Functional correction of RDEB in vitro and in vivo

Eva M Murauer, Laboratory for Molecular Therapy, EB House Austria, Department of Dermatology, University Hospital Salzburg, Salzburg, Austria

Yannick Gache2, Fernando Larcher3, Marcela Del Rio3, Wolfgang Muss4, Guerrino Meneguzzi2, Helmut Hintner1, Johann W Bauer1
2INSERM U 634, Faculté de Médecine, Nice, France
3Cutaneous Disease Modeling Unit, Epithelial Biomedicine Division, Basic Research Department, CIEMAT-CIBERER, Madrid, Spain
4Institute of Pathology, Paracelsus Medical University of Salzburg, Salzburg, Austria.

Dystrophic Epidermolysis bullosa (DEB) is caused by inherited alterations in the collagen 7 gene that produces collagen 7 protein. Collagen 7 protein is the major component of structures called anchoring fibrils that hold the layers of the skin together. People with DEB have a reduced number of anchoring fibrils or completely lack these structures in their skin. Therefore minor mechanical trauma leads to the characteristic blistering of the skin of DEB patients.

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The aim of my research project is to develop a gene therapy for people suffering from DEB in order to cure the skin disease. Using a special gene therapy technique, called SMaRT, we want to repair the altered collagen 7 gene in skin cells so that the cells recover their ability to produce a normal functional collagen 7 protein for skin adhesion. During the last years this method was established in our laboratory. To repair a gene of interest, we first have to generate a repair molecule which brings in the normal copy of the gene portion to be replaced in the cells. Furthermore, the repair molecule has to contain a recognition site that ensures the identification of the target gene within a cell. We have constructed a repair molecule, which accounts for the precise binding to the collagen 7 gene and subsequent replacement of the altered part by a correct copy. We have already successfully introduced this repair molecule into skin cells of an RDEB affected person in the laboratory. As a consequence, the treated cells regained their ability to produce collagen 7 protein. Further, we have constructed an artificial skin using the repaired patient cells, which showed a normal coherence of the skin layers due to restoration of collagen 7 production.

As a next step we wanted to verify if the correction of the collagen 7 gene lasts over a longer period of time. For this purpose we grafted the cultivated skin which was made with the SMaRT corrected cells onto a so-called nude mouse. This mouse completely lack an immune system, thus no graft rejection was expected. Five weeks later the human skin was analyzed according to its cohesiveness. No blistering was observed and collagen 7 protein was still present between the skin layers after 5 weeks, which indicates a long-term effect of the gene correction.

Based on these results, our long-term goal is to develop an ex vivo gene therapy for DEB patients. For that reason cells are removed from the patient, grown in the laboratory and then treated with the repair molecule. Cells corrected by the repair molecule are then cultivated to generate a thin skin layer, which is grafted back onto non-healing wounds of the patient's skin. In the natural environment of the skin, the transplanted cells should develop into normal skin cells reproducing the lacking protein. As a consequence, the wounds of the treated skin areas will be closed and further the new skin will show the properties of healthy skin.

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