Clinically relevant correction of recessive dystrophic epidermolysis bullosa by dual sgRNA CRISPR/Cas9-mediated gene editing

• Verfasst von: Bonafont, Jose [VerfasserIn]
• Verfasst von: Haußer-Siller, Ingrid [VerfasserIn]
• Titel: Clinically relevant correction of recessive dystrophic epidermolysis bullosa by dual sgRNA CRISPR/Cas9-mediated gene editing
• Verf.angabe: Jose Bonafont, Ángeles Mencía, Marta García, Raúl Torres, Sandra Rodríguez, Marta Carretero, Esteban Chacón-Solano, Silvia Modamio-Høybjør, Lucía Marinas, Carlos León, María J. Escamez, Ingrid Hausser, Marcela Del Río, Rodolfo Murillas, and Fernando Larcher
• E-Jahr: 2019
• Jahr: 15 March 2019
• Umfang: 13 S.
• Fussnoten: Gesehen am 17.02.2020
• Titel Quelle: Enthalten in: Molecular therapy
• Ort Quelle: Amsterdam : Elsevier, 2000
• Jahr Quelle: 2019
• Band/Heft Quelle: 27(2019), 5, Seite 986-998
• ISSN Quelle: 1525-0024
• DOI: doi:10.1016/j.ymthe.2019.03.007
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: CRISPR/Cas9
• Sach-SW: epidermal stem cells
• Sach-SW: epidermolysis bullosa
• Sach-SW: gene therapy
• K10plus-PPN: 1690133171

Abstract

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.