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Abstract: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant ( TRAC ) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC -replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC -integrated CAR + T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells., Competing Interests: As part of a collaboration agreement between Charité Universitätsmedizin Berlin and Integrated DNA Technologies (IDT), IDT provided certain reagents (HDR enhancer v.2 and TRAC sgRNA used in some experiments) and performed GUIDE-seq analysis, HDR-enhancing small-molecule screen in Jurkat cells, and targeted sequencing of potential off-target sites. R.T., B.T., M.L.S., G.L.K., and A.M.J. are employees of IDT, which offers reagents for sale similar to some of the compounds described in the manuscript. Products and tools supplied by IDT are for research use only and not intended for diagnostic or therapeutic purposes. Purchaser and/or user are solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Lonza GmbH provided 96-well 4D-Nucleofector unit and some nucleofection reagents. A.W. and L. Akyüz are part-time employees of CheckImmune GmbH. A.R. and U.E.H. filed a patent application WO 2017211900A1 “Chimeric antigen receptor and CAR T cells that bind BCMA” related to the work with the BCMA-CAR disclosed in this paper. A.R. and U.E.H. have received research funding from Fate Therapeutics for work unrelated to the data generated in the manuscript., (© 2022 The Author(s).)