A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa
Integrin α3β1 Is a Key Regulator of Several Protumorigenic Pathways during Skin Carcinogenesis
1 April 2021
In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial
28 March 2022

 Mol Ther Nucleic Acids. 2021 May 29;25:237-250. doi: 10.1016/j.omtn.2021.05.015. PMID: 34458008; PMCID: PMC8368800.

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

Authors: Thomas Kocher 1Johannes Bischof 1Simone Alexandra Haas 2 3Oliver Patrick March 1Bernadette Liemberger 1Stefan Hainzl 1Julia Illmer 1Anna Hoog 4Katharina Muigg 4Heide-Marie Binder 4Alfred Klausegger 1Dirk Strunk 4Johann Wolfgang Bauer 1 5Toni Cathomen 2 3 6Ulrich Koller 1

Affiliation:

  • 1EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
  • 2Institute for Transfusion Medicine and Gene Therapy, Medical Center – University of Freiburg, 79106 Freiburg, Germany.
  • 3Center for Chronic Immunodeficiency, Medical Center – University of Freiburg, 79106 Freiburg, Germany.
  • 4Cell Therapy Institute, SCI-TReCS, Paracelsus Medical University, 5020 Salzburg, Austria.
  • 5Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
  • 6Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany.

Abstract:

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.

Keywords: 3D skin equivalents; COL7A1; CRISPR/Cas9; Cas9 nickase; double-nicking; electroporation; epidermolysis bullosa; primary fibroblasts; primary keratinocytes; single-stranded oligonucleotides.

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