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Antisense RNA-mediated improvement of SMaRT therapy for KRT14 correction

Project lead Dr. Ulrich Koller
Organisation EB House Austria
Project budget EUR 196,339,00
Start date / Duration 01. Jan 2015 / 60 months
Funder(s) / Co-Funder(s) PMU, Others, DEBRA Austria
Other funder(s) FWF Austria
Research area Molecular therapy, Cellular therapy

Project details

Short lay summary

Dominant KRT14 mutations associated with EBS, lead to the accumulation and aggregation of mutated Keratin 14 protein within the cell, impairing proper cell function of basal keratinocytes and consequently the stability of the skin. We have previously employed SMaRT (Spliceosome-Mediated RNA Trans-splicing) technology to achieve partial reversal of the EBS phenotype following treatment of patient-derived keratinocytes in vitro. Building on this previous result, we aimed to improve the efficiency of SMaRT therapy by including antisense oligonucleotides that would inhibit competing cis-splicing events.

Scientific summary

Dominant negative mutations within keratin 14 (KRT14) are the underlying cause of EBS generalized severe (EBS gen sev). The incorporation of mutant K14 protein into the keratinocyte intermediate (KIF) filament network greatly compromises its integrity, leading to KIF collapse, cell cytolysis, and blistering of the skin upon minor mechanical trauma. In this project, we employed SMaRT, an RNA-based therapy that exploits naturally occurring cell-endogenous splicing processes, to correct KRT14 mutations in EBS-gen sev patient cells at the RNA level. To achieve this, the mutated KRT14 mRNA is recombined with an exogenously added repair molecule (RTM, RNA trans-splicing molecule), to generate a chimeric mRNA in which the mutated region has been replaced by a healthy copy. The engineered RTM provides the KRT14 wild-type sequence, core elements for the splicing reaction, and a binding sequence to guide the RTM to the affected region on the KRT14 pre-mRNA in a sequence-specific manner. For EBS, SMaRT represents an elegant strategy to simultaneously reduce levels of the dominant negative protein while increasing those of the functional wildtype protein. However, low trans-splicing efficiencies hinder further development of this therapeutic strategy. To enhance the trans-splicing repair efficiency, we co-delivered antisense oligonucleotides (ASOs) together with a KRT14 RTM into EBS cells. Different ASOs were designed to bind competing splice sites and enhancers within the KRT14 pre-mRNA, in order to induce a shift from the predominant cis-splicing reaction to the less efficient RTM-mediated trans-splicing. We screened several ASOs for functionality in our established fluorescence reporter-based screening system and successfully identified two that significantly increased SMaRT-mediated correction efficiency in vitro.

Strategic relevance

In contrast to other technologies which require the design of unique therapeutic molecules for each mutation to be repaired, SMaRT technology enables the correction of multiple mutations with only one repair molecule. Furthermore, SMaRT therapy mediates its effects on two fronts, by decreasing the number of mutant transcripts and concurrently increasing wild-type levels, which is ideal for correcting dominant mutations frequent in EBS. 

What did this project achieve?

With this project we were able to confirm the capability of a rationally designed KRT14 RTM in combination with short antisense molecules to correct a dominant KRT14 mutation in vitro in a highly efficient manner.


EB House Austria
EB House
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