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CRISPR/Cas9-based editing to treat recessive dystrophic epidermolysis bullosa

Ongoing
Project leadProf. Alain Hovnanian
Organisation INSERM UMR1163, Institut Imagine
Partner organizations & collaboratorsDépartement de Génétique, Hôpital Necker-Enfants malades, Paris
Project budgetEUR 132,600.00
Start date / Duration01. Sep 2018 / 24 months
Funder(s) / Co-Funder(s) Others, DEBRA Austria
Other funder(s)EB-loppet/Run4EBSweden
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

The purpose of this pre-clinical research project is to investigate the therapeutic potential of gene editing to correct two frequent mutations in the gene that codes for the protein type VII collagen. Its primary goal is to set the stage for future therapeutic approaches that would ultimately restore adherence between epidermis and dermis in RDEB patients. To achieve this objective, researchers will develop two strategies in parallel. One will focus on correcting RDEB keratinocytes and fibroblasts directly isolated from patients. The second strategy involves the generation of induced pluripotent stem cells (iPSCs) from patients’ fibroblasts. In these experiments, the plan is to reprogram biopsied adult fibroblasts to become undifferentiated cells with the capacity to give rise to a diversity of cell types of interest. This step might help yield a higher correction efficiency, according to previous work. Once genetically corrected, iPSCs will be re-routed to become skin keratinocytes, fibroblasts and MSCs. Skin made out of functionally corrected cells will be then grown in culture dishes and grafted on mice. In parallel, gene-corrected fibroblasts will be locally injected into the dermis to treat grafted skin.

Scientific summary

This research program aims at investigating the therapeutic potential of CRISPR/Cas9-mediated HDR to correct recurrent COL7A1 mutations (c.425A>G; p.Lys142Arg and c.6508C>T; p.Gln2170* in exon 3 and 80 respectively) in RDEB patient’s primary and IPSCs-derived keratinocytes, fibroblasts and MSCs respectively. Gene-edited RDEB cells will be used to demonstrate that restored type VII collagen is functional by grafting either gene-edited skin equivalents onto nude mice or by performing intradermal injections of primary or IPSCs-derived fibroblasts or MSCs into nude mice grafted with RDEB skin equivalents. The most efficient strategy will orientate future therapeutic choices towards clinical application.

Strategic relevance

This project novelty is manifold primarily because the targeted mutations are frequent in RDEB patients of European ancestry. Secondly, their correction will take place in a diversity of cell types, including adult primary fibroblasts and keratinocytes as well as iPSC. Additionally, alternative modes of delivery of the genetic material will be considered. Based on these two assets, this project has the potential to accelerate the clinical translation of gene-editing strategies for the treatment of RDEB patients.


Keywords

iPSC
gene-editing
RDEB
type VII collagen
CRISPR/Cas9
skin equivalent
Hovnanian
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