Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing

Bonafont, JoseMencía, ÁngelesGarcía, MartaTorres-Ruiz, RaulRodriguez Perales, SandraCarretero, MartaChacón-Solano, EstebanModamio-Høybjør, SilviaMarinas, LucíaLeón, CarlosEscamez, María JHausser, IngridDel Río, MarcelaMurillas, RodolfoLarcher, Fernando2019-05-202019-05-202019-05-08Mol Ther. 2019 ;27(5):986-998.15250016http://hdl.handle.net/20.500.12105/7611Gene 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.engVoRhttp://creativecommons.org/licenses/by-nc-sa/4.0/CRISPR/Cas9Epidermal stem cellsEpidermolysis bullosaGene therapyClinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene EditingAtribución-NoComercial-CompartirIgual 4.0 Internacional30930113275986-99810.1016/j.ymthe.2019.03.0071525-0024Molecular therapy : the journal of the American Society of Gene Therapyopen access