Treatment for dystrophic epidermolysis bullosa using an LNP delivery system and an mRNA-based gene editing to correct COL7A1

Summary: Abstract Body: Dystrophic Epidermolysis Bullosa (DEB) is caused by COL7A1 loss-of-function variants leading to dysfunctional type VII collagen (C7) and chronic blistering. Fibroblasts (FB) and keratinocytes (KC), which produce C7, differentiate from epidermal stem cells that maintain skin homeostasis. Current treatments offer only temporary wound healing improvements. Our study focuses on a permanent genetic therapy that corrects all proliferative skin cells. We developed efficient gene editing approaches using single-stranded DNA cleavage techniques that avoid homology-directed repair, minimizing indel risks. Our strategies include base editing (BE), prime editing (PE), and a novel technique called eePASSIGE, which integrates whole genes to deliver wild-type COL7A1. By comparing the effectiveness of BE, PE, and eePASSIGE in primary RDEB patient-derived cells, we aim first to create a “gene editing cream” for topical application using lipid nanoparticle (LNP)-mRNA systems. In vitro studies have shown 40% to 70% transfection efficiencies in FB and KC. Our best formulation, combining DTDTMA (C14) cationic lipid and DOPE phospholipid (C14/DOPE), achieved 84% correction of the c.5047C>T variant in COL7A1, increasing C7 production while reducing cytotoxicity. Prime Editor PE5bmax showed 43% overall editing efficiency, with 32% variant correction confirmed by Sanger Sequencing. We are currently optimizing the delivery of C14/DOPE-mCherry in 3D organotypics and a novel DEB mouse model while assessing safety and genomic effects through off-target and whole genome sequencing. If successful, our strategy could enable long-term skin regeneration and permanent closure of RDEB wounds. Joanna Jacków-Malinowska¹, Stephan Hart², Lukasz Laczmanski³, Emanuel Rognoni⁴, Matthew Caley⁴, John McGrath¹ 1. St John’s Institute of Dermatology, King's College London, London, England, United Kingdom. 2. Great Ormond Street Hospital for Children NHS Foundation Trust, London, England, United Kingdom. 3. Instytut Immunologii i Terapii Doswiadczalnej im Ludwika Hirszfelda Polskiej Akademii Nauk, Wroclaw, Lower Silesian Voivodeship, Poland. 4. Queen Mary University of London Blizard Institute, London, England, United Kingdom. Genetic Disease, Gene Regulation, Gene Therapy & Epigenetics