FP06 Optimized engineered prime editing guide RNAs unlock efficient prime editing for recessive dystrophic epidermolysis bullosa gene correction

Introduction and aims Recessive dystrophic epidermolysis bullosa (RDEB) is caused by pathogenic variants in COL7A1, resulting in severe skin fragility and remains without a curative treatment. Prime editing (PE), a versatile CRISPR-derived gene editing tool, capable of inducing insertions/deletions (indels) and all types of substitutions holds a great potential to provide a cure for RDEB. In this study, we hypothesized that installation of silent modifications integrated in the engineered PE guide RNA (epegRNA) could make on-target corrections evade DNA mismatch repair process, thereby increasing PE editing efficacy. Methods We first used PE5bmax with rationally designed epegRNAs to correct two pathogenic variants from patients with RDEB harbouring heterozygous COL7A1 c.5047C>T and c.1732C>T. Based on mechanistic insights from these experiments, an additional pathogenic variant, heterozygous c.7786delG leading to null collagen VII (C7), was targeted using PE6c. Following electroporation of PE mRNA into primary patient fibroblasts and a 7-day incubation, editing was validated by Sanger and Nanopore sequencing. Translation of gene correction into C7 restoration was assessed by Western blot analysis. Results On-target correction efficiencies of 32% for c.5047C>T and 16% for c.1732C>T were achieved across three technical replicates. In both cases, editing efficiency was strongly influenced by the number of silent modifications incorporated into the epegRNA. Using a similar approach, PE-mediated correction of the c.7786delG variant reached 65% across three independent experiments, representing the highest efficiency reported to date for COL7A1-targeting PE. Western blot analysis demonstrated 50% restoration of C7 in cell lysates and 20% in the supernatant. Conclusions This study demonstrates that rational epegRNA design incorporating silent modifications significantly enhances PE efficiency for COL7A1 correction in fibroblasts of patients with RDEB. High and reproducible editing efficiencies were achieved across multiple pathogenic variants, accompanied by substantial restoration of collagen VII protein. Building on these results, functional assessment in three-dimensional human skin constructs will be performed to evaluate phenotypic rescue.