Dual-AAV vector-mediated expression of MYO7A improves vestibular function in a mouse model of Usher syndrome 1B.

Usher syndrome is the most common cause of deafness-blindness in the world. Usher syndrome type 1B (USH1B) is associated with mutations in MYO7A. Patients with USH1B experience deafness, blindness, and vestibular dysfunction. In this study, we applied adeno-associated virus (AAV)-mediated gene therapy to the shaker-1 (Myo7a4626SB/4626SB) mouse, a model of USH1B. The shaker-1 mouse has a nonsense mutation in Myo7a, is profoundly deaf throughout life, and has significant vestibular dysfunction. Because of the ∼6.7-kb size of the MYO7A cDNA, a dual-AAV approach was used for gene delivery, which involves splitting human MYO7A cDNA into 5' and 3' halves and cloning them into two separate AAV8(Y733F) vectors. When MYO7A cDNA was delivered to shaker-1 inner ears using the dual-AAV approach, cochlear hair cell survival was improved. However, stereocilium organization and auditory function were not improved. In contrast, in the vestibular system, dual-AAV-mediated MYO7A delivery significantly rescued hair cell stereocilium morphology and improved vestibular function, as reflected in a reduction of circling behavior and improved vestibular sensory-evoked potential (VsEP) thresholds. Our data indicate that dual-AAV-mediated MYO7A expression improves vestibular function in shaker-1 mice and supports further development of this approach for the treatment of disabling dizziness from vestibular dysfunction in USH1B patients.

Function of bidirectional sensitivity in the otolith organs established by transcription factor Emx2.

Otolith organs of the inner ear are innervated by two parallel afferent projections to the brainstem and cerebellum. These innervations were proposed to segregate across the line of polarity reversal (LPR) within each otolith organ, which divides the organ into two regions of hair cells (HC) with opposite stereociliary orientation. The relationship and functional significance of these anatomical features are not known. Here, we show regional expression of Emx2 in otolith organs, which establishes LPR, mediates the neuronal segregation across LPR and constitutes the bidirectional sensitivity function. Conditional knockout (cKO) of Emx2 in HCs lacks LPR. Tmie cKO, in which mechanotransduction was abolished selectively in HCs within the Emx2 expression domain also lacks bidirectional sensitivity. Analyses of both mutants indicate that LPR is specifically required for mice to swim comfortably and to traverse a balance beam efficiently, but LPR is not required for mice to stay on a rotating rod.

Single-Cell RNA-Seq of Cisplatin-Treated Adult Stria Vascularis Identifies Cell Type-Specific Regulatory Networks and Novel Therapeutic Gene Targets.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

Assessment of auditory and vestibular damage in a mouse model after single and triple blast exposures.

The use of explosive devices in war and terrorism has increased exposure to concussive blasts among both military personnel and civilians, which can cause permanent hearing and balance deficits that adversely affect survivors' quality of life. Significant knowledge gaps on the underlying etiology of blast-induced hearing loss and balance disorders remain, especially with regard to the effect of blast exposure on the vestibular system, the impact of multiple blast exposures, and long-term recovery. To address this, we investigated the effects of blast exposure on the inner ear using a mouse model in conjunction with a high-fidelity blast simulator. Anesthetized animals were subjected to single or triple blast exposures, and physiological measurements and tissue were collected over the course of recovery for up to 180 days. Auditory brainstem responses (ABRs) indicated significantly elevated thresholds across multiple frequencies. Limited recovery was observed at low frequencies in single-blasted mice. Distortion Product Otoacoustic Emissions (DPOAEs) were initially absent in all blast-exposed mice, but low-amplitude DPOAEs could be detected at low frequencies in some single-blast mice by 30 days post-blast, and in some triple-blast mice at 180 days post-blast. All blast-exposed mice showed signs of Tympanic Membrane (TM) rupture immediately following exposure and loss of outer hair cells (OHCs) in the basal cochlear turn. In contrast, the number of Inner Hair Cells (IHCs) and spiral ganglion neurons was unchanged following blast-exposure. A significant reduction in IHC pre-synaptic puncta was observed in the upper turns of blast-exposed cochleae. Finally, we found no significant loss of utricular hair cells or changes in vestibular function as assessed by vestibular evoked potentials. Our results suggest that (1) blast exposure can cause severe, long-term hearing loss which may be partially due to slow TM healing or altered mechanical properties of healed TMs, (2) traumatic levels of sound can still reach the inner ear and cause basal OHC loss despite middle ear dysfunction caused by TM rupture, (3) blast exposure may result in synaptopathy in humans, and (4) balance deficits after blast exposure may be primarily due to traumatic brain injury, rather than damage to the peripheral vestibular system.

Expression of a membrane-targeted fluorescent reporter disrupts auditory hair cell mechanoelectrical transduction and causes profound deafness.

The reporter mT/mG mice expressing a membrane-targeted fluorescent protein are becoming widely used to study the auditory and vestibular system due to its versatility. Here we show that high expression levels of the fluorescent mtdTomato reporter affect the function of the sensory hair cells and the auditory performance of mT/mG transgenic mice. Auditory brainstem responses and distortion product otoacoustic emissions revealed that adult mT/mG homozygous mice are profoundly deaf, whereas heterozygous mice present high frequency loss. We explore whether this line would be useful for studying and visualizing the membrane of auditory hair cells by airyscan super-resolution confocal microscopy. Membrane localization of the reporter was observed in hair cells of the cochlea, facilitating imaging of both cell bodies and stereocilia bundles without altering cellular architecture or the expression of the integral membrane motor protein prestin. Remarkably, hair cells from mT/mG homozygous mice failed to uptake the FM1-43 dye and to locate TMC1 at the stereocilia, indicating defective mechanotransduction machinery. Our work emphasizes that precautions must be considered when working with reporter mice and highlights the potential role of the cellular membrane in maintaining functional hair cells and ensuring proper hearing.

Blast-induced hearing impairment in rats is associated with structural and molecular changes of the inner ear.

Auditory dysfunction is the most prevalent injury associated with blast overpressure exposure (BOP) in Warfighters and civilians, yet little is known about the underlying pathophysiological mechanisms. To gain insights into these injuries, an advanced blast simulator was used to expose rats to BOP and assessments were made to identify structural and molecular changes in the middle/inner ears utilizing otoscopy, RNA sequencing (RNA-seq), and histopathological analysis. Deficits persisting up to 1 month after blast exposure were observed in the distortion product otoacoustic emissions (DPOAEs) and the auditory brainstem responses (ABRs) across the entire range of tested frequencies (4-40 kHz). During the recovery phase at sub-acute time points, low frequency (e.g. 4-8 kHz) hearing improved relatively earlier than for high frequency (e.g. 32-40 kHz). Perforation of tympanic membranes and middle ear hemorrhage were observed at 1 and 7 days, and were restored by day 28 post-blast. A total of 1,158 differentially expressed genes (DEGs) were significantly altered in the cochlea on day 1 (40% up-regulated and 60% down-regulated), whereas only 49 DEGs were identified on day 28 (63% up-regulated and 37% down-regulated). Seven common DEGs were identified at both days 1 and 28 following blast, and are associated with inner ear mechanotransduction, cytoskeletal reorganization, myelin development and axon survival. Further studies on altered gene expression in the blast-injured rat cochlea may provide insights into new therapeutic targets and approaches to prevent or treat similar cases of blast-induced auditory damage in human subjects.

Ototoxicity and Platinum Uptake Following Cyclic Administration of Platinum-Based Chemotherapeutic Agents.

Cisplatin is a widely used anti-cancer drug used to treat a variety of cancer types. One of the side effects of this life-saving drug is irreversible ototoxicity, resulting in permanent hearing loss in many patients. In order to understand why cisplatin is particularly toxic to the inner ear, we compared the hearing loss and cochlear uptake of cisplatin to that of two related drugs, carboplatin and oxaliplatin. These three drugs are similar in that each contains a core platinum atom; however, carboplatin and oxaliplatin are considered less ototoxic than cisplatin. We delivered these three drugs to mice using a 6-week cyclic drug administration protocol. We performed the experiment twice, once using equimolar concentrations of the drugs and once using concentrations of the drugs more proportional to those used in the clinic. For both concentrations, we detected a significant hearing loss caused by cisplatin and no hearing loss caused by carboplatin or oxaliplatin. Cochlear uptake of each drug was measured using inductively coupled plasma mass spectrometry (ICP-MS) to detect platinum. Cochlear platinum levels were highest in mice treated with cisplatin followed by oxaliplatin, while carboplatin was largely excluded from the cochlea. Even when the drug doses were increased, cochlear platinum remained low in mice treated with oxaliplatin or carboplatin. We also examined drug clearance from the inner ear by measuring platinum levels at 1 h and 24 h after drug administration. Our findings suggest that the reduced cochlear platinum we observed with oxaliplatin and carboplatin were not due to increased clearance of these drugs relative to cisplatin. Taken together, our data indicate that the differential ototoxicity among cisplatin, carboplatin, and oxaliplatin is attributable to differences in cochlear uptake of these three drugs.

Noncoding Microdeletion in Mouse Hgf Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in HGF are associated with nonsyndromic deafness DFNB39 However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Hgf Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in Hgf leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness.SIGNIFICANCE STATEMENT Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of HGF encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a Hgf mutant mouse with a small 10 bp deletion recapitulating a human DFNB39 noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.

Short-term NAD+ supplementation prevents hearing loss in mouse models of Cockayne syndrome.

Age-related hearing loss (ARHL) is one of the most common disorders affecting elderly individuals. There is an urgent need for effective preventive measures for ARHL because none are currently available. Cockayne syndrome (CS) is a premature aging disease that presents with progressive hearing loss at a young age, but is otherwise similar to ARHL. There are two human genetic complementation groups of CS, A and B. While the clinical phenotypes in patients are similar, the proteins have very diverse functions, and insight into their convergence is of great interest. Here, we use mouse models for CS (CSA -/- and CSB m/m ) that recapitulate the hearing loss in human CS patients. We previously showed that NAD+, a key metabolite with various essential functions, is reduced in CS and associated with multiple CS phenotypes. In this study, we report that NAD+ levels are reduced in the cochlea of CSB m/m mice and that short-term treatment (10 days) with the NAD+ precursor nicotinamide riboside (NR), prevents hearing loss, restores outer hair cell loss, and improves cochlear health in CSB m/m mice. Similar, but more modest effects were observed in CSA -/- mice. Remarkably, we observed a reduction in synaptic ribbon counts in the presynaptic zones of inner hair cells in both CSA -/- and CSB m/m mice, pointing to a converging mechanism for cochlear defects in CS. Ribbon synapses facilitate rapid and sustained synaptic transmission over long periods of time. Ribeye, a core protein of synaptic ribbons, possesses an NAD(H) binding pocket which regulates its activity. Intriguingly, NAD+ supplementation rescues reduced synaptic ribbon formation in both CSA -/- and CSB m/m mutant cochleae. These findings provide valuable insight into the mechanism of CS- and ARHL-associated hearing loss, and suggest a possible intervention.

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

Pou4f1 Defines a Subgroup of Type I Spiral Ganglion Neurons and Is Necessary for Normal Inner Hair Cell Presynaptic Ca2+ Signaling.

Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells (IHCs) and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the IHC. More recently, single-cell profiling has identified multiple groups of SGNs based on transcriptional profiling; however, correlations between any of these groups and distinct neuronal physiology have not been determined. In this study, we show that expression of the POU (Pit-Oct-Unc) transcription factor Pou4f1 in type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early path-finding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca2+ channels in IHCs primarily by increasing their voltage sensitivity. Moreover, the modiolar to pillar gradient of active zone Ca2+ influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca2+ signaling in IHCs.SIGNIFICANCE STATEMENT Physiologically distinct classes of type I spiral ganglion neurons (SGNs) are necessary to encode sound intensities spanning the audible range. Although anatomical studies have demonstrated structural correlates for some physiologically defined classes of type I SGNs, an understanding of the molecular pathways that specify each type is only now emerging. Here, we demonstrate that expression of the transcription factor Pou4f1 corresponds to a distinct subgroup of type I SGNs that synapse on the modiolar side of inner hair cells. The conditional deletion of Pou4f1 after SGN formation does not disrupt ganglion size or morphology, change the distribution of IHC synaptic locations, or affect the creation of synapses, but it does influence the voltage dependence and strength of Ca2+ influx at presynaptic active zones in inner hair cells.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Tmc2 expression partially restores auditory function in a mouse model of DFNB7/B11 deafness caused by loss of Tmc1 function.

Mouse Tmc1 and Tmc2 are required for sensory transduction in cochlear and vestibular hair cells. Homozygous Tmc1∆/∆ mice are deaf, Tmc2∆/∆ mice have normal hearing, and double homozygous Tmc1∆/∆; Tmc2∆/∆ mice have deafness and profound vestibular dysfunction. These phenotypes are consistent with their different spatiotemporal expression patterns. Tmc1 expression is persistent in cochlear and vestibular hair cells, whereas Tmc2 expression is transient in cochlear hair cells but persistent in vestibular hair cells. On the basis of these findings, we hypothesized that persistent Tmc2 expression in mature cochlear hair cells could restore auditory function in Tmc1∆/∆ mice. To express Tmc2 in mature cochlear hair cells, we generated a transgenic mouse line, Tg[PTmc1::Tmc2], in which Tmc2 cDNA is expressed under the control of the Tmc1 promoter. The Tg[PTmc1::Tmc2] transgene slightly but significantly restored hearing in young Tmc1∆/∆ mice, though hearing thresholds were elevated with age. The elevation of hearing thresholds was associated with deterioration of sensory transduction in inner hair cells and loss of outer hair cell function. Although sensory transduction was retained in outer hair cells, their stereocilia eventually degenerated. These results indicate distinct roles and requirements for Tmc1 and Tmc2 in mature cochlear hair cells.

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.

Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.

Harnessing molecular motors for nanoscale pulldown in live cells.

Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.

Slc26a4 expression prevents fluctuation of hearing in a mouse model of large vestibular aqueduct syndrome.

SLC26A4 mutations cause fluctuating and progressive hearing loss associated with enlargement of the vestibular aqueduct (EVA). SLC26A4 encodes a transmembrane anion exchanger called pendrin expressed in nonsensory epithelial cells of the lateral wall of cochlea, vestibular organs and endolymphatic sac. We previously described a transgenic mouse model of EVA with doxycycline (dox)-inducible expression of Slc26a4 in which administration of dox from conception to embryonic day 17.5 (DE17.5) resulted in hearing fluctuation between 1 and 3months of age. In the present study, we hypothesized that Slc26a4 is required to stabilize hearing in DE17.5 ears between 1 and 3months of age. We tested our hypothesis by evaluating the effect of postnatal re-induction of Slc26a4 expression on hearing. Readministration of dox to DE17.5 mice at postnatal day 6 (P6), but not at 1month of age, resulted in reduced click-evoked auditory brainstem response (ABR) thresholds, less fluctuation of hearing and a higher surface density of pendrin expression in spindle-shaped cells of the stria vascularis. Pendrin expression in spindle-shaped cells was inversely correlated with ABR thresholds. These findings suggest that stabilization of hearing by readministration of dox at P6 is mediated by pendrin expression in spindle-shaped cells. We conclude that early re-induction of Slc26a4 expression can prevent fluctuation of hearing in our Slc26a4-insufficient mouse model. Restoration of SLC26A4 expression and function could reduce or prevent fluctuation of hearing in EVA patients.

Gene Therapy Restores Hair Cell Stereocilia Morphology in Inner Ears of Deaf Whirler Mice.

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Cochlear gene transfer mediated by adeno-associated virus: Comparison of two surgical approaches.

OBJECTIVES/HYPOTHESIS: Gene therapy offers the possibility of delivering corrective genetic materials to the cochlea, potentially improving hearing. In animals, the most commonly used surgical methods for viral gene therapy delivery to the cochlea are the round window and the cochleostomy approaches. However, the patterns of viral infection and the effects on hearing have not been directly compared between these two approaches. In this study, we compare the patterns of cochlear infection and effects on hearing between these two surgical approaches using adeno-associated virus serotype 2/8 (AAV8) as the gene delivery vehicle. STUDY DESIGN: Animal study and basic science research. METHODS: One- to two-month-old CBA/J mice were used in this study. AAV8-green fluorescent protein (GFP) was delivered to the cochlea by either the round window or the cochleostomy approach (described below). Auditory brainstem response was used to examine hearing thresholds before and after surgery. Animals were examined at 1, 2, 3, and 4 weeks after surgery for the patterns of cochlear infection and hearing loss. RESULTS: Cochlear gene transfer was successful through both surgical approaches. In both approaches, AAV8-GFP mostly infected the inner hair cells. There was occasional low-level infection of the outer hair cells and supporting cells. The two surgical approaches resulted in comparable viral infection efficiencies. The round window approach resulted in less surgical trauma, as indicated by hearing loss, than the cochleostomy approach. CONCLUSIONS: Adeno-associated virus-mediated gene transfer to the cochlea can be accomplished using either the round window or the cochleostomy surgical approach. The round window approach resulted in less hearing loss compared to the cochleostomy approach. LEVEL OF EVIDENCE: NA.