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

Authors :
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
Ministerio de Economía y Competitividad (España)
Instituto de Salud Carlos III
DEBRA Austria
European Commission
Source :
Repisalud, Instituto de Salud Carlos III (ISCIII), Molecular Therapy, e-Archivo. Repositorio Institucional de la Universidad Carlos III de Madrid, instname
Publication Year :
2019
Publisher :
Elsevier, 2019.

Abstract

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.<br />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.

Details

Database :
OpenAIRE
Journal :
Repisalud, Instituto de Salud Carlos III (ISCIII), Molecular Therapy, e-Archivo. Repositorio Institucional de la Universidad Carlos III de Madrid, instname
Accession number :
edsair.doi.dedup.....2f903e859eaa2881c8d991d6f99881dc