P13 Ionizable lipid nanoparticles for cutaneous delivery of mRNA-based gene editors in dystrophic epidermolysis bullosa

Abstract Introduction and aims Lipid nanoparticles (LNPs) are an attractive platform for delivering nucleic acid-based therapeutics, including gene editors (GEs), but their potential in treating skin diseases remains underinvestigated. Dystrophic epidermolysis bullosa (DEB) is a genetic skin disease caused by pathogenic variants in COL7A1, leading to collagen VII deficiency, chronic blistering, nonhealing wounds and eventually skin cancer. We hypothesize that LNP-mediated delivery of GEs, either topically or systemically, can treat DEB. Building on previous work demonstrating successful delivery of GEs to DEB fibroblasts using cationic LNPs, this work investigates the use of ionizable LNPs for mRNA-based GE delivery. Methods Ionizable LNPs encapsulating fluorescent reporter mRNA were formulated using ionizable lipids (MC3, DODMA, or ALC-0315), phospholipids (DOPE or DSPC), cholesterol, and PEG-2000-DMG with varying component ratios. The LNP’s ability to deliver fluorescent reporter mRNA was evaluated in vitro in primary human fibroblasts and NTERT keratinocytes, and three-dimensional (3D) skin constructs and in vivo onto wounds on a DEB mouse model. Results The LNPs exhibited favourable physicochemical properties, with small particle sizes, low polydispersity and high encapsulation efficiency. We identified several candidate LNPs, with ALC0315 or MC3 as ionizable lipids, capable of delivering fluorescent reporter mRNA to cells in vitro. Moreover, we have shown that efficacy and cell type selectivity is influenced by the choice of helper lipid and component ratios. For example, ALC0315/DOPE performs best in fibroblasts while ALC0315/DSPC favours keratinocyte delivery. We have begun applying the top LNP candidates in 3D human skin equivalents and into wounds on a DEB mouse model suitable for GE testing. Our preliminary results indicate successful delivery of LNPs into basal keratinocytes in the 3D constructs and into migrating cells within the mouse wound bed during healing. Conclusions These findings support the potential of LNPs as a versatile and translatable platform for GE therapies in DEB, providing a foundation for future therapeutic development.