@article {Muellere004446, author = {Katherine P Mueller and Nicole J Piscopo and Matthew H Forsberg and Louise A Saraspe and Amritava Das and Brittany Russell and Madeline Smerchansky and Dan Cappabianca and Lei Shi and Keerthana Shankar and Lauren Sarko and Namita Khajanchi and Nina La Vonne Denne and Apoorva Ramamurthy and Adeela Ali and Cicera R Lazzarotto and Shengdar Q Tsai and Christian M Capitini and Krishanu Saha}, title = {Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression}, volume = {10}, number = {9}, elocation-id = {e004446}, year = {2022}, doi = {10.1136/jitc-2021-004446}, publisher = {BMJ Specialist Journals}, abstract = {Background Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.Methods Anti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. 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, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG{\texttrademark} xenograft neuroblastoma model.Results In comparison to 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-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.Conclusions This study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.Data are available on reasonable request.}, URL = {https://jitc.bmj.com/content/10/9/e004446}, eprint = {https://jitc.bmj.com/content/10/9/e004446.full.pdf}, journal = {Journal for ImmunoTherapy of Cancer} }