biostudies-literatureWebber BREmployee of Intima BioscienceSponsored Research Agreement funded by Intima BiosciencesNIAID NIH HHSNHLBI NIH HHSFoundation for the National Institutes of HealthFoundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)NCI NIH HHS1553-1570https://www.ebi.ac.uk/biostudies/studies/S-EPMC11169092biostudies-literatureUnknown8(12)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 prNature biomedical engineeringCas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.PMC11169092R01 AI146009T32 HL007062P01 CA254849R37 CA276345R01 AI161017T32HL007062-46Skeate JGStarr TKHenley TDiers MDMcIvor RSSlipek NJWebber BRJohnson MJLahr WSMoriarity BSQiu XRathmann BChoudhry MWick BDeFeo APMills LJfalseCas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.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 pr2024-01-01T00:00:00Z2024 Dec2026-06-02T18:12:46.702Z2025-04-06T17:33:34.857ZS-EPMC111690923809285710.1038/s41551-023-01157-4