Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa

Abstract: 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

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Molecular therapy. - 30, 8 (2022) , 2680-2692, ISSN: 1525-0016

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2022
• Creator: Bischof, Johannes ; March, Oliver Patrick ; Liemberger, Bernadette ; Haas, Simone ; 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 ; Piñón Hofbauer, Josefina ; Bauer, Johann ; Cathomen, Anton ; Kocher, Thomas ; Koller, Ulrich

Further information

• DOI: 10.1016/j.ymthe.2022.04.020
• URN: urn:nbn:de:bsz:25-freidok-2267778
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:44 PM CET