Suzanne de Bruijn

156 Chapter 3.2 Vehicle 50 nM 100 nM 150 nM 200 nM 250 nM 0.0 0.5 1.0 1.5 RIPOR2 transcripts (fold change) Dose-response AON 6 Mutant Wildtype 100% 76% 71% 39% 47% 32% 100% 80% 86% 66% 77% 78% ns ns ns ns ns ns ns *** *** ** Figure 3. Dose-response analysis of AON 6. To validate the potency of the previously identified lead molecule, AON 6, the efficacy and specificity of the molecule was evaluated at different concentrations (50 nM – 250 nM in the medium) in DFNA21 patient-derived fibroblast cells. A significant decrease in mutant RIPOR2 transcripts 24 hours after transfection was observed at concentrations ≥ 150 nM. No significant reduction of wildtype transcripts was observed for any of the doses. Data are expressed as mean ± SEM of three replicate transfections, normalized to the expression of GUSB and displayed as the fold change compared to cells treated with transfection reagent only (vehicle). **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Tukey’s post-test. Although this experimental paradigm does not optimally reflect the patient situation, the ability to assess the effect of the AONs on protein translation is an important advantage over patient-derived fibroblasts. We co-supplemented HEK293T cells with DNA constructs encoding FLAG-tagged mutant or wildtype RIPOR2, together with AON 6. The maximum AON concentration (250 nM) that was tested in fibroblast cells was selected for this experiment. Again, an allele-specific knockdown was observed on the transcript level (88% mutant transcript reduction (p-value <0.0001, ****) versus 8% wildtype transcript reduction (p-value 0.5591, not significant) ( Figure 4A ). Additionally, we performed a western blot analysis to confirm the effect of the AON molecule on RIPOR2 translation. In three replicate AON deliveries, western blot analysis of RIPOR2 revealed a strong decrease in mutant RIPOR2 translation (95% reduction). However, also a decrease in wildtype RIPOR2 protein levels was observed (80% reduction), larger than anticipated based on the knockdown of wildtype transcript levels. DISCUSSION DFNA21, resulting from an in-frame deletion in the RIPOR2 gene (c.1696_1707del), is a frequent form of dominantly inherited HL, and is estimated to affect thousands of individuals within Europe. 1 The DFNA21 HL phenotype is highly variable, but in