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Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells

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Item Type:Article
Title:Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells
Creators Name:Kath, J. and Du, W. and Pruene, A. and Braun, T. and Thommandru, B. and Turk, R. and Sturgeon, M.L. and Kurgan, G.L. and Amini, L. and Stein, M. and Zittel, T. and Martini, S. and Ostendorf, L. and Wilhelm, A. and Akyüz, L. and Rehm, A. and Hoepken, U.E. and Pruß, A. and Künkele, A. and Jacobi, A.M. and Volk, H.D. and Schmueck-Henneresse, M. and Stripecke, R. and Reinke, P. and Wagner, D.L.
Abstract: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 knock-in rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knock-in 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.
Keywords:Gene Editing, CRISPR-Cas9, Chimeric Antigen Receptor, Non-Viral Cell Manufacturing, CAR T Cells, TRAC, Knock-in, Adoptive T Cell Therapy, HDR
Source:Molecular Therapy - Methods and Clinical Development
Publisher:Cell Press
Page Range:311-330
Date:9 June 2022
Official Publication:https://doi.org/10.1016/j.omtm.2022.03.018
PubMed:View item in PubMed
Related to:
https://edoc.mdc-berlin.de/20007/Preprint version

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