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Novel approach to gene therapy for severe dermatological disorders: autografts of genetically corrected epidermal stem cells for the treatment of recessive dystrophic epidermolysis bullosa


- Characterise COL7A1 mutations and their consequences on type VII collagen polypeptide in RDEB patients.
- Generate a library of primary epidermal stem cells from RDEB patients.
- Develop new DNA vectors for COL7A1 expression and design replacement constructs for homologous recombination.
- Optimise transfection procedures for stable transformation of epidermal stem cells using micro-injection, biolistic transformation and other approaches.
- Develop long-term selection procedures to isolate stably transfected cells using antibiotic resistance, enhanced green fluorescent protein detection and/or type VII collagen secretion.
- Analyse type VII collagen expression in stably transfected cells.
- Demonstrate long term expression of type VII collagen and anchoring fibrils formation in a skin equivalent model and in transfected epidermal sheets grafted on athymic mice (pre-clinical testing).
- Perspective: autografts on RDEB patients (phase I clinical testing).

The aim of this project is to develop a European collaboration on gene therapy between three leading groups of complementary expertise, for one of the most severe inherited skin diseases affecting children and adults, e.g. recessive dystrophic epiderrnolysis bullosa (RDEB). Dystrophic epidermolysis bullosa (DEB) is a group of autosomal diseases characterized by blistering of the skin since birth due to loss of dermo-epidermal adhesion. The overall incidence of DEB is estimated to be1: 25000, but the social impact of the disease is high, owing to the extreme severity of the recessive mutilating forms (Hallopeau-Siemens, HS-RDEB). We previously showed close linkage between RDEB and the type Vll collagen gene (COL7AI), which codes for the anchoring fibrils. We have now characterized more than 70 COL7AI mutations in a wide patient population affected with HS-RDEB, the majority of which predicts COL7AI null alleles. Because no specific treatment is available for this life-threatening form of EB, the laboratories participating in the study propose to join their effort to develop an ex vivo gene therapy aiming to transfect a normal COL7AI gene into-cultured primary keratinocytes from RDEB patients suitable for grafting.

For this purpose, we have modified (retrofitted) a large genomic clone of 170kb containing the entire COL7AI gene, by insertion of a new DNA segmented compassing a selective marker and a reporter gene. The different phases of the project include:
1) Further characterization of COL7AI mutations in RDEB patients, study of their consequences on anchoring fibrils formation, and establishment of primary culture keratinocytes from RDEB patients;
2) Transfection of the COL7AI construct developed in our laboratory into primary epidermal stem cells using biolistic transformation and microinjection;
3) Long term selection of transfected keratinocytes from RDEB patients. Depending on the results of this phase, other constructs and transfection methods will be used to achieve stable transfection of primary keratinocytes;
4) Study of type VII collagen expression in stably transfected cells and in "skin equivalent" models;
5) Pre-clinical testing on athymic mice using grafts of genetically modified RDEB epiderrnal stem cells stably expressing type VII collagen;
6) Follow up of type VII collagen expression in transplants. At all stages, safety regulation applying to gene therapy will be strictly followed, with a perspective of engaging phase I clinical testing as soon as feasible. RDEB represents a model for human therapy using genetically modified keratinocytes, and achievement of this project would be the first specific therapeutic approach to the disease. We also believe that this project will create a powerful tool for studying gene function and regulation, and that it will contribute to the development of gene therapy for other dermatological or non dermatological diseases.

Funding Scheme

CSC - Cost-sharing contracts


Roosevelt Drive
OX3 7BN Oxford/headington
United Kingdom

Participants (2)

Ecole Normale Supérieure
46,Rue D'ulm
75230 Paris
Westfälische Wilhelms - Universität Münster
48149 Münster