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.
Bibliographical noteFunding Information:
This work was supported by grants from the Children’s Cancer Research fund, the Sobiech Osteosarcoma Fund Award, and the University of Minnesota Department of Pediatrics. B.R.W was supported by the Emerging Scientist award from the Children’s Cancer Research Fund. We thank Kenny Beckman and John Garbe from the University of Minnesota Genomics Center for advice on performing NGS; Madison Vignes and Lindsey Sumstad from the University of Minnesota for assistance in preparing NGS samples; Jason Gehrke and Darrell Johnson for advice on ddPCR assays; and Ezequiel Marron at the University of Minnesota Viral Vector & Cloning Core for producing lentivirus. The cytogenetic analyses were performed in the Cytogenomics Shared Resource at the University of Minnesota with support from the comprehensive Masonic Cancer Center NIH Grant #P30 CA077598.