Kath, Jonas ORCID: 0000-0001-7721-7978, Du, Weijie ORCID: 0000-0001-5924-8631, Pruene, Alina, Braun, Tobias ORCID: 0000-0002-3910-2886, Thommandru, Bernice, Turk, Rolf ORCID: 0000-0003-1283-9594, Sturgeon, Morgan L., Kurgan, Gavin L., Amini, Leila ORCID: 0000-0003-4855-5905, Stein, Maik, Zittel, Tatiana, Martini, Stefania, Ostendorf, Lennard, Wilhelm, Andreas, Akyuez, Levent, Rehm, Armin, Hoepken, Uta E., Pruss, Axel, Kunkele, Annette ORCID: 0000-0002-8406-5412, Jacobi, Ashley M., Volk, Hans-Dieter ORCID: 0000-0002-7743-6668, Schmueck-Henneresse, Michael ORCID: 0000-0001-5964-9179, Stripecke, Renata, Reinke, Petra and Wagner, Dimitrios L. (2022). Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Mol.Ther.-Methods Clin. Dev., 25. S. 311 - 331. CAMBRIDGE: CELL PRESS. ISSN 2329-0501

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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 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.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Kath, JonasUNSPECIFIEDorcid.org/0000-0001-7721-7978UNSPECIFIED
Du, WeijieUNSPECIFIEDorcid.org/0000-0001-5924-8631UNSPECIFIED
Pruene, AlinaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Braun, TobiasUNSPECIFIEDorcid.org/0000-0002-3910-2886UNSPECIFIED
Thommandru, BerniceUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Turk, RolfUNSPECIFIEDorcid.org/0000-0003-1283-9594UNSPECIFIED
Sturgeon, Morgan L.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kurgan, Gavin L.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Amini, LeilaUNSPECIFIEDorcid.org/0000-0003-4855-5905UNSPECIFIED
Stein, MaikUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Zittel, TatianaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Martini, StefaniaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Ostendorf, LennardUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wilhelm, AndreasUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Akyuez, LeventUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rehm, ArminUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hoepken, Uta E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Pruss, AxelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kunkele, AnnetteUNSPECIFIEDorcid.org/0000-0002-8406-5412UNSPECIFIED
Jacobi, Ashley M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Volk, Hans-DieterUNSPECIFIEDorcid.org/0000-0002-7743-6668UNSPECIFIED
Schmueck-Henneresse, MichaelUNSPECIFIEDorcid.org/0000-0001-5964-9179UNSPECIFIED
Stripecke, RenataUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Reinke, PetraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Wagner, Dimitrios L.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
URN: urn:nbn:de:hbz:38-692047
DOI: 10.1016/j.omtm.2022.03.018
Journal or Publication Title: Mol.Ther.-Methods Clin. Dev.
Volume: 25
Page Range: S. 311 - 331
Date: 2022
Publisher: CELL PRESS
Place of Publication: CAMBRIDGE
ISSN: 2329-0501
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
CHIMERIC ANTIGEN RECEPTORS; DNA-REPAIR; EFFICIENCY; THERAPY; IDENTIFICATION; INTEGRATION; INHIBITOR; DELIVERY; LOCUS; CGASMultiple languages
Medicine, Research & ExperimentalMultiple languages
URI: http://kups.ub.uni-koeln.de/id/eprint/69204

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