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)Completed
|Dr Fernando Larcher
|CIEMAT - Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Madrid, SPAIN
|Start date / Duration
|15. Feb 2017 / 23 months
|Funder(s) / Co-Funder(s)
|DEBRA Austria, MSAP/EBEP Recommended
|Molecular therapy, Cellular therapy
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.
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.
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.