Ultra-large targeted DNA integrations in primary human cells.
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Kernick Courtney, Chow Lauren, Alejandro Mikail, Li Ke, Foisey Maxwell G, Yang Xiaoyu, Hilburger Claire, Lu Johnathan, Wu Lujing, McClellan Alison, Takacsi-Nagy Oliver, Brajenovic Rocco, Theberath Nicole E, Fuentes Emily Celallos, Lin Erin, Hartman Austin, Truong Tina, Lee Jae Hyun Jenny, Workley Luke, Ha Alvin S, Ji Yongchang, Putnam Nicholas, Andronikou Nektaria, Fatima Nujhat, Dotson Max, Wong Kimberly A, Burns Christian H, Engelhard Floris A S, Stoyanova Elena, Vukovic Milica, Adie Tom, Lim Wendell A, Roybal Kole, Santostefano Katherine, Almeida Ricardo, Allen Greg M, Shy Brian R, Roth Theodore L
Аннотация
Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cell's genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond ~5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large-up to 10 kb DNA-integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.