TMC function, dysfunction, and restoration in mouse vestibular organs.

Tmc1 and Tmc2 are essential pore-forming subunits of mechanosensory transduction channels localized to the tips of stereovilli in auditory and vestibular hair cells of the inner ear. To investigate expression and function of Tmc1 and Tmc2 in vestibular organs, we used quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization - hairpin chain reaction (FISH-HCR), immunostaining, FM1-43 uptake and we measured vestibular evoked potentials (VsEPs) and vestibular ocular reflexes (VORs). We found that Tmc1 and Tmc2 showed dynamic developmental changes, differences in regional expression patterns, and overall expression levels which differed between the utricle and saccule. These underlying changes contributed to unanticipated phenotypic loss of VsEPs and VORs in Tmc1 KO mice. In contrast, Tmc2 KO mice retained VsEPs despite the loss of the calcium buffering protein calretinin, a characteristic biomarker of mature striolar calyx-only afferents. Lastly, we found that neonatal Tmc1 gene replacement therapy is sufficient to restore VsEP in Tmc1 KO mice for up to six months post-injection.

TMEM63 proteins function as monomeric high-threshold mechanosensitive ion channels.

OSCA/TMEM63s form mechanically activated (MA) ion channels in plants and animals, respectively. OSCAs and related TMEM16s and transmembrane channel-like (TMC) proteins form homodimers with two pores. Here, we uncover an unanticipated monomeric configuration of TMEM63 proteins. Structures of TMEM63A and TMEM63B (referred to as TMEM63s) revealed a single highly restricted pore. Functional analyses demonstrated that TMEM63s are bona fide mechanosensitive ion channels, characterized by small conductance and high thresholds. TMEM63s possess evolutionary variations in the intracellular linker IL2, which mediates dimerization in OSCAs. Replacement of OSCA1.2 IL2 with TMEM63A IL2 or mutations to key variable residues resulted in monomeric OSCA1.2 and MA currents with significantly higher thresholds. Structural analyses revealed substantial conformational differences in the mechano-sensing domain IL2 and gating helix TM6 between TMEM63s and OSCA1.2. Our studies reveal that mechanosensitivity in OSCA/TMEM63 channels is affected by oligomerization and suggest gating mechanisms that may be shared by OSCA/TMEM63, TMEM16, and TMC channels.

Complexes of vertebrate TMC1/2 and CIB2/3 proteins form hair-cell mechanotransduction cation channels.

Calcium and integrin-binding protein 2 (CIB2) and CIB3 bind to transmembrane channel-like 1 (TMC1) and TMC2, the pore-forming subunits of the inner-ear mechanoelectrical transduction (MET) apparatus. Whether these interactions are functionally relevant across mechanosensory organs and vertebrate species is unclear. Here we show that both CIB2 and CIB3 can form heteromeric complexes with TMC1 and TMC2 and are integral for MET function in mouse cochlea and vestibular end organs as well as in zebrafish inner ear and lateral line. Our AlphaFold 2 models suggest that vertebrate CIB proteins can simultaneously interact with at least two cytoplasmic domains of TMC1 and TMC2 as validated using nuclear magnetic resonance spectroscopy of TMC1 fragments interacting with CIB2 and CIB3. Molecular dynamics simulations of TMC1/2 complexes with CIB2/3 predict that TMCs are structurally stabilized by CIB proteins to form cation channels. Overall, our work demonstrates that intact CIB2/3 and TMC1/2 complexes are integral to hair-cell MET function in vertebrate mechanosensory epithelia.

Genotype-Phenotype Correlations in TMPRSS3 (DFNB10/DFNB8) with Emphasis on Natural History.

BACKGROUND: Mutations in TMPRSS3 are an important cause of autosomal recessive non-syndromic hearing loss. The hearing loss associated with mutations in TMPRSS3 is characterized by phenotypic heterogeneity, ranging from mild to profound hearing loss, and is generally progressive. Clinical presentation and natural history of TMPRSS3 mutations vary significantly based on the location and type of mutation in the gene. Understanding these genotype-phenotype relationships and associated natural disease histories is necessary for the successful development and application of gene-based therapies and precision medicine approaches to DFNB8/10. The heterogeneous presentation of TMPRSS3-associated disease makes it difficult to identify patients clinically. As the body of literature on TMPRSS3-associated deafness grows, there is need for better categorization of the hearing phenotypes associated with specific mutations in the gene. SUMMARY: In this review, we summarize TMPRSS3 genotype-phenotype relationships including a thorough description of the natural history of patients with TMPRSS3-associated hearing loss to lay the groundwork for the future of TMPRSS3 treatment using molecular therapy. KEY MESSAGES: TMPRSS3 mutation is a significant cause of genetic hearing loss. All patients with TMPRSS3 mutation display severe-to-profound prelingual (DFNB10) or a postlingual (DFNB8) progressive sensorineural hearing loss. Importantly, TMPRSS3 mutations have not been associated with middle ear or vestibular deficits. The c.916G>A (p.Ala306Thr) missense mutation is the most frequently reported mutation across populations and should be further explored as a target for molecular therapy.

Delivery of gene therapy through a cerebrospinal fluid conduit to rescue hearing in adult mice.

Inner ear gene therapy has recently effectively restored hearing in neonatal mice, but it is complicated in adulthood by the structural inaccessibility of the cochlea, which is embedded within the temporal bone. Alternative delivery routes may advance auditory research and also prove useful when translated to humans with progressive genetic-mediated hearing loss. Cerebrospinal fluid flow via the glymphatic system is emerging as a new approach for brain-wide drug delivery in rodents as well as humans. The cerebrospinal fluid and the fluid of the inner ear are connected via a bony channel called the cochlear aqueduct, but previous studies have not explored the possibility of delivering gene therapy via the cerebrospinal fluid to restore hearing in adult deaf mice. Here, we showed that the cochlear aqueduct in mice exhibits lymphatic-like characteristics. In vivo time-lapse magnetic resonance imaging, computed tomography, and optical fluorescence microscopy showed that large-particle tracers injected into the cerebrospinal fluid reached the inner ear by dispersive transport via the cochlear aqueduct in adult mice. A single intracisternal injection of adeno-associated virus carrying solute carrier family 17, member 8 (Slc17A8), which encodes vesicular glutamate transporter-3 (VGLUT3), rescued hearing in adult deaf Slc17A8-/- mice by restoring VGLUT3 protein expression in inner hair cells, with minimal ectopic expression in the brain and none in the liver. Our findings demonstrate that cerebrospinal fluid transport comprises an accessible route for gene delivery to the adult inner ear and may represent an important step toward using gene therapy to restore hearing in humans.

Hearing Preservation and Spatial Hearing Outcomes After Cochlear Implantation in Children With TMPRSS3 Mutations.

OBJECTIVE: Investigate hearing preservation and spatial hearing outcomes in children with TMPRSS3 mutations who received bilateral cochlear implantation. STUDY DESIGN AND METHODS: Longitudinal case series report. Two siblings (ages, 7 and 4 yr) with TMPRSS3 mutations with down-sloping audiograms received sequential bilateral cochlear implantation with hearing preservation with low-frequency acoustic amplification and high-frequency electrical stimulation. Spatial hearing, including speech perception and localization, was assessed at three time points: preoperative, postoperative of first and second cochlear implant (CI). RESULTS: Both children showed low-frequency hearing preservation in unaided, acoustic-only audiograms. Both children demonstrated improvements in speech perception in both quiet and noise after CI activations. The emergence of spatial hearing was observed. Each child's overall speech perception and spatial hearing when listening with bilateral CIs were within the range or better than published group data from children with bilateral CIs of other etiology. CONCLUSION: Bilateral cochlear implantation with hearing preservation is a viable option for managing hearing loss for pediatric patients with TMPRSS3 mutations.

Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11.

Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene (Tmc1) and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of Tmc1Δ/Δ mice and the new humanized Tmc1-p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in Tmc1-p.N193I mice injected with AAV9-PHP.B-CB6-hTMC1-WPRE. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B-CB6-hTMC1-WPRE construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive TMC1 mutations.

Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.

pH regulates potassium conductance and drives a constitutive proton current in human TMEM175.

Human TMEM175, a noncanonical potassium (K+) channel in endolysosomes, contributes to their pH stability and is implicated in the pathogenesis of Parkinson's disease (PD). Structurally, the TMEM175 family exhibits an architecture distinct from canonical potassium channels, as it lacks the typical TVGYG selectivity filter. Here, we show that human TMEM175 not only exhibits pH-dependent structural changes that reduce K+ permeation at acidic pH but also displays proton permeation. TMEM175 constitutively conducts K+ at pH 7.4 but displays reduced K+ permeation at lower pH. In contrast, proton current through TMEM175 increases with decreasing pH because of the increased proton gradient. Molecular dynamics simulation, structure-based mutagenesis, and electrophysiological analysis suggest that K+ ions and protons share the same permeation pathway. The M393T variant of human TMEM175 associated with PD shows reduced function in both K+ and proton permeation. Together, our structural and electrophysiological analysis reveals a mechanism of TMEM175 regulation by pH.

Dual-vector gene therapy restores cochlear amplification and auditory sensitivity in a mouse model of DFNB16 hearing loss.

Hearing loss affects an estimated 466 million people worldwide, with a substantial fraction due to genetic causes. Approximately 16% of genetic hearing loss is caused by pathogenic mutations in STRC, a gene that encodes the protein stereocilin. To develop gene therapy strategies for patients with STRC hearing loss, we generated a mouse model with a targeted deletion in the Strc gene. We devised a novel dual-vector approach to circumvent the size limitation of AAV vectors and drive expression of full-length STRC protein. To target outer hair cells, which are difficult to transduce, we used synthetic AAV9-PHP.B vectors for efficient dual-vector transduction. We report robust recovery of exogenous STRC expression in outer hair cells of Strc-deficient mice, recovery of hair bundle morphology, substantially improved cochlear amplification, and enhanced auditory sensitivity. The data raise the prospect that our strategy could benefit ~2.3 million patients worldwide affected by STRC mutations.

Sensory transduction is required for normal development and maturation of cochlear inner hair cell synapses.

Acoustic overexposure and aging can damage auditory synapses in the inner ear by a process known as synaptopathy. These insults may also damage hair bundles and the sensory transduction apparatus in auditory hair cells. However, a connection between sensory transduction and synaptopathy has not been established. To evaluate potential contributions of sensory transduction to synapse formation and development, we assessed inner hair cell synapses in several genetic models of dysfunctional sensory transduction, including mice lacking transmembrane channel-like (Tmc) 1, Tmc2, or both, in Beethoven mice which carry a dominant Tmc1 mutation and in Spinner mice which carry a recessive mutation in transmembrane inner ear (Tmie). Our analyses reveal loss of synapses in the absence of sensory transduction and preservation of synapses in Tmc1-null mice following restoration of sensory transduction via Tmc1 gene therapy. These results provide insight into the requirement of sensory transduction for hair cell synapse development and maturation.

Putting the Pieces Together: the Hair Cell Transduction Complex.

Identification of the components of the mechanosensory transduction complex in hair cells has been a major research interest for many auditory and vestibular scientists and has attracted attention from outside the field. The past two decades have witnessed a number of significant advances with emergence of compelling evidence implicating at least a dozen distinct molecular components of the transduction machinery. Yet, how the pieces of this ensemble fit together and function in harmony to enable the senses of hearing and balance has not been clarified. The goal of this review is to summarize a 2021 symposium presented at the annual mid-winter meeting of the Association for Research in Otolaryngology. The symposium brought together the latest insights from within and beyond the field to examine individual components of the transduction complex and how these elements interact at molecular, structural, and biophysical levels to gate mechanosensitive channels and initiate sensory transduction in the inner ear. The review includes a brief historical background to set the stage for topics to follow that focus on structure, properties, and interactions of proteins such as CDH23, PCDH15, LHFPL5, TMIE, TMC1/2, and CIB2/3. We aim to present the diversity of ideas in this field and highlight emerging theories and concepts. This review will not only provide readers with a deeper appreciation of the components of the transduction apparatus and how they function together, but also bring to light areas of broad agreement, areas of scientific controversy, and opportunities for future scientific discovery.

Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness.

Genetic variants account for approximately half the cases of congenital and early-onset deafness. Methods and technologies for viral delivery of genes into the inner ear have evolved over the past decade to render gene therapy a viable and attractive approach for treatment. Variants in SYNE4, encoding the protein nesprin-4, a member of the linker of nucleoskeleton and cytoskeleton (LINC), lead to DFNB76 human deafness. Syne4-/- mice have severe-to-profound progressive hearing loss and exhibit mislocalization of hair cell nuclei and hair cell degeneration. We used AAV9-PHP.B, a recently developed synthetic adeno-associated virus, to deliver the coding sequence of Syne4 into the inner ears of neonatal Syne4-/- mice. Here we report rescue of hair cell morphology and survival, nearly complete recovery of auditory function, and restoration of auditory-associated behaviors, without observed adverse effects. Uncertainties remain regarding the durability of the treatment and the time window for intervention in humans, but our results suggest that gene therapy has the potential to prevent hearing loss in humans with SYNE4 mutations.

The Mechanosensory Transduction Machinery in Inner Ear Hair Cells.

Sound-induced mechanical stimuli are detected by elaborate mechanosensory transduction (MT) machinery in highly specialized hair cells of the inner ear. Genetic studies of inherited deafness in the past decades have uncovered several molecular constituents of the MT complex, and intense debate has surrounded the molecular identity of the pore-forming subunits. How the MT components function in concert in response to physical stimulation is not fully understood. In this review, we summarize and discuss multiple lines of evidence supporting the hypothesis that transmembrane channel-like 1 is a long-sought MT channel subunit. We also review specific roles of other components of the MT complex, including protocadherin 15, cadherin 23, lipoma HMGIC fusion partner-like 5, transmembrane inner ear, calcium and integrin-binding family member 2, and ankyrins. Based on these recent advances, we propose a unifying theory of hair cell MT that may reconcile most of the functional discoveries obtained to date. Finally, we discuss key questions that need to be addressed for a comprehensive understanding of hair cell MT at molecular and atomic levels.

Single and Dual Vector Gene Therapy with AAV9-PHP.B Rescues Hearing in Tmc1 Mutant Mice.

AAV-mediated gene therapy is a promising approach for treating genetic hearing loss. Replacement or editing of the Tmc1 gene, encoding hair cell mechanosensory ion channels, is effective for hearing restoration in mice with some limitations. Efficient rescue of outer hair cell function and lack of hearing recovery with later-stage treatment remain issues to be solved. Exogenous genes delivered with the adeno-associated virus (AAV)9-PHP.B capsid via the utricle transduce both inner and outer hair cells of the mouse cochlea with high efficacy. Here, we demonstrate that AAV9-PHP.B gene therapy can promote hair cell survival and successfully rescues hearing in three distinct mouse models of hearing loss. Tmc1 replacement with AAV9-PHP.B in a Tmc1 knockout mouse rescues hearing and promotes hair cell survival with equal efficacy in inner and outer hair cells. The same treatment in a recessive Tmc1 hearing-loss model, Baringo, partially recovers hearing even with later-stage treatment. Finally, dual delivery of Streptococcus pyogenes Cas9 (SpCas9) and guide RNA (gRNA) in separate AAV9-PHP.B vectors selectively disrupts a dominant Tmc1 allele and preserves hearing in Beethoven mice, a model of dominant, progressive hearing loss. Tmc1-targeted gene therapies using single or dual AAV9-PHP.B vectors offer potent and versatile approaches for treating dominant and recessive deafness.

In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness.

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.