Suzanne de Bruijn

148 Chapter 3.2 ABSTRACT DFNA21 is a type of dominantly inherited adult-onset hearing loss and caused by a 12-nucleotide deletion in the RIPOR2 gene. Previously, the c.1696_1707del RIPOR2 variant was reported to be the most frequent cause of inherited adult-onset HL in Northwest Europe. There are strong indications that the RIPOR2 deletion acts via a dominant, non-haploinsufficiency disease mechanism. Mutant RIPOR2 is aberrantly localized in the stereocilia of murine auditory hair cells, suggesting a toxic gain-of- function effect. Both humans and mice carrying heterozygous loss-of-function alleles do not display hearing loss. This implies that inhibiting the synthesis of mutant RIPOR2 protein, by selectively degrading the (pre)-mRNA transcribed from the mutant allele, can alleviate the negative consequences of mutant RIPOR2 on auditory function. In this study, gapmer antisense oligonucleotides (AONs) were designed to specifically target mutant RIPOR2 transcripts for degradation by the endogenous RNase H1 enzyme. The molecular efficacy of the AONs was validated in DFNA21 patient-derived fibroblasts and HEK293T cells. This revealed a lead AON molecule that was able to significantly reduce mutant RIPOR2 transcript levels (up to ~90% in HEK293T cells), whilst leaving the level of wildtype RIPOR2 mRNA intact. Additionally, western blot analyses showed that the decrease in mutant RIPOR2 transcripts leads to a marked decrease in mutant protein synthesis. These studies provide proof-of-concept for the induction of rapid and specific degradation of mutant- RIPOR2 transcripts by gapmer AONs. The identified lead molecule is a strong candidate for further preclinical development to ultimately establish a treatment for DFNA21.