Suzanne de Bruijn

153 The development of a genetic therapy for DFNA21 RESULTS Design of allele-specific RNase H1-dependent AONs We first employed several in silico analyses in order to design RNase H1-dependent AONs complementary to transcripts of the c.1696_1707del mutant, but not wildtype, RIPOR2 allele. First, internal hybridization of the (pre-)mRNA of mutant RIPOR2 exon 14 was predicted in silico to determine accessibility of the target region ( Figure 1A ). In the putative AON target region, 17 of the 30 nucleotides are unpaired in the most probable 3D structure. Several candidate AONs were selected that span the 12nt-deletion region, which is a prerequisite for allele-discrimination. Next, thermodynamic properties were calculated, among which the free energy of on- and off-target AON binding, and the potential of the AONs for hairpin formation and dimerization. Based on these in silico predictions ( Table S1 ), three AON molecules were ordered as phosphorothioate (PS)- linked DNA-bases for an initial screening. Figure 1B provides a schematic overview of the target regions of all AONs that are used in this study. AONs induce efficient knockdown of mutant RIPOR2 transcripts in patient-derived fibroblast cells To assess the potency of the AON strategy and the accessibility of the AON-target region of the mutant RIPOR2 transcript, DFNA21 patient-derived primary fibroblast cells were treated with PS-DNA oligos at a final concentration of 100 nM in the culture medium. This dose was selected based on earlier findings described by de Vrieze et al. 11 where significant reduction in mutant COCH transcripts was achieved with similarly designed AONs. The non-gapmer composition was selected for its strong ability to recruit RNase H1. All three c.1696_1707del-targeting AON molecules were able to induce a significant reduction in transcript levels of the mutant allele (ranging from 60-78%) compared to cells that were treated with transfection reagent only, indicating that the target region in mutant RIPOR2 is indeed accessible to AONs ( Figure 2A ). However, a reduction of wildtype RIPOR2 transcript levels was observed for all three AONs. The strongest concomitant reduction in wildtype RIPOR2 transcripts was observed for AON 3, for which a 78% reduction inmutant transcripts coincides with a 58% reduction of wildtype transcripts. As a next step, AONs 1 and 2 were ordered with a gapmer chemistry that included a central gap region of PS-DNA bases flanked by 2’-O-methyl RNA wings (AON 6 and AON 7, respectively). Although the gapmer composition is known to decrease RNase H1 cleavage efficiency, their improved stability and reduced toxicity, make them particularly attractive for clinical applications. In addition, the inability of RNase H1 to