Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells
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...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-06-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050122000511 |
| _version_ | 1818009495332192256 |
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| author | Jonas Kath Weijie Du Alina Pruene Tobias Braun Bernice Thommandru Rolf Turk Morgan L. Sturgeon Gavin L. Kurgan 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 Renata Stripecke Petra Reinke Dimitrios L. Wagner |
| author_facet | Jonas Kath Weijie Du Alina Pruene Tobias Braun Bernice Thommandru Rolf Turk Morgan L. Sturgeon Gavin L. Kurgan 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 Renata Stripecke Petra Reinke Dimitrios L. Wagner |
| author_sort | Jonas Kath |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-14T05:43:35Z |
| format | Article |
| id | doaj.art-007e0c658baf459b90751503a142b022 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-04-14T05:43:35Z |
| publishDate | 2022-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-007e0c658baf459b90751503a142b0222022-12-22T02:09:22ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012022-06-0125311330Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cellsJonas Kath0Weijie Du1Alina Pruene2Tobias Braun3Bernice Thommandru4Rolf Turk5Morgan L. Sturgeon6Gavin L. Kurgan7Leila Amini8Maik Stein9Tatiana Zittel10Stefania Martini11Lennard Ostendorf12Andreas Wilhelm13Levent Akyüz14Armin Rehm15Uta E. Höpken16Axel Pruß17Annette Künkele18Ashley M. Jacobi19Hans-Dieter Volk20Michael Schmueck-Henneresse21Renata Stripecke22Petra Reinke23Dimitrios L. Wagner24BIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyRegenerative Immune Therapies Applied, Clinics of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Region, GermanyRegenerative Immune Therapies Applied, Clinics of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Region, GermanyIntegrated DNA Technologies, Inc., Coralville, IA 52241, USAIntegrated DNA Technologies, Inc., Coralville, IA 52241, USAIntegrated DNA Technologies, Inc., Coralville, IA 52241, USAIntegrated DNA Technologies, Inc., Coralville, IA 52241, USABIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyBerlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyBerlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, GermanyDepartment 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; Deutsches Rheuma-Forschungszentrum (DRFZ), A Leibniz Institute, Berlin, GermanyCheckImmune GmbH, 13353 Berlin, GermanyCheckImmune GmbH, 13353 Berlin, GermanyDepartment of Translational Tumorimmunology, Max-Delbrück-Center for Molecular Medicine (MDC), 13125 Berlin, GermanyDepartment of Microenvironmental Regulation in Autoimmunity and Cancer, Max-Delbrück-Center for Molecular Medicine (MDC), 13125 Berlin, GermanyInstitute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, GermanyDepartment 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; Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; German Cancer Consortium (DKTK), 10117 Berlin, GermanyIntegrated DNA Technologies, Inc., Coralville, IA 52241, USABIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, Germany; Institute 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), Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyRegenerative Immune Therapies Applied, Clinics of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany; German Center for Infection Research (DZIF), Hannover-Braunschweig Region, Germany; Clinic I for Internal Medicine, Cancer Center Cologne Essen, University Hospital Cologne, Cologne, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, GermanyBIH Center for Regenerative Therapies (BCRT), Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; Berlin Center for Advanced Therapies (BeCAT), Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, Germany; Institute of Transfusion Medicine, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin, Germany; Institute 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), Augustenburger Platz 1, 13353 Berlin, Germany; Corresponding author Dimitrios Laurin Wagner, MD, PhD, Berlin Center for Advanced Therapies (BeCAT) BIH Center for Regenerative Therapies (BCRT) Charité Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health (BIH), Augustenburger Platz 1, 13353 Berlin, Germany.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.http://www.sciencedirect.com/science/article/pii/S2329050122000511gene editingCRISPR-Cas9chimeric antigen receptornon-viral cell manufacturingCAR T cellsTRAC |
| spellingShingle | Jonas Kath Weijie Du Alina Pruene Tobias Braun Bernice Thommandru Rolf Turk Morgan L. Sturgeon Gavin L. Kurgan 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 Renata Stripecke Petra Reinke Dimitrios L. Wagner Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells Molecular Therapy: Methods & Clinical Development gene editing CRISPR-Cas9 chimeric antigen receptor non-viral cell manufacturing CAR T cells TRAC |
| title | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| title_full | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| title_fullStr | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| title_full_unstemmed | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| title_short | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| title_sort | pharmacological interventions enhance virus free generation of trac replaced car t cells |
| topic | gene editing CRISPR-Cas9 chimeric antigen receptor non-viral cell manufacturing CAR T cells TRAC |
| url | http://www.sciencedirect.com/science/article/pii/S2329050122000511 |
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