A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa

• Published in: Molecular therapy. Nucleic acids Vol. 25; pp. 237 - 250
• Main Authors: Kocher, Thomas, Bischof, Johannes, Haas, Simone Alexandra, March, Oliver Patrick, Liemberger, Bernadette, Hainzl, Stefan, Illmer, Julia, Hoog, Anna, Muigg, Katharina, Binder, Heide-Marie, Klausegger, Alfred, Strunk, Dirk, Bauer, Johann Wolfgang, Cathomen, Toni, Koller, Ulrich
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 03.09.2021; American Society of Gene & Cell Therapy; Elsevier
• Subjects: 3D skin equivalents; Cas9 nickase; COL7A1; CRISPR/Cas9; double-nicking; electroporation; epidermolysis bullosa; Original; primary fibroblasts; primary keratinocytes; single-stranded oligonucleotides; primary fibroblasts; Cas9 nickase; CRISPR/Cas9; 3D skin equivalents; epidermolysis bullosa; COL7A1; electroporation; double-nicking; primary keratinocytes; single-stranded oligonucleotides
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2021.05.015

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses. [Display omitted] The study of Kocher et al. highlights an advanced virus- and selection-free HDR-based gene correction strategy with improved safety profile for severe dystrophic epidermolysis bullosa. Electroporation of paired Cas9 nickases together with single-stranded oligonucleotides was shown to efficiently restore C7 expression in patient-derived primary fibroblasts and keratinocytes.