Suzanne de Bruijn

158 Chapter 3.2 decrease in mutant RIPOR2 synthesis, but allow sufficient synthesis of wildtype protein to sustain normal inner ear function. We previously developed and in vitro validated an allele-specific AON for the single nucleotide change c.151C>T in COCH that is associated with hearing loss and vestibular dysfunction type DFNA9. 11 The 12-nt difference between mutant and wildtype RIPOR2 would offer, in theory, a better opportunity to design mutant allele-specific AONs. All of the AON molecules that were included in this study, both full PS-DNA AONs and gapmers, were able to significantly reduce RIPOR2 transcript levels. Surprisingly, a clear mutant allele-specific effect was only observed for gapmer AON 6. In patient-derived fibroblasts, 250 nM of AON 6 resulted in a 68% reduction of mutant RIPOR2 transcripts accompanied by a mild, statistically not significant, decrease in wildtype RIPOR2 transcripts. Upon in silico predictions of the 3D structure of the AON-target duplexes, a potential explanation of the lack of mutant allele-specificity of the PS-DNA AONs emerged. Unlike for AONs directed to a specific missense variant, all nucleotides of the AON can form base pairs with their complementary nucleotides of both the mutant and the wildtype transcript. Indeed, base pairing of the PS-DNA AONwith wildtype RIPOR2 is predicted by RNAfold 23 to lead to the formation of 12-nucleotide loop in the target RNA, with 11- to 15-nucleotide stretches of PS-DNA/RNA duplexes that can still recruit RNase H1. The slightly lower predicted binding affinity for the wildtype transcript is perfectly reflected in the slightly stronger decrease in mutant transcript levels than in wildtype transcript levels that is observed upon delivery of the PS-DNA AONs to patient-derived primary fibroblasts. 2’-O-methyl (2’-OMe) sugar modified RNA nucleotides are resistant to RNase H1 activity, and were used in the gapmer AONs to reduce the ability of the AONs to recruit RNase H1 to the stretches flanking the 12-nucleotide loop. The resulting gapmer AON 6 displayed stronger allele-specificity, indicating that the ability of the AONwings to recruit RNase H1 was indeed sufficiently decreased. The specificity of AON 6 for the mutant allele was confirmed in HEK293T cells. Co-transfection of AON 6 with a mutant RIPOR2 cDNA construct led to a decrease of 88% in mutant RIPOR2 transcripts, whereas co-transfection of AON 6 with a wildtype RIPOR2 cDNA construct did not significantly affect wildtype RIPOR2 transcript levels (8% reduction). Unfortunately, based on the western blot results, this effect was less specific at the protein level as a significant reduction of wildtype protein was observed (80%). A potential explanation could be that the delivery of an AON, independent of the sequence, has an effect on protein translation. While discrepancies between transcript and protein levels are common, the observed mutant RIPOR2 reduction (95%) is also higher than observed at the transcript level (88%). This indicates that the difference might be (partially) attributed to a general effect of AON delivery, and should be further investigated using a control AON treatment in future experiments.