Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa

• Published in: Molecular therapy Vol. 30; no. 8; pp. 2680 - 2692
• Main Authors: Bischof, Johannes, March, Oliver Patrick, Liemberger, Bernadette, Haas, Simone Alexandra, Hainzl, Stefan, Petković, Igor, Leb-Reichl, Victoria, Illmer, Julia, Korotchenko, Evgeniia, Klausegger, Alfred, Hoog, Anna, Binder, Heide-Marie, Garcia, Marta, Duarte, Blanca, Strunk, Dirk, Larcher, Fernando, Reichelt, Julia, Guttmann-Gruber, Christina, Wally, Verena, Hofbauer, Josefina Piñón, Bauer, Johann Wolfgang, Cathomen, Toni, Kocher, Thomas, Koller, Ulrich
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.08.2022; American Society of Gene & Cell Therapy
• Subjects: Autoantigens - genetics; Collagen Type XVII; CRISPR-Cas9; Deoxyribonuclease I - genetics; Epidermolysis Bullosa - metabolism; Epidermolysis Bullosa, Junctional - genetics; Epidermolysis Bullosa, Junctional - therapy; gene reframing; Homozygote; Humans; JEB; junctional epidermolysis bullosa; Laminin - genetics; Mutation; next-generation sequencing; Non-Fibrillar Collagens - genetics; Original; paired nicking; Sequence Deletion; skin equivalents; junctional epidermolysis bullosa; paired nicking; gene reframing; next-generation sequencing; skin equivalents; JEB; CRISPR-Cas9
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2022.04.020

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6β4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context. ▪ The study of Bischof et al. highlights a patient-specific Cas9-nickase-based targeting strategy for reframing a COL17A1 mutation in junctional epidermolysis bullosa. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing.