Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

ABSTRACT: The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for pr

SUBMITTER: Webber BR 

PROVIDER: S-EPMC11169092 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature