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A non-viral in vivo RNA therapy approach for dystrophic epidermolysis bullosa

Project lead Dr Ulrich Koller
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget EUR 200,000.00 + Extension 2 years + EUR 200,000.00
Start date / Duration 01. Mar 2018 / 72 months
Funder(s) / Co-Funder(s) Others
Other funder(s) Land Salzburg
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene that lead to functional impairment or loss of type VII collagen protein, the main component of anchoring fibrils in the skin. In principle, gene therapy aimed at correcting the effects of disease-causing mutation, can be applied at the genomic DNA or RNA level. Previously, we demonstrated that the RNA trans-splicing molecule RTMS6 was capable of functional correction of the DEB phenotype in an ex vivo setting (Murauer et al. 2011, Peking et al. 2017). Importantly, RTMS6 can potentially be applied for the correction of ~40% of all known COL7A1 mutations at the RNA level. Here, we focus on in situ delivery of this molecule into the skin using both 3D organotypic cultures and mouse models.

Scientific summary

This project aims to develop a non-viral in vivo RNA therapy approach for dystrophic EB patients. We employ spliceosome-mediated RNA trans-splicing (SMaRT) technology to recombine two RNA molecules: the cell-endogenous mutation-bearing pre-mRNA and an engineered therapeutic RTM that is introduced into the cell. The RTM encodes all necessary splice components and a binding domain that directs the molecule to its target. Accurate trans-splicing between these two molecules replaces the mutant sequence of the endogenous transcript with a wild type copy provided by the RTM. We have previously designed RTMS6, encoding wild type exons 1-64 of COL7A1. RTMS6 is therefore capable of correcting all mutations located within the corresponding region on the COL7A1 pre-mRNA, which amounts to ~40 % of all DEB-causing mutations. In the course of this project we will evaluate several in vivo delivery strategies to bring RTMS6-minicircle plasmids into the skin, including gene gun bombardment, a method we have already successfully applied in mouse models. Non-invasive (and thus less painful) alternatives will also be investigated, such as liposome-based delivery. In a first step, these strategies will be tested in vitro in 3D RDEB skin equivalents, with those giving promising results advanced to in vivo testing in appropriate xenograft mouse models. COL7A1 gene correction at the RNA level and functional type VII collagen protein restoration at the basement membrane zone will be analysed via RT-PCR, Western blot analysis, and immunofluorescence staining, in both in vitro and in vivo experiments.

Strategic relevance

In vivo delivery remains an obstacle to the application of gene therapeutic molecules. For RDEB, non-viral approaches are preferred in order to reduce potential mutagenic insertion in patients who are already prone to skin cancer development. The establishment of a non-viral in vivo delivery strategy for RTMS6 will provide the possibility to correct a high proportion of COL7A1 mutations associated with RDEB. The RNA trans-splicing approach offers several advantages over gene replacement strategies such as the delivery of a shorter transgene, the maintenance of the endogenous regulation and the possibility to correct both recessively and dominantly inherited mutations. Because SMaRT technology is used to repair genes at the RNA level, a high safety profile of the approach is also expected.


RNA trans-splicing
in-vivo therapy
EB House
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