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Development of improved trans-splicing molecules for major types of epidermolysis bullosa (Bauer 4)

Project lead Prof Johann Bauer
Organisation EB House Austria, Salzburg, AUSTRIA
Partner organizations & collaborators Dr. Eva Murauer and Dr. Verena Wally from EB House Austria
Project budget EUR 82,180.00
Start date / Duration 01. Mar 2012 / 36 months
Funder(s) / Co-Funder(s) DEBRA Austria, EB MSAP/EBEP Recommended
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

In this project the safety and efficiency of the RNA trans-splicing method was improved. Trans-splicing uses a designed repair molecule (RTM) that replaces a specific portion of the mutated mRNA with a healthy copy in order to produce a healthy protein in EB skin cells. An RTM has to contain a binding domain (BD) that facilitates the exchange on the right mRNA within a cell.

Little differences in the BD have great influence on the repair efficiency. Due to the lack of rules on how to design most potent BDs, we developed a fluorescence-based screening method where we can identify highly functional RTMs out of a big pool of randomly generated molecules. Cells that fluoresce intensively have undergone successful repair and can be separated using a FACS machine. Here we have been able to select optimal RTMs for four main genes affected in different types of EB.

Scientific summary

Spliceosome-mediated RNA trans-splicing (SMaRT) is a technology to repair genetic mutations by replacing a defined region of a mutated pre-mRNA by its wild-type copy using an RNA trans-splicing molecule (RTM). This molecule, besides the cDNA portion to be replaced consists of a binding domain (BD) hybridizing to the target region of interest, a process that influences the specificity and efficiency of the trans-splicing reaction.

A clinically useful causal therapy requires efficient and sustained expression of the therapeutic gene, which is met by the trans-splicing technology, providing that the corrective RTM shows high trans-splicing efficiency. As to date there are no reliable parameters for the design of BDs, we established a screening protocol, in order to define characteristics of highly potent BDs in terms of length, sequence and binding for specific target sites of the EB genes COL7A1, COL17A1, K5, PLEC.

For this screening system we generated RTM libraries containing randomly designed BDs and a green fluorescence protein (GFP) molecule split into a 5’, a 3’ portion or an internal portion, respectively. A target minigene that mimics the endogenous target pre-mRNA was constructed to contain the target intron/exon sequence as well as the remaining part(s) of the GFP and was stably integrated into HEK293 cells. In this system, transfection of the RTM library into the target cell lines generates a full-length GFP upon trans-splicing. A high GFP expression correlates with a high trans-splicing efficiency and can be measured by FACS analysis. After RTM library transfection into target cell lines, the cells were FACS sorted into three fractions of cells expressing a) high GFP b) low GFP and c) no GFP levels. Sequencing analysis revealed the composition of the BDs present in each fraction.

Strategic relevance

  • DEBRA strategic goal: ‘Develop disease-modifying and curative therapies’ through basic research, to develop therapies that target the underlying disease mechanism in EB.
  • Project goal: To improve the safety and efficiency of the SMaRT technology as a means to repair genetic mutations in different types of EB.

What did this project achieve?

Our group has developed the SMaRT technology over several years with the aim to establish a safe and effective technique for correcting the mutations in EB genes overcoming drawbacks of other causative therapy approaches.

Attractive features, provided by the SMaRT technology, are maintenance of endogenous regulation of the transgene. Using this technology the size of the transgene is reduced, allowing the use of non-viral plasmids for delivery. In case of an ex vivo approach where viral vectors are used, the risk of truncated protein expression due to generated rearranged provirus during the viral transductions is reduced. Further, it is possible to correct dominant negative mutations, thus allowing an application for recessively and dominantly inherited EB forms.

Here we have developed a robust and reliable screening system in order to improve the selection of potent RTMs for future clinical applications. 

We have statistically analyzed and characterized RTM binding domains. These experiments allowed us to identify preferred target regions for RTM binding and thus to select promising BDs for 3’, 5’ and internal trans-splicing approaches for our selected EB genes. 

The analysis of BD cluster isolated of high GFP cell fraction has come up with important information on the design of RTMs for the selected genes. 

Selected highly potent RTMs were adapted for endogenous experiments and the analysis of the trans-splicing specificity.


binding domain
causal therapy
EB House Austria
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