Clinically relevant correction of recessive dystrophic epidermolysis bullosa by dual sgRNA CRISPR/Cas9-mediated gene editing
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 targete...
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| Main Authors: | , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
15 March 2019
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| In: |
Molecular therapy
Year: 2019, Volume: 27, Issue: 5, Pages: 986-998 |
| ISSN: | 1525-0024 |
| DOI: | 10.1016/j.ymthe.2019.03.007 |
| Online Access: | Verlag, Volltext: https://doi.org/10.1016/j.ymthe.2019.03.007 Verlag, Volltext: http://www.sciencedirect.com/science/article/pii/S1525001619300930 
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| Author Notes: | 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 |
| Summary: | 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. |
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| Item Description: | Gesehen am 17.02.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1525-0024 |
| DOI: | 10.1016/j.ymthe.2019.03.007 |