P19 Proof of concept for a variant-agnostic, permanent DNA editing cure for dystrophic epidermolysis bullosa

Abstract Introduction and aims Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disease with over 800 causative mutations identified in the COL7A1 gene. eePASSIGE is a breakthrough DNA editing technology that pairs prime editing (PE) with serine integrase technology, allowing for targeted integration of large DNA constructs. We aim (i) to use eePASSIGE to integrate a full-length cDNA of COL7A1 into the AAVS1 safe harbour locus, and (ii) to develop lipid-based nanoparticles (LNPs) capable of delivering eePASSIGE machinery to human skin cells. Together, these two strategies form the basis for a proposed permanent ‘one-size-fits-all’ DEB cure. Methods We have used lipofection of HEK293T, N/TERT, and human fibroblast cells to provide proof of concept. For PE and eePASSIGE, lipofectamine was used to deliver plasmid DNA encoding editing constructs and pegRNAs. For PE, Sanger sequencing of target site amplicons was used to identify successful edits. For eePASSIGE, puromycin selection was used to isolate successfully edited cells. For LNP studies, fluorescent reporter mRNA was delivered to N/TERTs and fibroblasts; successful transfection was quantified with flow cytometry. Results In N/TERT cells, we achieved up to 17% PE efficiency in transfected cells, although only 1% of cells were successfully transfected. eePASSIGE was used to successfully integrate a puromycin resistance gene into HEK293T cells at the AAVS1 safe harbour locus. Lipid nanoparticles were used to successfully transfect human fibroblasts and keratinocytes in submerged culture with reporter constructs with up to 80% efficiency and with lower toxicity than lipofectamine. Conclusions Our experiments thus far have laid the groundwork for future study of eePASSIGE to integrate large coding constructs into disease-relevant cells. Our next steps are (i) to improve transfection of eePASSIGE machinery in cells of interest, and (ii) to optimize LNP formulations for use in three-dimensional culture and in vivo mouse skin.