Suzanne de Bruijn

157 The development of a genetic therapy for DFNA21 general displays an adult-onset of symptoms which provides a window of opportunity for therapeutic intervention. Inspired by the non-haploinsufficiency pathogenic mechanism of the variant, we explored the specific silencing of the mutant RIPOR2 allele as a potential therapeutic strategy for DFNA21. Several AON molecules were designed and evaluated for their ability to target mutant RIPOR2 transcripts for degradation by the RNase H1 enzyme, thereby blocking the synthesis of (toxic) mutant RIPOR2 proteins whilst leaving wildtype RIPOR2 transcripts intact. We identified a lead molecule that showed the potency to efficiently and specifically reduce mutant RIPOR2 transcript levels and, to a lesser extent, protein synthesis. 92% ns A RNA quantification Vehicle Plasmid AON 6 0.0 0.5 1.0 1.5 RIPOR2 transcripts (fold change) Mutant Wildtype 100% 100% 12% **** Protein quantification B Vehicle Plasmid AON 6 0.0 0.5 1.0 1.5 Relative ratio RIPOR2/Tubulin Mutant Wildtype *** ** 100% 100% 20% 5% RIPOR2wt Tubulin Vehicle Plasmid AON 6 RIPOR2mt Tubulin Vehicle Plasmid AON 6 C Figure 4. Evaluation of efficacy and specificity of AON 6 in HEK293T cells. HEK293T cells were co-transfected with mutant- or wildtype RIPOR2 cDNA constructs and AON 6 (250 nM in the medium). RNA and protein were isolated 24 hours afterwards. (A) RT-qPCR analysis revealed a significant reduction of mutant RIPOR2 transcript cells when HEK293T cells were treated with AON 6. Data are expressed as mean ± SEM of six replicate transfections, normalized to the expression of GUSB and displayed as the fold change compared to cells treated with RIPOR2 plasmid and transfection reagent only (plasmid). Vehicle transfected cells were transfected with transfection reagent only and show no endogenous RIPOR2 expression. (B- C) Western blot analyses was performed using anti-RIPOR2 and anti-tubulin antibodies. Analyses confirmed a reduction in mutant RIPOR2 protein synthesis in cells treated with AON 6. Also a reduction in wildtype RIPOR2 protein levels was observed. Quantification of western blot results was performed using the Fiji software (v1.47). Data are expressed as mean ± SEM of three replicate transfections, the relative ratio of RIPOR2 protein to tubulin protein was calculated and compared to cells treated with transfection reagent and RIPOR2 only (plasmid). **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s post-test. The ability of AONs to specifically target mutant transcripts for degradation is of key importance for the development of an AON-based therapy for the c.1696_1707del RIPOR2 gain-of-function allele. The therapeutic strategy must induce a large enough