Recent advancements in non-viral gene editing technologies offer precise genome engineering at significantly reduced costs and biosafety risks compared to viruses. CRISPR is a programmable genome targeting system, which relies on a donor DNA as a template for homology-directed repair (HDR) to introduce exogenous sequences for cell and gene therapy. Therefore, as viruses are increasingly being replaced by safer and cheaper technologies, there is a growing need for reliable and effective vectors for targeted gene insertions. Plasmid DNA (pDNA), which has traditionally been used as the incumbent template for gene-length knock-ins, however, is associated with high toxicities, poor immunogenicity profiles and low HDR efficiencies, all of which has served to limit the success of non-viral gene therapy.

Touchlight’s doggybone™ DNA (dbDNA™) is a fully synthetic, GMP-grade, covalently closed, linear vector, which directly addresses these challenges. The enzymatic manufacture eliminates bacterial sequences and produces a minimal vector of higher purity and lower immunogenicity compared to fermentation-derived DNA. Furthermore, the platform offers unparalleled scalability with reduced timelines overcoming pDNA manufacture bottlenecks. dbDNA has been robustly evaluated as an HDR template in ex vivo CRISPR gene editing platforms internally and by gene therapy industry leaders and is shown to maintain significantly better cell viability compared to pDNA. Improved cell survival leads to accelerated recovery and expansion in culture to produce higher edited primary immune cell counts. With its reduced size, dbDNA has a copy-number advantage over pDNA, allowing improved CRIPSR knock-in rates at a lower template concentration, further minimising the DNA bioburden. Importantly, the improved toxicity and immunogenicity profiles lead to better and more reliable knock-in efficiencies across primary T cells isolated from different blood donors, reducing undesired donor-to-donor variability. This altogether makes dbDNA highly suitable for autologous ex vivo gene and cell therapies, where cell numbers are typically limited and genome editing outcomes inconsistent.