RT Journal Article SR Electronic T1 Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.08.06.455489 DO 10.1101/2021.08.06.455489 A1 Katherine P. Mueller A1 Nicole J. Piscopo A1 Matthew H. Forsberg A1 Louise A. Saraspe A1 Amritava Das A1 Brittany Russell A1 Madeline Smerchansky A1 Lei Shi A1 Keerthana Shankar A1 Adeela Ali A1 Cicera R. Lazzarotto A1 Shengdar Q. Tsai A1 Christian M. Capitini A1 Krishanu Saha YR 2021 UL http://biorxiv.org/content/early/2021/12/29/2021.08.06.455489.abstract AB Background Chimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.Methods Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.Results In comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.Conclusions This proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.Competing Interest StatementConflicts of interest: K.P.M., N.J.P., M.H.F., L.A.S., A.D., C.M.C. and K.S. are inventors on a patent application related to this manuscript. C.M.C. receives honoraria for advisory board membership for Nektar Therapeutics and Novartis. No other conflicts of interest are reported.