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Splicing Therapies for Dystrophic Epidermolysis Bullosa

Project lead Dr Eva Murauer
Organisation EB House Austria, Salzburg, AUSTRIA
Project budget EUR 247,905.00
Start date / Duration 01. Apr 2013 / 48 months
Funder(s) / Co-Funder(s) Others
Other funder(s) FWF – Austrian Science Fund
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

The purpose of this project was to investigate trans-splicing strategies as a causative treatment option for DEB patients. Trans-splicing uses repair molecules that are introduced into skin cells in order to replace a portion of the mutated collagen 7 (C7) messenger RNA (mRNA) with a healthy copy. As a result, a functional C7 protein is produced.

The first strategy was an ex vivo approach, for which we have optimized a repair molecule that specifically replaces the back portion of the human C7 mRNA. RDEB skin cells were corrected and then transplanted onto mice.  A healthy C7 protein was detected in the human skin grafts.

In the second study, we developed an in vivo approach, in which a repair molecule, replacing the front portion of the mouse C7 mRNA, was introduced into the skin of mice using a gene-gun. The C7 repair took place directly in the skin.

Scientific summary

Mutations in the COL7A1 gene lead to malfunction, reduction, or absence of type VII collagen (C7) and anchoring fibrils in the basement membrane zone (BMZ) of the skin, causing DEB. Causative therapeutic approaches for the severe forms of RDEB have already been established, whereas studies regarding therapeutic options for DDEB are rare as yet. As a promising therapy option suitable for both, RDEB and DDEB we use the Spliceosome mediated RNA trans-splicing (SMaRT) technology in order to repair the mutated target pre-mRNA. Designed RNA trans-splicing molecules (RTM) are delivered into cells where they specifically replace selected exons during the natural occurring splicing process. In this project, we have implemented trans-splicing applications ex vivo and in situ using two different pre-clinical mouse models.

For the ex vivo approach, we used a self-inactivating lentiviral vector for stable integration of an RTM able to specifically substitute a 3.3 kb 3’ COL7A1 coding sequence into C7 deficient RDEB keratinocytes, carrying a homozygous mutation in exon 80. After selection of one single cell clone, showing functional C7 expression at similar levels to normal keratinocytes, skin equivalents were generated and grafted onto immunodeficient mice. Histological and immunohistological analysis of 12 week old grafts showed no signs of blistering and strong labelling of human C7 at the BMZ, with no expression in the suprabasal cell layers.

For direct in situ Col7a1 repair in a murine model we generated a non-viral minicircle plasmid expressing an RTM capable to specifically replace the 2.1 kb 5’ Col7a1 coding sequence. This molecule was transiently delivered into the skin of wild-type mice using a gene gun. Successful vector delivery and transgene expression within dermis and epidermis was shown via histological and immunofluorescence analysis of bombarded skin. Furthermore, trans-spliced C7 protein was detected at the BMZ.

Strategic relevance

  • DEBRA strategic goal: ‘Develop disease-modifying and curative therapies’ through basic research and pre-clinical studies, to develop therapies that target the underlying disease mechanism in EB.
  • Project goal: To investigate clinical applicability of the RNA trans-splicing application ex vivo and in vivo for DEB treatment.
  • Preceding/ follow-on projects, and related projects: This project builds on previous projects funded by the Austrian National Bank (‘No 10923: Developing Spliceosome Mediated RNA Trans-Splicing (SMaRT) for Gene Therapy in Epidermolysis Bullosa Dystrophica Patients’), Fondation René Touraine (‘Optimizing trans-splicing based gene therapy for patients suffering from recessive dystrophic epidermolysis bullosa’), and DEBRA International (Development of improved trans-splicing molecules for major types of epidermolysis bullosa)

What did this project achieve?

In this project the RNA trans-splicing technology was optimized and two different approaches were tested in pre-clinical studies, either suitable for an ex vivo therapy for DEB or for direct in situ repair of COL7A1.

The ex vivo study demonstrated the first trans-splicing approach leading to fully phenotypically reverted human RDEB keratinocytes in a xenograft mouse model fulfilling necessary safety criteria towards a clinical application. The results highlight that only a very low trans-splicing efficiency can lead to RDEB phenotype correction, provided, that single cell clones, producing high levels of repaired C7, are selected. The isolation of single clones further allows the determination of amount and location of the proviral integration in the cell’s genome prior to transplantation, thus reducing the risk of severe adverse events.

For the first time, the RNA trans-splicing technology was applied in an in situ approach in skin, using a minicircle plasmid as a novel non-viral RTM delivery system. Additionally, the use of a gene gun to deliver the RTM into the skin is unique, providing an easy, pain-reduced, efficient and reproducible way of in vivo application. Avoiding tissue damage, this strategy is an attractive alternative to dermal injections associated with severe pain.

Both therapy options demonstrate promising pre-clinical data that can be adapted for a clinical application in RDEB and DDEB patients.


causal therapy
ex vivo
in vivo
in situ
gene gun
mouse model
lentiviral vector
type VII collagen
EB House Austria
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