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

Authors

  • J. Kath
  • W. Du
  • A. Pruene
  • T. Braun
  • B. Thommandru
  • R. Turk
  • M.L. Sturgeon
  • G.L. Kurgan
  • L. Amini
  • M. Stein
  • T. Zittel
  • S. Martini
  • L. Ostendorf
  • A. Wilhelm
  • L. Akyüz
  • A. Rehm
  • U.E. Hoepken
  • A. Pruß
  • A. Künkele
  • A.M. Jacobi
  • H.D. Volk
  • M. Schmueck-Henneresse
  • R. Stripecke
  • P. Reinke
  • D.L. Wagner

Journal

  • Molecular Therapy - Methods and Clinical Development

Citation

  • Mol Ther Methods Clin Dev 25: 311-330

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.


DOI

doi:10.1016/j.omtm.2022.03.018