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Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss.

ABSTRACT: Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in TMC1, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approaches is limited by the infeasibility of development and validation of novel constructs for each variant. We developed an allele-non-specific approach consisting of mutation-agnostic RNAi suppression of both mutant and WT alleles, co-delivered with a knockdown-resistant engineered WT allele with or without the use of woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to augment transgene expression. This therapeutic construct was delivered into the mature murine model of DFNA36 with an AAV vector and achieved robust hair cell and auditory brainstem response preservation. However, WPRE-enhanced Tmc1 expression resulted in inferior outcomes, suggesting a role for gene dosage optimization in future TMC1 gene therapy development.

SUBMITTER: Iwasa Y

PROVIDER: S-EPMC9795038 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Mutation-agnostic RNA interference with engineered replacement rescues <i>Tmc1</i>-related hearing loss.

Iwasa Yoichiro Y Klimara Miles J MJ Yoshimura Hidekane H Walls William D WD Omichi Ryotaro R West Cody A CA Shibata Seiji B SB Ranum Paul T PT Smith Richard Jh RJ

Life science alliance 20221227 3

Hearing loss is the most common sensory deficit, of which genetic etiologies are a frequent cause. Dominant and recessive mutations in <i>TMC1</i>, a gene encoding the pore-forming subunit of the hair cell mechanotransduction channel, cause DFNA36 and DFNB7/11, respectively, accounting for ∼2% of genetic hearing loss. Previous work has established the efficacy of mutation-targeted RNAi in treatment of murine models of autosomal dominant non-syndromic deafness. However, application of such approa ...[more]

PMID: 36574989

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Project description:Variants in TMC1 (transmembrane channel-like 1) can cause both autosomal dominant and recessive hearing loss in human population. Mice with Tmc1 variants have been shown to be ideal animal models for gene therapy. In this article, we report four TMC1 variants in four different Chinese families and the follow-up auditory phenotype of a previously reported family. Four families with TMC1 variants, as well as a previously described family with TMC1 variant orthologous to the Beethoven mouse, were recruited in this study. A comprehensive auditory evaluation was performed on all ascertained family members. High-throughput sequencing was conducted using genomic DNA from the probands and other family members to identify probable deafness genes. We identified four TMC1 (NM_138691.2) variations, including two pathogenic variants, c.1714G>A, and c.1253T>A, one likely pathogenic variant, c.[797T>C];[797T>C], and one single nucleotide polymorphism (SNP), c.2276G>A. Among these variants, c.[797T>C];[797T>C] is a novel likely pathogenic variant, and c.1714G>A and c.1253T>A are known pathogenic variants at the DFNB7/11 (DFNA36) locus. Phenotype-genotype correlation analysis of TMC1 variants showed that the TMC1 dominant variation-related phenotype was late-onset, progressive, high frequency to all frequency sensorineural hearing loss, while the TMC1 recessive variant was related to congenital all frequency sensorineural hearing impairment. Two pathogenic, one likely pathogenic variants and one SNP of TMC1 were identified in four Chinese families with hereditary hearing loss, indicating that TMC1 may be a more frequent cause of hearing loss than expected. TMC1 variants related to hearing loss result in specific phenotypes. The TMC1 c.1253T>A (p.M418K) variation, homologous to the Tmc1 c. 1235 T> A (p.M412K) variant in Beethoven mice, was the second report of this variant in human patients with hearing loss, suggesting the possibility to translational gene therapy from Beethoven mice to human patients.

Project description:BackgroundTMC1, which encodes transmembrane channel-like protein 1, forms the mechanoelectrical transduction (MET) channel in auditory hair cells, necessary for auditory function. TMC1 variants are known to cause autosomal dominant (DFNA36) and autosomal recessive (DFNB7/11) non-syndromic hearing loss, but only a handful of TMC1 variants underlying DFNA36 have been reported, hampering analysis of genotype-phenotype correlations.MethodsIn this study, we retrospectively reviewed 338 probands in an in-house database of genetic hearing loss, evaluating the clinical phenotypes and genotypes of novel TMC1 variants associated with DFNA36. To analyze the structural impact of these variants, we generated two structural models of human TMC1, utilizing the Cryo-EM structure of C. elegans TMC1 as a template and AlphaFold protein structure database. Specifically, the lipid bilayer-embedded protein database was used to construct membrane-embedded models of TMC1. We then examined the effect of TMC1 variants on intramolecular interactions and predicted their potential pathogenicity.ResultsWe identified two novel TMC1 variants related to DFNA36 (c.1256T > C:p.Phe419Ser and c.1444T > C:p.Trp482Arg). The affected subjects had bilateral, moderate, late-onset, progressive sensorineural hearing loss with a down-sloping configuration. The Phe419 residue located in the transmembrane domain 4 of TMC1 faces outward towards the channel pore and is in close proximity to the hydrophobic tail of the lipid bilayer. The non-polar-to-polar variant (p.Phe419Ser) alters the hydrophobicity in the membrane, compromising protein-lipid interactions. On the other hand, the Trp482 residue located in the extracellular linker region between transmembrane domains 5 and 6 is anchored to the membrane interfaces via its aromatic rings, mediating several molecular interactions that stabilize the structure of TMC1. This type of aromatic ring-based anchoring is also observed in homologous transmembrane proteins such as OSCA1.2. Conversely, the substitution of Trp with Arg (Trp482Arg) disrupts the cation-π interaction with phospholipids located in the outer leaflet of the phospholipid bilayer, destabilizing protein-lipid interactions. Additionally, Trp482Arg collapses the CH-π interaction between Trp482 and Pro511, possibly reducing the overall stability of the protein. In parallel with the molecular modeling, the two mutants degraded significantly faster compared to the wild-type protein, compromising protein stability.ConclusionsThis results expand the genetic spectrum of disease-causing TMC1 variants related to DFNA36 and provide insight into TMC1 transmembrane protein-lipid interactions.

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Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss.

Publications

Mutation-agnostic RNA interference with engineered replacement rescues <i>Tmc1</i>-related hearing loss.

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