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.

doi:10.64898/2026.04.09.717505

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bioRxiv · 10.64898/2026.04.09.717505

Ultra-large targeted DNA integrations in primary human cells

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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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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.. 2026-04-09. Ultra-large targeted DNA integrations in primary human cells. https://doi.org/10.64898/2026.04.09.717505

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