Suzanne de Bruijn

159 The development of a genetic therapy for DFNA21 Interestingly, when tested at the same concentration of 100 nM, the target knockdown induced by AON 1 (non-gapmer sequence of AON 6) was much higher than that of AON 6 (73%mutant reduction AON 1, versus 29-33% AON 6). Because of their complete DNA chemistry, PS-DNA AONs are known to have a higher RNase H1-recruiting activity than gapmer sequences. In addition, the differences in effect between these two AONs could also be attributed to differences in transfection efficiency, chemical toxicity of the PS- DNA AON, subcellular distribution, or any combination of these factors. Although to the best of our knowledge not reported before, the strong and highly mutant allele-specific knockdown of RIPOR2 induced by AON 6 in cDNA construct-transfected HEK293T cells suggests that transfection efficiency may have well been a limitation in the patient- derived fibroblasts. The use of a fluorescently-conjugated AON, and gymnotic delivery of AON 6, will shed more light on this. Additionally, alternative AON chemistries could be explored in parallel to enhance the on-target efficiency of the gapmer molecule. In this study, 2’-O-methyl (2’-OMe) sugar modified RNA wings were employed in the gapmer design. In several studies, alternative sugar modifications have been described that can potentially increase the potency of the molecule. A study performed in HeLa cells indicated that the 2'-O-methoxyethyl (2’-MOE) modified AONs are consistently more effective in suppressing CTNNB1 RNA levels compared to the corresponding 2’OMe AONs. 24 Alternatively, Rukov et al. reported the use of locked-nucleic acid (LNA)- modified gapmer wings as a promising approach to increase binding affinity and stability of the molecule. 25 By extending the free binding energy of the AON and the intended RNA target, the observed knockdown was increased and even higher than observed using full PS-DNA AONs. However, increasing the binding affinity of the AON may also increase the affinity for the wildtype RIPOR2 transcript. Therefore, adjusting the AON wing design or gap size, may offer a better chance at optimizing efficiency and specificity for the mutant RIPOR2 transcript (reviewed in (26)). Importantly, it should be acknowledged that the fibroblast cell model is probably not ideal to study inner ear disease. While the patient-specific genotype is advantageous over engineered cell models such as plasmid-transfected HEK293T cells, or stable transgenic cell models, they poorly reflect the transcriptional and cellular nature of the RIPOR2 -expessing hair cells in the inner ear. Therefore, alternative more relevant cell models, such as otic progenitor cells or inner ear organoids 27 , should be considered in future studies to reliably determine the necessity to optimize the chemistry and gapmer design of the lead AON. The RNA profile of these cell types is more representative for the auditory hair cells, and therefore more potent to screen for any potential adverse effects.