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• W3135704865 abstract "The c.151C>T founder mutation in COCH is a frequent cause of late-onset, dominantly inherited hearing impairment and vestibular dysfunction (DFNA9) in the Dutch/Belgian population. The initial clinical symptoms only manifest between the 3rd and 5th decade of life, which leaves ample time for therapeutic intervention. The dominant inheritance pattern and established non-haploinsufficiency disease mechanism indicate that suppressing translation of mutant COCH transcripts has high therapeutic potential. Single-molecule real-time (SMRT) sequencing resulted in the identification of 11 variants with a low population frequency (<10%) that are specific to the c.151C>T mutant COCH allele. Proof of concept was obtained that gapmer antisense oligonucleotides (AONs), directed against the c.151C>T mutation or mutant allele-specific intronic variants, are able to induce mutant COCH transcript degradation when delivered to transgenic cells expressing COCH minigenes. The most potent AON, directed against the c.151C>T mutation, was able to induce a 60% decrease in mutant COCH transcripts without affecting wild-type COCH transcript levels. Allele specificity decreased when increasing concentrations of AON were delivered to the cells. With the proven safety of AONs in humans, and rapid advancements in inner ear drug delivery, our in vitro studies indicate that AONs offer a promising treatment modality for DFNA9. The c.151C>T founder mutation in COCH is a frequent cause of late-onset, dominantly inherited hearing impairment and vestibular dysfunction (DFNA9) in the Dutch/Belgian population. The initial clinical symptoms only manifest between the 3rd and 5th decade of life, which leaves ample time for therapeutic intervention. The dominant inheritance pattern and established non-haploinsufficiency disease mechanism indicate that suppressing translation of mutant COCH transcripts has high therapeutic potential. Single-molecule real-time (SMRT) sequencing resulted in the identification of 11 variants with a low population frequency (<10%) that are specific to the c.151C>T mutant COCH allele. Proof of concept was obtained that gapmer antisense oligonucleotides (AONs), directed against the c.151C>T mutation or mutant allele-specific intronic variants, are able to induce mutant COCH transcript degradation when delivered to transgenic cells expressing COCH minigenes. The most potent AON, directed against the c.151C>T mutation, was able to induce a 60% decrease in mutant COCH transcripts without affecting wild-type COCH transcript levels. Allele specificity decreased when increasing concentrations of AON were delivered to the cells. With the proven safety of AONs in humans, and rapid advancements in inner ear drug delivery, our in vitro studies indicate that AONs offer a promising treatment modality for DFNA9. DFNA9, caused by mutations in the COCH gene, is a relatively common form of dominantly inherited highly progressive hearing loss and vestibular dysfunction. It is characterized by adult-onset hearing loss, leading to complete deafness by the age of 50–70 years.1Robertson N.G. Cremers C.W.R.J. Huygen P.L.M. Ikezono T. Krastins B. Kremer H. Kuo S.F. Liberman M.C. Merchant S.N. Miller C.E. et al.Cochlin immunostaining of inner ear pathologic deposits and proteomic analysis in DFNA9 deafness and vestibular dysfunction.Hum. Mol. Genet. 2006; 15: 1071-1085Crossref PubMed Scopus (95) Google Scholar,2Bom S.J.H. Kemperman M.H. Huygen P.L.M. Luijendijk M.W.J. Cremers C.W.R.J. Cross-sectional analysis of hearing threshold in relation to age in a large family with cochleovestibular impairment thoroughly genotyped for DFNA9/COCH.Ann. Otol. Rhinol. Laryngol. 2003; 112: 280-286Crossref PubMed Scopus (18) Google Scholar With progression of the disease, speech perception and conversation become severely limited. DFNA9 patients furthermore suffer from balance problems, which severely hamper their daily activities. Overall, the problems associated with DFNA9 have a severe impact on the quality of life of patients and their relatives and friends.3De Belder J. Matthysen S. Claes A.J. Mertens G. Van de Heyning P. Van Rompaey V. Does Otovestibular Loss in the Autosomal Dominant Disorder DFNA9 Have an Impact of on Cognition? A Systematic Review.Front. Neurosci. 2018; 11: 735Crossref PubMed Scopus (10) Google Scholar The COCH gene is located on chromosome 14, and encodes cochlin, a protein that consists of 550 amino acids. Cochlin is predicted to contain a signal peptide, an LCCL (limulus factor C, Cochlin, and late gestation lung protein Lgl1) domain, two short intervening domains, and two vWFA (von Willebrand factor A) domains. Cochlin is expressed in fibrocytes of the spiral ligament and spiral limbus, where it has been reported to assist in structural support and sound processing, and in the vestibular fibrocytes that are important in the maintenance of balance.4Gallant E. Francey L. Fetting H. Kaur M. Hakonarson H. Clark D. Devoto M. Krantz I.D. Novel COCH mutation in a family with autosomal dominant late onset sensorineural hearing impairment and tinnitus.Am. J. Otolaryngol. 2013; 34: 230-235Crossref PubMed Google Scholar Proteolytic cleavage of cochlin, between the LCCL domain and the more C-terminal vWFA domains, results in a 16-kDa LCCL domain-containing peptide that is secreted and has been shown to play a role in innate immunity in the cochlea.5Jung J. Yoo J.E. Choe Y.H. Park S.C. Lee H.J. Lee H.J. Noh B. Kim S.H. Kang G.Y. Lee K.M. et al.Cleaved Cochlin Sequesters Pseudomonas aeruginosa and Activates Innate Immunity in the Inner Ear.Cell Host Microbe. 2019; 25: 513-525.e6Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar The vWFA domain-containing cochlin fragments are presumed to be extracellular matrix proteins, as cochlin vWFA2 was found to interact with collagens in vitro, and cochlin is a major component of the cochlear extracellular matrix.1Robertson N.G. Cremers C.W.R.J. Huygen P.L.M. Ikezono T. Krastins B. Kremer H. Kuo S.F. Liberman M.C. Merchant S.N. Miller C.E. et al.Cochlin immunostaining of inner ear pathologic deposits and proteomic analysis in DFNA9 deafness and vestibular dysfunction.Hum. Mol. Genet. 2006; 15: 1071-1085Crossref PubMed Scopus (95) Google Scholar,6Nagy I. Trexler M. Patthy L. The second von Willebrand type A domain of cochlin has high affinity for type I, type II and type IV collagens.FEBS Lett. 2008; 582: 4003-4007Crossref PubMed Scopus (18) Google Scholar The c.151C>T (p.Pro51Ser) founder mutation, affecting the LCCL domain, appears to be the most prevalent mutation in COCH, as it underlies hearing loss in >1,000 Dutch and Belgian individuals.7de Kok Y.J. Bom S.J. Brunt T.M. Kemperman M.H. van Beusekom E. van der Velde-Visser S.D. Robertson N.G. Morton C.C. Huygen P.L.M. Verhagen W.I.M. et al.A Pro51Ser mutation in the COCH gene is associated with late onset autosomal dominant progressive sensorineural hearing loss with vestibular defects.Hum. Mol. Genet. 1999; 8: 361-366Crossref PubMed Scopus (116) Google Scholar,8Fransen E. Verstreken M. Bom S.J. Lemaire F. Kemperman M.H. De Kok Y.J. Wuyts F.L. Verhagen W.I.M. Huygen P.L.M. McGuirt W.T. et al.A common ancestor for COCH related cochleovestibular (DFNA9) patients in Belgium and The Netherlands bearing the P51S mutation.J. Med. Genet. 2001; 38: 61-65Crossref PubMed Google Scholar Histopathology of a temporal bone from a p.Pro51Ser DFNA9 patient revealed significant loss and degeneration of fibrocytes in the cochlea.1Robertson N.G. Cremers C.W.R.J. Huygen P.L.M. Ikezono T. Krastins B. Kremer H. Kuo S.F. Liberman M.C. Merchant S.N. Miller C.E. et al.Cochlin immunostaining of inner ear pathologic deposits and proteomic analysis in DFNA9 deafness and vestibular dysfunction.Hum. Mol. Genet. 2006; 15: 1071-1085Crossref PubMed Scopus (95) Google Scholar Overexpression of murine cochlin containing the ortholog of the p.Pro51Ser variant in cultured cells previously revealed that this mutation results in the formation of cytotoxic cochlin dimers and oligomers that sequester wild-type cochlin.9Yao J. Py B.F. Zhu H. Bao J. Yuan J. Role of protein misfolding in DFNA9 hearing loss.J. Biol. Chem. 2010; 285: 14909-14919Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar While the proteolytic cleavage of cochlin was shown to be reduced by the p.Pro51Ser variant and abolished by several other DFNA9-associated variants,9Yao J. Py B.F. Zhu H. Bao J. Yuan J. Role of protein misfolding in DFNA9 hearing loss.J. Biol. Chem. 2010; 285: 14909-14919Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar the potential contribution of decreased proteolytic cleavage to DFNA9 pathology requires further investigation. All available data indicate that DFNA9 results from a gain-of-function and/or a dominant-negative disease mechanism, rather than from haploinsufficiency. Downregulation of the mutant allele, thereby alleviating the inner ear from the burden caused by the formation of cytotoxic cochlin dimers, therefore has high therapeutic potential. The lack of auditory and vestibular phenotypes in mice carrying a heterozygous protein-truncating mutation in Coch,10Jones S.M. Robertson N.G. Given S. Giersch A.B.S. Liberman M.C. Morton C.C. Hearing and vestibular deficits in the Coch(-/-) null mouse model: comparison to the Coch(G88E/G88E) mouse and to DFNA9 hearing and balance disorder.Hear. Res. 2011; 272: 42-48Crossref PubMed Scopus (32) Google Scholar and in heterozygous family members of patients with early-onset hearing impairment due to homozygous protein-truncating mutations in COCH,11JanssensdeVarebeke S.P.F. Van Camp G. Peeters N. Elinck E. Widdershoven J. Cox T. Deben K. Ketelslagers K. Crins T. Wuyts W. Bi-allelic inactivating variants in the COCH gene cause autosomal recessive prelingual hearing impairment.Eur. J. Hum. Genet. 2018; 26: 587-591Crossref PubMed Scopus (17) Google Scholar illustrate that sufficient functional cochlin proteins can be produced from a single healthy COCH allele. We speculate that a timely intervention might even prevent hearing impairment altogether. Antisense oligonucleotides (AONs) with DNA-like properties can be employed to target (pre-)mRNA molecules for degradation by the RNase H1 endonuclease.12Crooke S.T. Molecular mechanisms of action of antisense drugs.Biochim. Biophys. Acta. 1999; 1489: 31-44Crossref PubMed Scopus (343) Google Scholar,13Vickers T.A. Crooke S.T. The rates of the major steps in the molecular mechanism of RNase H1-dependent antisense oligonucleotide induced degradation of RNA.Nucleic Acids Res. 2015; 43: 8955-8963Crossref PubMed Scopus (39) Google Scholar Chemical modifications can be introduced in the 5′ and 3′ flanking nucleotides to increase stability and nuclease resistance, while maintaining a central gap region of oligo-deoxynucleotides to bind to the target RNA and thereby activate RNase H1.12Crooke S.T. Molecular mechanisms of action of antisense drugs.Biochim. Biophys. Acta. 1999; 1489: 31-44Crossref PubMed Scopus (343) Google Scholar These AONs are named gapmers, and their ability to specifically target mutant alleles for degradation has shown great promise in treatment strategies for non-haploinsufficiency disorders such as Huntington’s disease.14Vickers T.A. Crooke S.T. Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms.PLoS ONE. 2014; 9: e108625Crossref PubMed Scopus (44) Google Scholar,15Southwell A.L. Kordasiewicz H.B. Langbehn D. Skotte N.H. Parsons M.P. Villanueva E.B. Caron N.S. Østergaard M.E. Anderson L.M. Xie Y. et al.Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease.Sci. Transl. Med. 2018; 10: eaar3959Crossref PubMed Scopus (66) Google Scholar For a successful application of AON therapy for non-haploinsufficiency disorders such as DFNA9, it is of major importance that the designed AONs only target the mutant (pre-)mRNA, and not the wild-type (pre-)mRNA, for degradation. As the options to design allele-discriminating AONs based on a single nucleotide difference are limited, we used single-molecule real-time (SMRT) sequencing to identify additional allele-discriminating variants that can be exploited for AON design. This resulted in the identification of 11 variants with a low population frequency (<10%) that are specific to the c.151C>T mutant COCH haplotype. Our results show that both the c.151C>T mutation in COCH and low-frequency variants in cis with the DFNA9 mutation can be used to specifically target mutant COCH transcripts for degradation by RNase H1. Lead molecule c.151C>T AON-E appears to be the most promising molecule for further preclinical investigation. As this AON targets the DFNA9-causing mutation, future clinical application is not limited by the potential presence of the target on the patient’s wild-type allele. In order to develop a mutant-allele-specific therapy for DFNA9, reliable discrimination between the mutant and the wild-type allele is of vital importance. However, the single-nucleotide changes in COCH underlying most cases of DFNA9 restrict the design of allele-discriminating therapies. In search of additional variants that can be exploited to improve discrimination between the c.151C>T mutant and wild-type COCH allele, we subjected the genomic COCH sequence of three DFNA9 patients to long-read SMRT sequencing. We amplified the COCH gene in three fragments that contain overlapping single-nucleotide polymorphisms (SNPs) (c.151C>T and c.734-304T>G) to aid haplotype assembly (Figure 1A). The identified variants are annotated on transcript NM_001135058.1, which does not contain the extended second coding exon. To identify targetable allele-specific variants that potentially allow for the treatment of the majority of the Dutch/Belgian DFNA9 patients, we filtered the variants in cis with the c.151C>T mutation for a population frequency below 20%. This resulted in the identification of 11 deep-intronic variants that are specific for the c.151C>T mutant COCH allele and have population frequencies between 5% and 10% (Figure 1B; Table 1). The identified variants provide additional targets for the development of a mutant allele-specific genetic therapy. The identified variants were validated using Sanger sequencing and confirmed to segregate with the c.151C>T mutation in COCH in two branches of Dutch DFNA9 families (Figure S1).Table 1Identified low-frequency variants on the c.151C>T COCH haplotypeLocationSNP identifierNucleotide change (HGVS)Amino acid changeFrequency (percentage) gnomAD European non-Finnishe4rs28938175c.151C>TPro51SerT: 0.0032i4rs143609554c.240-239A>TT: 5.4i6rs7140538c.436+185G>TT: 5.5i6rs10701465c.436+368_436+369dupAGdupAG: 5.5i8rs186627205c.629+1186T>CC: 5.4i8rs200080665c.629+1779delCdelC: 5.4i8rs368638521c.629+1807delAdelA: 5.9aNo data in gnomAD; frequency data from dbSNP 153.i8rs554238963c.629+1809A>CC: 9.9aNo data in gnomAD; frequency data from dbSNP 153.i8rs184635675c.629+1812A>TT: 5.4i8rs2295128c.630-208A>CC: 5.3i9rs28362773c.734-304T>GG: 7.2i11rs17097458c.1477+9C>AA: 5.4HGVS, Human Genome Variant Society.a No data in gnomAD; frequency data from dbSNP 153. Open table in a new tab HGVS, Human Genome Variant Society. We selected the c.151C>T founder mutation and the intronic, mutant-allele-specific variant c.436+368_436+369dupAG as targets for AON-based therapy. In contrast with the identified single-nucleotide changes or deletions, the c.436+368_436+369dupAG variant is the only multi-nucleotide variant that is specific to the mutant allele. Based on this, we hypothesized that AONs directed against this variant can provide the highest allele specificity. To design AONs, we combined the criteria that are commonly used to design splice-switching AONs with the previously established notion that RNase H1-dependent AONs require a series of nucleotides with DNA-like properties in their central region.16Pallan P.S. Egli M. Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex.Cell Cycle. 2008; 7: 2562-2569Crossref PubMed Scopus (21) Google Scholar, 17Aartsma-Rus A. van Vliet L. Hirschi M. Janson A.A.M. Heemskerk H. de Winter C.L. de Kimpe S. van Deutekom J.C.T. ’t Hoen P.A.C. van Ommen G.J. Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms.Mol. Ther. 2009; 17: 548-553Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Slijkerman R. Kremer H. van Wijk E. Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays.Methods Mol. Biol. 2018; 1828: 519-530Crossref PubMed Scopus (3) Google Scholar All possible AONs were investigated for thermodynamic properties in silico, with particular attention to the difference in binding affinity between the mutant and wild-type COCH mRNA. Targeting regions of all AONs used in this study are shown in Figure 2A. Note that the difference in binding affinity between the mutant and wild-type COCH mRNA was predicted to be larger for the AONs directed against the dupAG variant (c.436+368_436+369dupAG) compared to those directed against the single nucleotide substation (c.151C>T) (Table S1). The recognition of RNA/DNA duplexes by RNase H1 relates to the nature of the carbohydrate moiety in the AON backbone (2′-ribose versus 2′-deoxyribose).16Pallan P.S. Egli M. Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex.Cell Cycle. 2008; 7: 2562-2569Crossref PubMed Scopus (21) Google Scholar Therefore, AONs were either comprised completely of phosphorothioate (PS)-linked DNA bases, or of a central “gap” region of PS-DNA bases flanked by wings of 2′-O-methyl-RNA bases (gapmers). The gapmer design is particularly suitable for clinical application, as the nuclease-resistant 2′-modified ribonucleotides provide an increased binding affinity and half-life time.12Crooke S.T. Molecular mechanisms of action of antisense drugs.Biochim. Biophys. Acta. 1999; 1489: 31-44Crossref PubMed Scopus (343) Google Scholar,19Lima W.F. Crooke S.T. Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids.Biochemistry. 1997; 36: 390-398Crossref PubMed Scopus (112) Google Scholar,20Bennett C.F. Swayze E.E. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform.Annu. Rev. Pharmacol. Toxicol. 2010; 50: 259-293Crossref PubMed Scopus (949) Google Scholar The COCH expression levels in patient-derived primary fibroblast and Epstein-Barr virus-transformed lymphoblastoid cells are too low to reliably determine the effect of RNase H1-dependent AONs. Therefore, we used the Flp-In system to generate two stable transgenic T-REx 293 cell lines, expressing either a mutant (including three deep-intronic allele-discriminating variants; Figure 1) or a wild-type COCH minigene construct under the control of a tetracycline-dependent promotor. The minigene constructs span the genomic COCH sequence between the transcription initiation site and the last complete nucleotide triplet of exon 7 (Figure S2A). For both alleles, several clones were expanded and investigated for inducible COCH expression (Figure S2B). Correct pre-mRNA splicing of both wild-type and mutant minigene COCH exons 1–7 was confirmed with RT-PCR (Figure S2C). In order to reliably quantify mutant and wild-type COCH transcript levels, we used a custom TaqMan assay (Applied Biosystems), in which different fluorophores are coupled to probes specific for either the mutant or the wild-type transcript. As the COCH gene is continuously expressed in the human cochlea, we opted for an experimental design in which COCH transcription remains active. To induce COCH transcription, seeded cells were treated overnight with tetracycline (0.25 μg/mL). Next morning, culture medium was replaced by fresh tetracycline-containing medium, and cells were transfected with the AONs. For the initial screening of AONs, we transfected AONs (n = 1) to a final concentration in the culture medium of 250 nM. This dose was selected based on the work of Naessens et al.,21Naessens S. Ruysschaert L. Lefever S. Coppieters F. De Baere E. Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa.Genes (Basel). 2019; 10: 363Crossref Scopus (12) Google Scholar who showed a 50%–70% decrease in target transcripts in an overexpression-based cell model to identified candidate AONs for the future treatment of NR2E3-associated retinitis pigmentosa. Six (out of seven) AONs directed against the c.151C>T mutation (Figure 2B) and four (out of seven) AONs directed against the dupAG variant (Figure 2C) were able to decrease the level of mutant COCH transcripts as compared to a scrambled control AON. Three of the most effective AONs directed against the c.151C>T mutation, and one AON directed against the dupAG variant, were analyzed in more detail using different concentrations of gapmer AONs and multiple technical replications (Figure 3). c.151C>T AON-A was able to induce a significant decrease in mutant COCH transcripts at a dose of 250 nM (p = 0.02, Tukey’s multiple comparison test), but not at 100 nM (Figure 3A). While AON-B showed a stronger effect in comparison to AON-A in the initial screening, the effect sizes of AON-A and -B were very similar in this replication experiment (Figure 3B). A significant decrease of mutant COCH transcripts was found at 100 nM and 250 nM (p < 0.0012, Tukey’s multiple comparison test). The third AON directed against the c.151C>T mutation that was investigated in more detail, AON-E, did show a typical dose-dependent decrease in mutant COCH transcript levels. At 100 nM, the level of mutant COCH transcripts was approximately half of the number of transcripts detected in cells treated with a scrambled control AON (p < 0.0002, Tukey’s multiple comparison test). Mutant COCH transcript levels were even further decreased in cells transfected with 250 nM of AON-E (p < 0.0001, Tukey’s multiple comparison test). While on average the AONs directed against the dupAG variants appeared slightly less effective in the initial AON screen, transfection of mutant COCH-minigene-expressing cells with dupAG AON-B resulted in a significant decrease in mutant COCH transcripts at the three concentrations tested (Figure 3D; p < 0.0009, Tukey’s multiple comparison test). The highest effect size appears already to be achieved at 25 nM. The maximum effect size of dupAG AON-B was similar to the effect observed for c.151C>T AON-A and -B, but it was much less when compared to c.151C>T AON-E. We next investigated the ability of these four AONs in discriminating between mutant and wild-type COCH transcripts (Figure 4). We chose to compare the AONs at a concentration of 100 nM, as three out of the four AONs were able to significantly reduce mutant COCH transcript levels at this concentration. As observed previously, transfection of mutant COCH minigene cells with c.151C>T AON-B, c.151C>T AON-E, and dupAG AON-B significantly decreased mutant COCH transcript levels as compared to a scrambled control AON (Figure 4A). None of the four AONs induced a significant decrease of wild-type COCH transcripts when transfected in wild-type COCH-expressing transgenic cells, although we did observe a marked decrease in both mutant and wild-type COCH transcript levels resulting from the transfection of c.151C>T AON-A (Figure 4B). Likely, the correction for multiple comparisons explains the lack of a significant difference between c.151C>T AON-A-treated and scrambled AON-treated wild-type COCH minigene cells. While both cell lines display the same tetracycline-induced 18-fold increase in COCH minigene expression (Figure 4, left y axis), the expression levels relative to housekeeping gene RPS18 differ between clones (Figure 4, right y axis). The higher normalized expression of wild-type COCH minigene transcripts as compared to mutant COCH minigene transcripts may lead to an overestimation of allele specificity of the AONs. Furthermore, the lack of mutant COCH transcripts in these cells poorly resembles the situation in patients. To better determine allele specificity, we searched for a mutant COCH minigene clone with similar expression levels of tetracycline-induced mutant COCH minigene and endogenous (wild-type) COCH. Subjecting these cells to three different AON concentrations revealed that c.151C>T AON-E can induce a significant and allele-specific reduction in mutant COCH transcript level at 25 nM (Figure 5A) (p < 0.0001, Tukey’s multiple comparison test). At 100 nM and 250 nM, c.151C>T AON-E induces a concomitant decrease in endogenous wild-type COCH levels. Nevertheless, the 4- to 6-fold lower levels of mutant COCH transcripts as compared to wild-type COCH transcripts indicate that c.151C>T AON-E has a stronger affinity for the mutant transcript. In the same cell model, transfection of 25 nM of dupAG AON-B did not result in significant differences in mutant and wild-type COCH transcript levels (Figure 5B). A scrambled control AON was included in this experiment to confirm that the observed effect is specific to the AON sequence. Transfection of the cells with the scrambled AON mildly increases endogenous wild-type COCH expression but not the expression of the tetracycline-induced mutant COCH minigene. Although compared to the delivery of scrambled AON, c.151C>T AON-E does reduce the levels of wild-type COCH transcripts with 30% (p = 0.14, Tukey’s multiple comparison test), the comparison with untransfected cells expressing both mutant and wild-type COCH transcripts is most relevant from a therapeutic point of view. The c.151C>T founder mutation in COCH is estimated to be one of the most prevalent causes of dominantly inherited, adult-onset hearing loss and vestibular dysfunction in Northwest Europe, affecting >1,000 individuals in the Dutch/Belgian population. In this work, we present 11 intronic variants in cis with the c.151C>T mutation and show that these variants can be exploited for the development of a mutant-allele-specific therapy using RNase H1-dependent AONs. We identified a highly effective AON, directed against the c.151C>T mutation, as the most promising candidate for further preclinical development. The ability of AONs to specifically target mutant transcripts for degradation is of key importance for the development of an AON-based therapy for dominantly inherited disorders with a dominant-negative or gain-of-function disease mechanism such as DFNA9. The therapeutic strategy must be potent enough to prevent the synthesis of proteins from the mutant allele but allow sufficient protein synthesis from the wild-type allele for normal inner ear function. For any antisense-based approach, discrimination between alleles based on a single-nucleotide difference presents as a potential pitfall in terms of concomitant downregulation of the wild-type allele.22Zaleta-Rivera K. Dainis A. Ribeiro A.J.S. Cordero P. Rubio G. Shang C. Liu J. Finsterbach T. Parikh V.N. Sutton S. et al.Allele-Specific Silencing Ameliorates Restrictive Cardiomyopathy Attributable to a Human Myosin Regulatory Light Chain Mutation.Circulation. 2019; 140: 765-778Crossref PubMed Scopus (17) Google Scholar, 23Jiang J. Wakimoto H. Seidman J.G. Seidman C.E. Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy.Science. 2013; 342: 111-114Crossref PubMed Scopus (125) Google Scholar, 24Southwell A.L. Skotte N.H. Kordasiewicz H.B. Østergaard M.E. Watt A.T. Carroll J.B. Doty C.N. Villanueva E.B. Petoukhov E. Vaid K. et al.In vivo evaluation of candidate allele-specific mutant huntingtin gene silencing antisense oligonucleotides.Mol. Ther. 2014; 22: 2093-2106Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar Recently published AONs directed against a mutation in NR2E3, causative for dominantly inherited retinitis pigmentosa, also significantly reduced the wild-type transcript and protein levels.21Naessens S. Ruysschaert L. Lefever S. Coppieters F. De Baere E. Antisense Oligonucleotide-Based Downregulation of the G56R Pathogenic Variant Causing NR2E3-Associated Autosomal Dominant Retinitis Pigmentosa.Genes (Basel). 2019; 10: 363Crossref Scopus (12) Google Scholar In contrast, for Huntington’s disease (HTT gene), also resulting from a non-haploinsufficiency disease mechanism, the use of gapmer AONs to target a SNP specific to the mutant allele emerged as a promising therapeutic strategy in vitro and in vivo.15Southwell A.L. Kordasiewicz H.B. Langbehn D. Skotte N.H. Parsons M.P. Villanueva E.B. Caron N.S. Østergaard M.E. Anderson L.M. Xie Y. et al.Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease.Sci. Transl. Med. 2018; 10: eaar3959Crossref PubMed Scopus (66) Google Scholar As nearly all cases of DFNA9 are caused by single-nucleotide changes,25Bae S.-H. Robertson N.G. Cho H.-J. Morton C.C. Jung D.J. Baek J.-I. Choi S.-Y. Lee J. Lee K.-Y. Kim U.-K. Identification of pathogenic mechanisms of COCH mutations, abolished cochlin secretion, and intracellular aggregate formation: genotype-phenotype correlations in DFNA9 deafness and vestibular disorder.Hum. Mutat. 2014; 35: 1506-1513Crossref PubMed Scopus (32) Google Scholar we explored the presence of mutant-allele-specific variants that can serve as additional targets to develop a therapeutic strategy for the most frequently occurring DFNA9 mutation, c.151C>T. Here, we employed SMRT sequencing26Eid J. Fehr A. Gray J. Luong K. Lyle J. Otto G. Peluso P. Rank D. Baybayan P. Bettman B. et al.Real-time DNA sequencin" @default.
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• W3135704865 title "AON-based degradation of c.151C>T mutant COCH transcripts associated with dominantly inherited hearing impairment DFNA9" @default.
• W3135704865 cites W1525023123 @default.
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