Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.
Mol Ther
; 27(5): 986-998, 2019 05 08.
Article
en En
| MEDLINE
| ID: mdl-30930113
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Epidermólisis Ampollosa Distrófica
/
Colágeno Tipo VII
/
Sistemas CRISPR-Cas
/
Edición Génica
Límite:
Animals
/
Humans
Idioma:
En
Revista:
Mol Ther
Asunto de la revista:
BIOLOGIA MOLECULAR
/
TERAPEUTICA
Año:
2019
Tipo del documento:
Article
País de afiliación:
España