2026-05-10T21:08:10Zhttps://repisalud.isciii.es/rest/oai/request

oai:repisalud.isciii.es:20.500.12105/76112024-11-29T17:28:14Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597

00925njm 22002777a 4500 dc Bonafont, Jose author Mencía, Ángeles author García, Marta author Torres-Ruiz, Raul author Rodriguez Perales, Sandra author Carretero, Marta author Chacón-Solano, Esteban author Modamio-Høybjør, Silvia author Marinas, Lucía author León, Carlos author Escamez, María J author Hausser, Ingrid author Del Río, Marcela author Murillas, Rodolfo author Larcher, Fernando author 2019-05-08 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. Mol Ther. 2019 ;27(5):986-998. 10.1016/j.ymthe.2019.03.007 1525-0024 15250016 Molecular therapy : the journal of the American Society of Gene Therapy 30930113 http://hdl.handle.net/20.500.12105/7611 CRISPR/Cas9 Epidermal stem cells Epidermolysis bullosa Gene therapy Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing