COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa.

Autor:	Petković I; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Bischof J; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Kocher T; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., March OP; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Liemberger B; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Hainzl S; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Strunk D; Cell Therapy Institute, SCI-TReCS, Paracelsus Medical University, Salzburg, Austria., Raninger AM; Cell Therapy Institute, SCI-TReCS, Paracelsus Medical University, Salzburg, Austria., Binder HM; Cell Therapy Institute, SCI-TReCS, Paracelsus Medical University, Salzburg, Austria., Reichelt J; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Guttmann-Gruber C; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Wally V; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Piñón Hofbauer J; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Bauer JW; Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria., Koller U; EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, Salzburg, Austria.
Jazyk:	angličtina
Zdroj:	Frontiers in medicine [Front Med (Lausanne)] 2022 Aug 25; Vol. 9, pp. 976604. Date of Electronic Publication: 2022 Aug 25 (Print Publication: 2022).
DOI:	10.3389/fmed.2022.976604
Abstrakt:	Background: Epidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB). Methods: In this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene. Results: As analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents. Conclusion: Here we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2022 Petković, Bischof, Kocher, March, Liemberger, Hainzl, Strunk, Raninger, Binder, Reichelt, Guttmann-Gruber, Wally, Piñón Hofbauer, Bauer and Koller.)
Databáze:	MEDLINE
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