Ultra-large targeted DNA integrations in primary human cells
Kernick, C.; Chow, L.; Alejandro, M.; Li, K.; Foisey, M.; Yang, X.; Hilburger, C.; Lu, J.; Wu, L.; McClellan, A.; Takacsi-Nagy, O.; Brajenovic, R.; Theberath, N.; Celallos, E.; Lin, E.; Hartman, A.; Truong, T.; Lee, J. H. J.; Ji, Y.; Workley, L.; Ha, A.; Putnam, N.; Andronikou, N.; Fatima, N.; Dotson, M.; Wong, K. A.; Burns, C. H.; Engelhardt, F. A. S.; Stoyanova, E.; Vukovic, M.; Adie, T.; Khan, O.; Lim, W.; Roybal, K.; Santostefano, K.; Almeida, R.; Allen, G.; Shy, B. R.; Roth, T. L.
Show abstract
Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells 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.
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