RT Journal Article SR Electronic T1 Fast, efficient and virus-free generation of TRAC-replaced CAR T cells JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.02.14.431017 DO 10.1101/2021.02.14.431017 A1 Jonas Kath A1 Weijie Du A1 Bernice Thommandru A1 Rolf Turk A1 Leila Amini A1 Maik Stein A1 Tatiana Zittel A1 Stefania Martini A1 Lennard Ostendorf A1 Andreas Wilhelm A1 Levent Akyüz A1 Armin Rehm A1 Uta E. Höpken A1 Axel Pruß A1 Annette Künkele A1 Ashley M. Jacobi A1 Hans-Dieter Volk A1 Michael Schmueck-Henneresse A1 Petra Reinke A1 Dimitrios L. Wagner YR 2021 UL http://biorxiv.org/content/early/2021/02/14/2021.02.14.431017.abstract AB Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.Competing Interest StatementAs part of a collaboration agreement between Charite Universitatsmedizin Berlin and Integrated DNA Technologies (IDT), IDT provided certain reagents (HDR enhancer V2, TRAC sgRNA used in some experiments) and performed GUIDE-seq analysis. R.T., B.T. and A.J. are employees of Integrated DNA Technologies, which offers reagents for sale similar to some of the compounds described in the manuscript. Lonza GmbH provided 96-Well-4D-Nucleofector unit and some nucleofection reagents. A.W. and L.Ak. are part-time employees of Check-Immune 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.