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| Item Type: | Article |
|---|---|
| Title: | Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells |
| Creators Name: | Kath, J., Du, W., Pruene, A., Braun, T., Thommandru, B., Turk, R., Sturgeon, M.L., Kurgan, G.L., Amini, L., Stein, M., Zittel, T., Martini, S., Ostendorf, L., Wilhelm, A., Akyüz, L., Rehm, A., Hoepken, U.E., Pruß, A., Künkele, A., Jacobi, A.M., Volk, H.D., Schmueck-Henneresse, M., Stripecke, R., 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 |
| ISSN: | 2329-0501 |
| Publisher: | Cell Press |
| Volume: | 25 |
| 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 |
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