Suzanne de Bruijn

150 Chapter 3.2 normal inner ear function. Antisense oligonucleotides (AON)-mediated therapy can be considered a suitable strategy to modulate mutant RIPOR2 expression. AONs with DNA- like properties can be specifically designed to bind transcripts harboring pathogenic variants, and will subsequently recruit RNase H1 endonuclease. This endonuclease degrades RNA molecules that are part of RNA:DNA duplexes, which will lead to a sequence-specific decrease in protein synthesis. 7,8 The 5’ and 3’ wings of the RNase H1- dependent AONs can be chemically modified to increase thermodynamic stability and nuclease resistance, while maintaining a central gap region of DNA nucleotides that ensures RNase H1 activity. 9 These modified AONs are referred to as gapmers, and have shown great therapeutic potential in treatment strategies for other inherited disorders including amyotrophic lateral sclerosis (ALS) and Huntington disease. 10 More recently, gapmers were designed to specifically decrease mutant COCH transcript levels as a potential future treatment for HL type DFNA9. 11 To date, there are four AON-gapmers on the market that have been FDA- or EMA-approved and many more gapmers are under investigation in clinical trials. 12 An AON-mediated therapy holds important advantages over other genetic therapy strategies suchas CRISPR/Cas9-mediatedDNAediting.The effect of theAONs is transient, and there is no risk for the introduction of non-reversible DNA changes (reviewed in (13,14)). The transient nature of AONs also significantly decreases the risk of severe adverse effects. Treatments can be halted when adverse effects occur, and off-target transcript degradation is only temporary, unlikely to induce biologically significant changes in protein levels. 13 Additionally, previous studies achieved successful delivery of AONs to and uptake of AONs in relevant cochlear cells after intratympanic delivery. 15,16 This method of AON delivery could successfully restore Ush1c pre-mRNA splicing in adult Ush1c knockin mice 15 , which confirms the feasibility of an AON approach as a potential treatment for (inherited) hearing disorders. In the current study, we designed several (gapmer) AONs aimed to specifically target the transcripts of the c.1696_1707del RIPOR2 allele for degradation. The designed molecules were evaluated in both patient-derived fibroblast cells and in transiently- transfected HEK293T cells. We successfully identified an AON with a strong potency to downregulate mutant RIPOR2 transcript levels and thereby reduce the synthesis of mutant RIPOR2 protein, whilst leaving the level of wildtype RIPOR2 transcript intact. The identified lead molecule is a strong candidate for further preclinical development to ultimately establish a treatment for DFNA21.