Splicing therapies for Dystrophic Epidermolysis Bullosa (SpliceEB)

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare devastating skin disease without current effective treatment. The clinical management of this genetic disease is mainly supportive with symptomatic treatment. Gene therapy represents a compelling strategy for RDEB. However, critical shortcomings associated to peculiarities of the therapeutic gene (COL7A1) could compromise the use of standard ex vivo gene transfer strategy shown to be successful in a patient with the junctional form of Epidermolysis Bullosa reported by Mavilio and co-workers. This project thus aims at developing a novel, safer, efficacious and long-lasting ex vivo gene therapy approach. In this study we will test the 3` trans-splicing correction approach for the COL7A1 pre-mRNA on keratinocytes from patients carrying in homozygosis the c.6527dupC mutation in exon 80, which is highly recurrent in RDEB patients in Southern Europe. Gene correction will be assessed in vitro and in vivo. We will pay great attention to safety issues at the molecular level (e.g. transgene-host genome interactions) as well as to sustained regeneration of gene- corrected skin using a robust skin-humanized mouse model. Another part of the proposal suggests to develop a COL7A1 humanized RDEB mouse model suitable for testing our 5` and 3` trans-splicing therapies. In this grant application we also aim to validate the 5` trans-splicing approach for the correction of mutations in the murine Col7a1 gene using a suitable hypomorphic EB mouse model. The SpliceEB consortium is formed by young scientists in the Skin Gene Therapy field from The Netherlands (Dr. M. Pasmooij, University Medical Center Groningen; coordinator), Germany (Dr. A. Nyström, University Medical Center Freiburg), Austria (Dr. E. Murauer, Univ. Hospital Salzburg, EB House) and an expert in the development of Antisense-mediated exon (AON) skipping for genetic diseases from The Neterhlands (Dr. A. Aartsma-Rus, Leiden University Medical Center).

In this project, we successfully tested new therapy approaches for the treatment of the severe genetic skin disease dystrophic epidermolysis bullosa (DEB) in mouse models. DEB is caused by mutations in the collagen 7 gene, whose transcript produces the collagen VII protein, which in healthy humans provides a firm anchoring of the upper and lower skin. In the case of DEB, this cohesion of the skin layers is disturbed, and therefore the characteristic blisters form after mild mechanical trauma. Patients with severe DEB forms characterized by a complete absence of collagen VII protein and therefore by severe blistering, often develop chronic wounds. In addition to severe pain, these wounds also present a major risk for the development of associated aggressive skin cancers. Thus, therapeutic approaches for this devastating disease are urgently needed. In the course of the project Type VII collagen correction by RNA trans-splicing, we used the RNA trans-splicing technology to specifically repair the collagen 7 transcript in the affected skin cells. As a result, a normally functioning collagen VII protein should be produced, leading to a firm cohesion of the skin layers. For this, a specially designed repair molecule, called RNA trans-splicing molecule (RTM), is introduced into the cells, where it specifically replaces a defective portion of the collagen 7 transcript by its correct copy. In this project, we have developed a human RTM specifically replacing a defective collagen 7 section in DEB patient cells, as well as a murine RTM to correct a defective section in the collagen 7 transcript from DEB mice. After introduction of the human RTM into DEB patient skin cells, we were able to detect a successfully corrected transcript as well as the formation of functional protein. We then used the corrected human skin cells to generate skin equivalents and transplanted these onto immunodeficient mice. 12 weeks after transplantation, normal human skin had been formed, showing corrected collagen VII protein between the skin layers, which led to prevention of blistering of the grafted human skin. Aiming to introduce the human RTM directly into the murine skin of living healthy and DEB mice in a safe and pain-reduced manner, we used a "gene gun" device. 7 days after application, we were able to show corrected collagen VII protein in the skin of the mice, proving that the collagen 7 transcript was repaired directly in the skin cells by the RTM. In this study we have developed preclinical therapeutic approaches for the RNA trans-splicing technology, which can now be adapted for application in DEB patients.