In vitro repair of a deletion mutation within a model substrate of the oncogenic CTNNB1 mRNA

Therapeutic nucleic acids such as DNAzymes, siRNAs and trans-cleaving ribozymes have been shown to be capable of inhibiting undesired gene expression by cleavage of a particular target sequence [1,2]. Here we report a novel strategy that aims at the application of twin ribozymes for site-directed modification of RNA.

Twin ribozymes are small RNAs derived from the hairpin ribozyme by tandem duplication and mediate two chain cleavages and two ligation events in a strictly controlled fashion. This activity can be used to cut a target sequence out of a suitable RNA substrate followed by ligating a new fragment into the gap left behind. Depending on the specific design of the twin ribozyme-substrate complex, fragments of equal lengths are exchanged, or a shorter patch is replaced with a longer one and vice versa. Thus, the system enables the repair of short deletions [3], insertions [4], and base replacement mutations [5], making twin ribozymes potential tools for phenotypic correction of genetic disorders.

In order to demonstrate the potential of twin ribozymes for functional repair of genetically relevant substrates, we have developed a twin ribozyme targeting the mutated transcript of the CTNNB1 gene, encoding β-Catenin, an effector of the canonical Wnt signalling pathway. Using a model substrate with a three base deletion, in vitro repair could be successfully demonstrated with a yield of up to 24% of corrected RNA. Further experiments towards activity tests in cell culture are in progress.