CRISPR/Cas9-based editing to treat recessive dystrophic epidermolysis bullosa (Hovnanian 7 + Ext)
Completed| Project lead | Prof Alain Hovnanian |
| Organisation | INSERM UMR1163, Institut Imagine, Paris, FRANCE |
| Partner organizations & collaborators | Département de Génétique, Hôpital Necker-Enfants malades, Paris |
| Project budget | EUR 132,600.00 + EUR 77,000.00 extension |
| Start date / Duration | 01. Sep 2018 / 40 months |
| Funder(s) / Co-Funder(s) | DEBRA Austria, MSAP/EBEP Recommended, DEBRA France |
| 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.
What did this project achieve?
The specific aim of this research project was to investigate the therapeutic potential of CRISPR/Cas9-mediated Homology Directed Repair (HDR) to correct two recurrent COL7A1 mutations in cells derived from two RDEB patients. In this project, A. Hovnanian used primary cells and IPSCs derived from two RDEB probands who were homozygous for the common null mutation c.425A>G in exon 3 or the c.6508C>T mutation in exon 80. To achieve this goal, they developed two strategies in parallel: The first strategy targeted correction of RDEB in primary keratinocytes and fibroblasts, and the second targeted iPSCs derived from the patients' cells.
Functional correction of RDEB cells was investigated ex vivo in a xenograft model using skin equivalents derived from gene-edited RDEB cells. They use a platform based on ribonucleoprotein particles delivering HiFi Cas9 and the corresponding gRNAs.The strategy is not based on fluorescence detection or antibiotic selection. For C425>G they obtained a lower efficiency than for C6508>T. The future goal is to increase the efficiency of the correction for C425>G. To achieve this goal, new ssODNs will be synthesised and tested. This follow-up project is now fully funded by DEBRA France and AFM Téléthon.