Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing
| Autor: | Raul M. Torres, María José Escámez, Carlos León, E. Chacón-Solano, Marcela Del Rio, Lucía Marinas, Marta García, Silvia Modamio-Høybjør, Jose Bonafont, Ángeles Mencía, Sandra Rodriguez, Marta Carretero, Fernando Larcher, Ingrid Hausser, Rodolfo Murillas |
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| Přispěvatelé: | Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, DEBRA Austria, European Commission |
| Rok vydání: | 2019 |
| Předmět: |
Keratinocytes
Collagen Type VII Medicina Population Biology Gene mutation Frameshift mutation Mice 03 medical and health sciences Exon 0302 clinical medicine Genome editing epidermal stem cells Drug Discovery Genetics medicine Animals Humans CRISPR epidermolysis bullosa Frameshift Mutation education Molecular Biology Gene CRISPR/Cas9 030304 developmental biology Gene Editing Pharmacology 0303 health sciences education.field_of_study High-Throughput Nucleotide Sequencing Exons medicine.disease gene therapy Epidermolysis Bullosa Dystrophica Disease Models Animal 030220 oncology & carcinogenesis Molecular Medicine Original Article Epidermolysis bullosa CRISPR-Cas Systems RNA Guide Kinetoplastida |
| Zdroj: | Repisalud Instituto de Salud Carlos III (ISCIII) Molecular Therapy e-Archivo. Repositorio Institucional de la Universidad Carlos III de Madrid instname |
| Popis: | 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. Highly efficient and safe approaches for precise correction of pathogenic mutations are required to realize the full potential of gene-editing protocols for clinical practice. Here we describe a single-step NHEJ-based CRISPR/Cas9 strategy of COL7A1 mutation-bearing exon deletion with unprecedented efficacy for ex vivo correction of recessive dystrophic epidermolysis bullosa. |
| Databáze: | OpenAIRE |
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