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

Mueller, Katherine P.; Piscopo, Nicole J.; Forsberg, Matthew H.; Saraspe, Louise A.; Das, Amritava; Russell, Brittany; Smerchansky, Madeline; Shi, Lei; Ali, Adeela; Lazzarotto, Cicera R.; Tsai, Shengdar Q.; Capitini, Christian M.; Saha, Krishanu

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.