To advance utilization of allogeneic T cells for CAR therapy, Webber and Lonetree et al. showed that the CRISPR-CAS9 base editing technology could overcome the undesirable effects of double-strand breaks initiated with CAS9 nuclease, and focused on altering critical splice sites to eliminate gene expression. Maximal efficiency was observed with codon-optimized mRNA encoding the editing machinery. Simultaneous multiplex knockdout of the TRAC, β2M, and PDCD1 (encoding PD-1) loci in primary human T cells approached 90% efficiency; the cells retained polyfunctionality and cytotoxicity, including toward a PD-L1-overexpressing target.
The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.
Author Info: (1) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, Un
Author Info: (1) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA. (2) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (3) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (4) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (5) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (6) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (7) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (8) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (9) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (10) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. (11) Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. (12) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA. (13) Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. (14) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA. (15) Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu. Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu. Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA. mori0164@umn.edu.
