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Designer nucleases-based NHEJ-mediated gene editing for the correction of a highly recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa epidermal stem cells. (Larcher 1)

Project lead Dr Fernando Larcher
Organisation CIEMAT - Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Madrid, SPAIN
Project budget EUR 96,500.00
Start date / Duration 15. Feb 2017 / 23 months
Funder(s) / Co-Funder(s) DEBRA Austria, EB MSAP/EBEP Recommended
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

RDEB patients lack the protein Collagen VII (C7), one of the main constituents of anchoring fibrils, which firmly hold epithelia to the underlying connective tissue. Mutations in its coding gene, COL7A1, result in the lack of C7 and generalized blistering. This project focuses on cutting-edge gene-editing techniques to repair certain types of genetic mutations that impair collagen production. The ultimate goal of this fundamental research is to grow healthy skin for use in grafting of blistering areas in RDEB patients. It is expected that such gene-corrected skin grafts should have adhesion properties similar to healthy skin since the production of Collagen VII would be restored. The gene-corrected skin transplants would be generated using cells from the recipient patient, thus avoiding or minimizing potential immune rejection problems. This project may be a proof of principle study for the development of similar therapies for other RDEB-causing mutations.

Scientific summary

A high percentage of RDEB patients from Spain (46%) and likely from Latin America, harbour a frame-shift mutation (c.6527insC) in exon 80 of COL7A1 gene leading to the absence of type VII collagen (C7). C7 expression can be efficiently restored in keratinocytes from these patients ex vivo by introducing either small deletions in the vicinity of this COL7A1 mutation or whole mutant exons using designer nucleases. Bulk populations or clones carrying frame-restoring deletions can be isolated, expanded, characterized for biosafety features and used to generate transplantable skin with long-term persistence ability. Analysis of the structural and biomechanical properties of corrected clonal skin grafts will assess the therapeutic potential of this gene-editing strategy.

Strategic relevance

On-going gene-therapy clinical trials for RDEB involve grafting of bioengineered skin generated with patients’ cells modified to express C7. In these trials, the expression of C7 is brought about by the addition of the normal cDNA coding for COL7A1 using viral vectors. The goal here is to improve the genetic correction step by restoring the functionality of the defective gene. Furthermore, gene editing will be undertaken directly on keratinocytes, which is the chief clinically relevant cell type for skin transplantation. Although gene addition methods are in theory applicable to a larger number of patients, the ease and precision of this methodology in combination with its safe profile, permit to imagine its future adoption in clinical trials, and if successful, its use in personalized medicine.

What did this project achieve?

In this study, Fernando Larcher and collaborators achieved efficient and safe restoration of Collagen VII expression in RDEB keratinocytes bearing a frame-shift mutation in exon 80 of the COL7A1 gene.  This non-viral approach, based on the electroporation of ribonucleoprotein complexes of sgRNA-guided CRISPR/Cas9 into keratinocytes, resulted in 95 % of edited alleles. Off-target editing events remained near the detection threshold. When assessed by flow cytometry, 81% of edited RDEB keratinocytes expressed C7, a figure that was confirmed by Western Blot and immuno-fluorescence. Engraftment of epidermal sheets derived from corrected RDEB keratinocytes onto mice revealed a normal dermo-epidermal junction with anchoring fibrils, as verified by electron microscopy examination of the basement membrane. Long-term persistence of the correction as well as the targeting of the epidermal stem-cell compartment was confirmed 20 weeks post-grafting.  These results support the feasibility and translatability of this strategy and pave the way for similar studies adopting precision medicine in the EB field.

Orphan Drug Designation: news article


ribonucleoprotein electroporation
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