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Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

Jonas Kath, Weijie Du, Bernice Thommandru, Rolf Turk, View ORCID ProfileLeila Amini, Maik Stein, Tatiana Zittel, Stefania Martini, View ORCID ProfileLennard Ostendorf, Andreas Wilhelm, Levent Akyüz, Armin Rehm, Uta E. Höpken, Axel Pruß, View ORCID ProfileAnnette Künkele, View ORCID ProfileAshley M. Jacobi, View ORCID ProfileHans-Dieter Volk, View ORCID ProfileMichael Schmueck-Henneresse, View ORCID ProfilePetra Reinke, View ORCID ProfileDimitrios L. Wagner
doi: https://doi.org/10.1101/2021.02.14.431017
Jonas Kath
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Weijie Du
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
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Bernice Thommandru
3Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA
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Rolf Turk
3Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA
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Leila Amini
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Maik Stein
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Tatiana Zittel
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Stefania Martini
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
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Lennard Ostendorf
4Department of Nephrology and Intensive Care Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
5Deutsches Rheuma-Forschungszentrum (DRFZ), A Leibniz Institute, Berlin, Germany
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Andreas Wilhelm
6CheckImmune GmbH, 13353 Berlin, Germany
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Levent Akyüz
6CheckImmune GmbH, 13353 Berlin, Germany
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Armin Rehm
7Department of Translational Tumorimmunology, Max-Delbrück-Center for Molecular Medicine (MDC), 13125 Berlin, Germany
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Uta E. Höpken
8Department of Microenvironmental Regulation in Autoimmunity and Cancer, Max-Delbrück-Center for Molecular Medicine (MDC), 13125 Berlin, Germany
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Axel Pruß
9Institute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Annette Künkele
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
10Department of Pediatric Oncology and Hematology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Ashley M. Jacobi
3Integrated DNA Technologies, Inc., Coralville, IA, 52241, USA
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Hans-Dieter Volk
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
11Institute of Medical Immunology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Michael Schmueck-Henneresse
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
11Institute of Medical Immunology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Petra Reinke
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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Dimitrios L. Wagner
1BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH), Berlin, Germany
2Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
9Institute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
11Institute of Medical Immunology, Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany
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  • For correspondence: dimitrios-l.wagner@charite.de
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Abstract

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 Statement

As 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted February 14, 2021.
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Fast, efficient and virus-free generation of TRAC-replaced CAR T cells
Jonas Kath, Weijie Du, Bernice Thommandru, Rolf Turk, Leila Amini, Maik Stein, Tatiana Zittel, Stefania Martini, Lennard Ostendorf, Andreas Wilhelm, Levent Akyüz, Armin Rehm, Uta E. Höpken, Axel Pruß, Annette Künkele, Ashley M. Jacobi, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Dimitrios L. Wagner
bioRxiv 2021.02.14.431017; doi: https://doi.org/10.1101/2021.02.14.431017
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Fast, efficient and virus-free generation of TRAC-replaced CAR T cells
Jonas Kath, Weijie Du, Bernice Thommandru, Rolf Turk, Leila Amini, Maik Stein, Tatiana Zittel, Stefania Martini, Lennard Ostendorf, Andreas Wilhelm, Levent Akyüz, Armin Rehm, Uta E. Höpken, Axel Pruß, Annette Künkele, Ashley M. Jacobi, Hans-Dieter Volk, Michael Schmueck-Henneresse, Petra Reinke, Dimitrios L. Wagner
bioRxiv 2021.02.14.431017; doi: https://doi.org/10.1101/2021.02.14.431017

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