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Mol Ther. 2017 Nov 1;25(11):2573-2584. doi: 10.1016/j.ymthe.2017.07.005. Epub 2017 Jul 13.

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa.

Author information

1
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria.
2
Epithelial Biomedicine Division, CIEMAT-CIBERER, Department of Bioengineering, UC3M, Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, 28040 Madrid, Spain.
3
Laboratory for Immunological and Molecular Cancer Research, 3rd Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases and Rheumatology, Oncologic Center, Paracelsus Medical University, 5020 Salzburg, Austria.
4
Department of Dermatology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria.
5
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University, 5020 Salzburg, Austria. Electronic address: u.koller@salk.at.

Abstract

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

KEYWORDS:

COL7A1; CRISPR/Cas9; epidermolysis bullosa; homology-directed repair

PMID:
28800953
PMCID:
PMC5675435
DOI:
10.1016/j.ymthe.2017.07.005
[Indexed for MEDLINE]
Free PMC Article

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