, Zhang, C.P.^#, Qiu, H.Y.^#, Zhang, H.X.^#, Chen, Q.B., Zhang, Y.M., Lei, X.L., Zhang, C.X., Yin, H.*, and Zhang, Y.* (2023). Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS-STING pathway. Nat Biomed Eng 10.1038/s41551-023-01073-7.（殷昊、张楹）

Abstract

Viral delivery of DNA for the targeted reprogramming of human T cells can lead to random genomic integration, and electroporation is inefficient and can be toxic. Here we show that electroporation-induced toxicity in primary human T cells is mediated by the cytosolic pathway cGAS-STING (cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase-stimulator of interferon genes). We also show that an isotonic buffer, identified by screening electroporation conditions, that reduces cGAS-STING surveillance allowed for the production of chimaeric antigen receptor (CAR) T cells with up to 20-fold higher CAR T cell numbers than standard electroporation and with higher antitumour activity in vivo than lentivirally generated CAR T cells. The osmotic pressure of the electroporation buffer dampened cGAS-DNA interactions, affecting the production of the STING activator 2'3'-cGAMP. The buffer also led to superior efficiencies in the transfection of therapeutically relevant primary T cells and human haematopoietic stem cells. Our findings may facilitate the optimization of electroporation-mediated DNA delivery for the production of genome-engineered T cells.

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https://www.nature.com/articles/s41551-023-01073-7