10087211Enabling gene therapy through the manufacture of a novel circular single-stranded CRISPR editing templateClosedCollaborative R&DInnovate UKRecent 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, using DNA as a template for homology-directed repair (HDR) to introduce exogenous sequences for cell and gene therapy. ssDNA is more efficient, less toxic and more specific than double-stranded DNA (dsDNA) when used as HDR template for gene editing. However, due to difficulties in its manufacture, GMP lssDNA at scale is still unavailable, forcing non-viral gene editing to largely rely on plasmid DNA as donor for gene-length insertions, despite the high immunogenicity, low knock-in efficiencies and off-target effects associated with it. In response to the growing need for reliable and effective vectors for targeted gene insertions, Touchlight has developed a platform, producing a novel lssDNA, MegaBulb DNA (mbDNATM), which overcomes the size limitations and manufacturing scalability bottlenecks of existing lssDNA and viruses. mbDNA is a closed single-stranded fully synthetic CRISPR gene editing template comprising a short complementary stem region with a target site for a guided endonuclease. Extensive evaluations have shown that mbDNA exhibits extremely low toxicities and outperforms dsDNA and open lssDNA as a CRISPR HDR template in primary cells. Importantly, unlike other lssDNA manufacture platforms, mbDNA's production relies on Touchlight's well established, rapidly scalable, quick turnaround technology -- doggybone DNA (dbDNATM). By leveraging Touchlight's CDMO facilities and technical capabilities, the mbDNA platform promises to be significantly more efficient and affordable, allowing production of lssDNA at the multi-milligram scales required by the gene editing market for the first time. This will facilitate access to lssDNA and advance gene therapies making them better, safer and cheaper, allowing to tackle unmet clinical needs. Through mbDNA commercialisation, this work will enable seminal advances in gene therapy. To achieve this, we will extensively characterise mbDNA, develop relevant analytical methods and scale up its manufacture. Molecular characterisation will facilitate mbDNA's functional understanding, inform and improve production pipelines and quality control development of the novel GMP-compatible process, thus facilitating commercialisation. Through this collaborative work we aim to establish and develop innovative analytical methods to enable mbDNA process development and scale up to supply it as a vital ingredient for the gene editing industry. Taking mbDNA to market will make it available for use by the industry in cell and gene therapy pipelines, where with its improved gene editing profile mbDNA commercial availability promises to revolutionise the field.