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

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.

• Authors: Stefan Hainzl, Patricia Peking, Thomas Kocher, Eva M Murauer, Fernando Larcher, Marcela Del Rio, Blanca Duarte, Markus Steiner, Alfred Klausegger, Johann W Bauer, Julia Reichelt, Ulrich Koller
• Journal: Molecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 25 Issue 11 Pg. 2573-2584 (11 01 2017) ISSN: 1525-0024 [Electronic] United States
• PMID: 28800953 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Chemical References

• COL7A1 protein, human
• Collagen Type VII

Topics

• Animals
• Base Sequence
• CRISPR-Cas Systems
• Collagen Type VII (genetics, metabolism)
• Epidermolysis Bullosa Dystrophica (genetics, metabolism, pathology, therapy)
• Exons
• Gene Editing (methods)
• Gene Expression
• High-Throughput Nucleotide Sequencing
• Humans
• Keratinocytes (metabolism, pathology, transplantation)
• Mice
• Mice, Nude
• Molecular Targeted Therapy
• Mutation
• Plasmids (chemistry, metabolism)
• Primary Cell Culture
• Transplantation, Heterologous
• Treatment Outcome