Suzanne de Bruijn

155 The development of a genetic therapy for DFNA21 PS-DNA AONs (100 nM) Vehicle AON 1 AON 2 AON 3 0.0 0.5 1.0 1.5 Mutant Wildtype RIPOR2 transcripts (fold change) 0.0 0.5 1.0 1.5 Mutant Wildtype RIPOR2 transcripts (fold change) Gapmer AONs (100 nM) Vehicle AON 6 AON 7 A B 100% 64% 100% 27% 75% 42% 100% 97% 60% 40% 22% 100% 67% 49% **** **** **** **** **** ns ns ** **** ** Figure 2. Screening of candidate antisense oligonucleotides. Degradation of mutant and wildtype RIPOR2 transcripts in DFNA21 patient-derived fibroblast cells by antisense oligonucleotides (AONs) (100 nM in the culture medium), directed against the c.1696_1707del RIPOR2 variant. (A) All three phosphorothioate (PS)-modified DNA AONs (1-3) were able to significantly reduce mutant transcript levels, 24 hours after transfection. (B) Gapmer AONs (AON 6 and AON 7) were designed based on sequences of AON 1 and AON 2, respectively. Both AONs were able to significantly decrease mutant RIPOR2 transcript levels, but only AON 6 showed allele-discriminative potential as it did not significantly reduce wildtype transcript levels, 24 hours after transfection. 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 < 0.0001, one-way ANOVA with Tukey’s post-test. AON 6 induces a dose-dependent and specific decrease in mutant RIPOR2 transcript levels To further investigate the efficacy and allele-specificity of AON 6, a dose-response analysis was performed. The AONs were transfected in patient-derived fibroblast cells, with a final concentration in the culture medium ranging from 50 nM to 250 nM. A significant reduction in transcripts of the mutant allele could be observed when treated with concentrations ≥150 nM ( Figure 3 ). A maximum knockdown of 68% was achieved anda clear dose-dependent response couldbeobserved. For noneof the concentrations, a significant reduction in transcripts of the wildtype allele was observed. Although a non-significant decrease in wildtype transcripts at 50 nM compared to transfection reagent only was found, this change was not dose-dependent. This suggests that AON 6 has the ability to specifically target the mutant RIPOR2 allele for RNase H1-mediated degradation. Validation of the lead AON molecule in HEK293T cells We questioned whether the lower knockdown efficiency of AON 6 as compared to the full PS-DNA AON is, in part, due to a lower transfection efficiency. To overcome this potential limitation, we co-transfected AON 6 with vectors encoding FLAG-tagged mutant or wildtype RIPOR2 in HEK293T cells.