Dotson, M. | bioRxiv Science

bioRxiv2026

Ultra-large targeted DNA integrations in primary human cells

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.

genetics↗

bioRxiv2024

Accelerating CAR T cell manufacturing with an automated next-day process

The traditional method of CAR T cell production involves lengthy ex vivo culture times which can result in the reduction of crucial naive T cell subsets. Moreover, traditional CAR T cell therapy manufacturing processes can prolong time-to-patient and contribute to disease progression. In this study, we describe an innovative and automated 24-hour CAR T manufacturing process that yields a higher percentage of naive/stem-cell like T cells which have increased cytotoxic activity and cytokine release. The data supports the feasibility of implementing this streamlined manufacturing process in clinics. This approach also has the potential to enhance CAR T therapy efficacy and improve patient access to therapy.

bioengineering↗