2026-05-10T21:07:44Zhttps://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
Repisalud author 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 funder Ministerio de Economía y Competitividad (España) funder Instituto de Salud Carlos III funder DEBRA Austria 2019-05-20T10:00:37Z 2019-05-20T10:00:37Z 2019-05-08 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 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. eng 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 journal article 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 URL https://repisalud.isciii.es/bitstreams/f49af786-d1ba-488f-896c-90b5d242d481/download File MD5 49b6eac4b97d17a2aa096e1782748f71 3911201 application/pdf ClinicallyRelevantCorrection_2019.pdf URL https://repisalud.isciii.es/bitstreams/b89d379c-edff-439b-8520-7b81d771fbdc/download File MD5 a3a51a5d1fda5b3edf537cf812ddb291 813358 application/pdf ClinicallyRelevantCorrection-mmc1_2019_supl.pdf URL https://repisalud.isciii.es/bitstreams/6a805d74-bc0a-42fe-b807-6f71108eae1e/download File MD5 f29a462f913ad318cc299dcc6e37b4ba 4718576 application/pdf ClinicallyRelevantCorrection-mmc2_supl_2019.pdf URL https://repisalud.isciii.es/bitstreams/05a05cb8-d5db-4436-84e8-150360428203/download File MD5 ee9372b8a987164e9d77cb2dde820969 62411 text/plain ClinicallyRelevantCorrection_2019.pdf.txt URL https://repisalud.isciii.es/bitstreams/5277056b-3fd6-47de-b410-aa7fc0454a16/download File MD5 908d92087dfd3356d6ae6de2fd766601 4519 text/plain ClinicallyRelevantCorrection-mmc1_2019_supl.pdf.txt URL https://repisalud.isciii.es/bitstreams/62f5cc47-fc28-4967-9e5f-0b2afc96c501/download File MD5 56f1b38985a7e0c6bc1bf4076c3f80b7 66930 text/plain ClinicallyRelevantCorrection-mmc2_supl_2019.pdf.txt