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CRISPR-mediated insertion of a chimeric antigen receptor produces nonviral T cell products capable of inducing solid tumor regression

By Katherine Mueller, Nicole Piscopo, Matthew Forsberg, Louise Saraspe, Amritava Das, Brittany Russell, Madeline Smerchansky, Lei Shi, Adeela Ali, Cicera Lazzarotto, Shengdar Tsai, Christian M Capitini, Krishanu Saha

Posted 08 Aug 2021
bioRxiv DOI: 10.1101/2021.08.06.455489

Chimeric antigen receptor (CAR) T cells traditionally harbor viral vectors that encode the CAR transgene in the genome. However, viral vector manufacturing typically is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures. Here, CAR T cells were generated within nine days using recombinant SpCas9 protein and nucleic acids, without any viral vectors or animal components. In comparison to traditional retroviral CAR T cells, nonviral CAR T cells exhibit TRAC-targeted genomic integration of the CAR transgene, gene expression signatures associated with a memory phenotype, and low receptor signaling prior to infusion. Upon exposure to the GD2 target antigen, the nonviral anti-GD2 CAR cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing and persistence within a murine neuroblastoma xenograft model. This proof-of-principle study eliminating viral vectors and animal components during CAR gene transfer could enable more flexible and scalable manufacturing of clinically-relevant, high-quality CAR T cells to treat cancers, including solid tumors.

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