This proposal aims to develop gene therapy for Recessive Dystrophic Epidermolysis Bullosa (RDEB), one of the most severe inherited skin diseases affecting children and young adults. Dystrophic Epidermolysis Bullosa (DEB) is a group of genetic diseases characterized by blistering of the skin from birth due to loss of dermo-epidermal adhesion. Approximately one person in 25000 is affected, but the social impact of the disease is high and those affected require specific educational, social and medical care owing to the extreme severity of the recessive mutilating forms. No specific treatment is available for this life threatening form of EB, but recent progress in the understanding of the molecular basis of RDEB is providing the foundation for new therapeutic approaches. The development of gene therapy for these severe forms of EB is obviously the major goal for research applied to these diseases in the near future.

Dr Hovnanian's group has previously shown that the disease is due to genetic defects in the type VII collagen gene (COL7A1), which codes for the anchoring fibrils. Anchoring fibrils secure the top layer of the skin (epidermis) to the layer underneath (dermis) and these fibrils are either not formed or are rare and very abnormal in RDEB patients. He and others has now characterized a large number of COL7A1 mutations in a wide population of patients affected with severe RDEB, thus providing direct evidence for the implication of this gene in RDEB. The majority of these defects lead to absence of synthesis of type VII collagen resulting in lack of anchoring fibrils and loss of dermo-epidermal adhesion. Studies of the parents of these patients show that a single copy of COL7A1 permits normal anchoring fibril formation suggesting that inducing the production of a normal type VII collagen collaborative.

This research seeks to develop gene therapy for RDEB with the aim of restoring normal type VII collagen expression into transplantable cultured primary keratinocytes from RDEB patients. Because the stem cell of the epidermis has a self-renewal capacity sufficient for covering the surface of the entire body, grafting of genetically modified stem cells is potentially a very powerful system for gene therapy for EB.

Although grafting the entire surface body is not feasible in RDEB patients it is believed that autografts using genetically modified keratinocytes which stably secrete normal type VII collagen may restore dermo-epidermal adhesion locally in treated areas. This, in turn would improve mechanical strength in selected areas such as hands and feet which are severely or predominantly affected. Such treatment would also be expected to prevent webbing of fingers and toes mitten-like deformities and joint contractures as well as the development of aggressive skin cancers, and would thus improve the quality of life and life expectancy in these severely affected patients.

The aim is to undertake genetic correction of COL7A1 defects by targeting correction of the disease mutation in one of the mutated COL7A1 genes, or by introducing a copy of the normal COL7A1 gene into keratinocytes from RDEB patients both approaches will be developed in this project. For this purpose he will use a new technique which has been shown to provide a very efficient method for site-directed correction of point mutations in targeted genes. He will target COL7A1 mutations with hybrid RND-DNA oligonucleotides containing the normal sequence. Replacement of the mutated sequence by the normal sequence should occur at high frequence and corrected cells will be expanded and tested for type VII collagen expression. In parallel to this approach he will also transfer the entire COL7A1 gene and its regulatory elements in epidermal stem cells from RDEB patients and select and analyse permanently corrected cells. RDEB cells stably expressing normal type VII collagen will be assayed for anchoring fibrils formation in skin equivalent models prior to preclinical and clinical testing.

This project would be the first specific therapeutic approach to the disease and could significantly change the way the disease is managed and improve the quality of life of RDEB patients. Although tremendous technical problems will still have to be overcome, this will constitute a breakthrough in the treatment of the disease and will provide new hope for improved health for these patients. It is believed that this project will create a powerful tool for studying the efficiency of gene correction procedures in epidermal stem cells and that it will contribute to the knowledge leading to the development of gene therapy for Dominant Dystrophic EB, for other forms of inherited EB, and for other dermatological or non dermatological diseases.