Repurposable Drugs That Interact with Steroid Responsive Gene Targets for Inner Ear Disease.

Corticosteroids, oral or transtympanic, remain the mainstay for inner ear diseases characterized by hearing fluctuation or sudden changes in hearing, including sudden sensorineural hearing loss (SSNHL), Meniere's disease (MD), and autoimmune inner ear disease (AIED). Despite their use across these diseases, the rate of complete recovery remains low, and results across the literature demonstrates significant heterogeneity with respect to the effect of corticosteroids, suggesting a need to identify more efficacious treatment options. Previously, our group has cross-referenced steroid-responsive genes in the cochlea with published single-cell and single-nucleus transcriptome datasets to demonstrate that steroid-responsive differentially regulated genes are expressed in spiral ganglion neurons (SGN) and stria vascularis (SV) cell types. These differentially regulated genes represent potential druggable gene targets. We utilized multiple gene target databases (DrugBank, Pharos, and LINCS) to identify orally administered, FDA approved medications that potentially target these genes. We identified 42 candidate drugs that have been shown to interact with these genes, with an emphasis on safety profile, and tolerability. This study utilizes multiple databases to identify drugs that can target a number of druggable genes in otologic disorders that are commonly treated with steroids, providing a basis for establishing novel repurposing treatment trials.

A genome-wide meta-analysis uncovers six sequence variants conferring risk of vertigo.

Vertigo is the leading symptom of vestibular disorders and a major risk factor for falls. In a genome-wide association study of vertigo (Ncases = 48,072, Ncontrols = 894,541), we uncovered an association with six common sequence variants in individuals of European ancestry, including missense variants in ZNF91, OTOG, OTOGL, and TECTA, and a cis-eQTL for ARMC9. The association of variants in ZNF91, OTOGL, and OTOP1 was driven by an association with benign paroxysmal positional vertigo. Using previous reports of sequence variants associating with age-related hearing impairment and motion sickness, we found eight additional variants that associate with vertigo. Although disorders of the auditory and the vestibular system may co-occur, none of the six genome-wide significant vertigo variants were associated with hearing loss and only one was associated with age-related hearing impairment. Our results uncovered sequence variants associating with vertigo in a genome-wide association study and implicated genes with known roles in inner ear development, maintenance, and disease.

Phosphatidylinositol 4-kinase ß mutations cause nonsyndromic sensorineural deafness and inner ear malformation.

Congenital hearing loss is a common disorder worldwide. Heterogeneous gene variation accounts for approximately 20-25% of such patients. We investigated a five-generation Chinese family with autosomal-dominant nonsyndromic sensorineural hearing loss (SNHL). No wave was detected in the pure-tone audiometry, and the auditory brainstem response was absent in all patients. Computed tomography of the patients, as well as of two sporadic SNHL cases, showed bilateral inner ear anomaly, cochlear maldevelopment, absence of the osseous spiral lamina, and an enlarged vestibular aqueduct. Such findings were absent in nonaffected persons. We used linkage analysis and exome sequencing and uncovered a heterozygous missense mutation in the PI4KB gene (p.Gln121Arg) encoding phosphatidylinositol 4-kinase ß (PI4KB) from the patients in this family. In addition, 3 missense PI4KB (p.Val434Gly, p.Glu667Lys, and p.Met739Arg) mutations were identified in five patients with nonsyndromic SNHL from 57 sporadic cases. No such mutations were present within 600 Chinese controls, the 1000 genome project, gnomAD, or similar databases. Depleting pi4kb mRNA expression in zebrafish caused inner ear abnormalities and audiosensory impairment, mimicking the patient phenotypes. Moreover, overexpression of 4 human missense PI4KB mutant mRNAs in zebrafish embryos resulted in impaired hearing function, suggesting dominant-negative effects. Taken together, our results reveal that PI4KB mutations can cause SNHL and inner ear malformation. PI4KB should be included in neonatal deafness screening.

Adeno-associated virus gene replacement for recessive inner ear dysfunction: Progress and challenges.

Approximately 3 in 1000 children in the US under 4 years of age are affected by hearing loss. Currently, cochlear implants represent the only line of treatment for patients with severe to profound hearing loss, and there are no targeted drug or biological based therapies available. Gene replacement is a promising therapeutic approach for hereditary hearing loss, where viral vectors are used to deliver functional cDNA to "replace" defective genes in dysfunctional cells in the inner ear. Proof-of-concept studies have successfully used this approach to improve auditory function in mouse models of hereditary hearing loss, and human clinical trials are on the immediate horizon. The success of this method is ultimately determined by the underlying biology of the defective gene and design of the treatment strategy, relying on intervention before degeneration of the sensory structures occurs. A challenge will be the delivery of a corrective gene to the proper target within the therapeutic window of opportunity, which may be unique for each specific defective gene. Although rescue of pre-lingual forms of recessive deafness have been explored in animal models thus far, future identification of genes with post-lingual onset that are amenable to gene replacement holds even greater promise for treatment, since the therapeutic window is likely open for a much longer period of time. This review summarizes the current state of adeno-associated virus (AAV) gene replacement therapy for recessive hereditary hearing loss and discusses potential challenges and opportunities for translating inner ear gene replacement therapy for patients with hereditary hearing loss.

Practical aspects of inner ear gene delivery for research and clinical applications.

The application of gene therapy is widely expanding in research and continuously improving in preparation for clinical applications. The inner ear is an attractive target for gene therapy for treating environmental and genetic diseases in both the auditory and vestibular systems. With the lack of spontaneous cochlear hair cell replacement, hair cell regeneration in adult mammals is among the most important goals of gene therapy. In addition, correcting gene defects can open up a new era for treating inner ear diseases. The relative isolation and small size of the inner ear dictate local administration routes and carefully calculated small volumes of reagents. In the current review, we will cover effective timing, injection routes and types of vectors for successful gene delivery to specific target cells within the inner ear. Differences between research purposes and clinical applications are also discussed.

Hypothalamic hamartomas and inner ear diverticula with X-linked stapes gusher syndrome - new associations?

X-linked stapes gusher syndrome is a genetic form of deafness with distinct radiographic features on temporal bone CT. Hypothalamic hamartoma is a congenital glioneuronal anomaly of the hypothalamus. We report a potential association between these two rare anomalies that, to our knowledge, has not been reported. Two brothers presented with sensorineural hearing loss and almost identical inner ear and hypothalamic abnormalities, consistent with a diagnosis of X-linked stapes gusher syndrome and hypothalamic hamartoma. Genetic testing revealed identical mutations in the POU3F4 gene associated with X-linked stapes gusher syndrome. Furthermore, multiple vestibular diverticula were seen in both brothers, which have also not been reported with X-linked stapes gusher syndrome. This case suggests that POU3F4 mediated X-linked stapes gusher syndrome may also lead to multiple vestibular diverticula and hypothalamic hamartoma and, therefore, brain magnetic resonance imaging (MRI) could be considered in patients presenting with these inner ear findings.

miR­10b­3p, miR­8112 and let­7j as potential biomarkers for autoimmune inner ear diseases.

Circulating microRNAs (miRNAs) have been suggested as non­invasive biomarkers for the diagnosis of several autoimmune diseases. However, to the best of our knowledge, no studies have yet examined the miRNA expression profiles in autoimmune inner ear disease (AIED). The present study aimed to use an miRNA sequencing assay to detect the miRNA expression profiles of serum samples from 3 control mice and 3 antigen­induced AIED model mice. Differentially expressed miRNAs (DE­miRNAs) were screened using a t­test. miRNA target prediction was performed using TargetScan Mouse. Then, the miRNA­target gene interaction network was constructed and visualized using Cytoscape software. The underlying functions of the target genes of the DE­miRNAs were predicted using the clusterProfiler package. As a result, 22 miRNAs were identified as DE­miRNAs between AIED and control mice, including 10 upregulated and 12 downregulated genes. Based on the TargetScan Mouse prediction, 1,958 genes were identified as the targets for the 22 DE­miRNAs. Functional analysis indicated that only the target genes of 8 miRNAs were respectively enriched for Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways, among which miR­10b­3p, let­7j and miR­8112 were shared between the two pathway analyses. These 3 miRNAs may be involved in AIED by affecting inflammatory chemokine (miR­10b­3p­C­C motif chemokine 12), Wnt signaling (miR­8112­Wnt9b/Wnt 3a/Wnt2b) and Mucin type O­glycan biosynthesis pathways (let­7j­Galnt2/Galnt12). In conclusion, miR­10b­3p, miR­8112 and let­7j may be underlying biomarkers for diagnosing AIED.

Genetic Causes of Inner Ear Anomalies: a Review from the Turkish Study Group for Inner Ear Anomalies

Inner ear anomalies diagnosed using a radiological study are detected in almost 30% of cases with congenital or prelingual-onset sensorineural hearing loss. Inner ear anomalies can be isolated or occur along with a part of a syndrome involving other systems. Although astonishing progress has been made in research aimed at revealing the genetic causes of hearing loss in the past few decades, only a few genes have been linked to inner ear anomalies. The aim of this review is to discuss the known genetic causes of inner ear anomalies. Identifying the genetic causes of inner ear anomalies is important for guiding clinical care that includes empowered reproductive decisions provided to the affected individuals. Furthermore, understanding the molecular underpinnings of the development of the inner ear in humans is important to develop novel treatment strategies for people with hearing loss.

Improved TMC1 gene therapy restores hearing and balance in mice with genetic inner ear disorders.

Fifty percent of inner ear disorders are caused by genetic mutations. To develop treatments for genetic inner ear disorders, we designed gene replacement therapies using synthetic adeno-associated viral vectors to deliver the coding sequence for Transmembrane Channel-Like (Tmc) 1 or 2 into sensory hair cells of mice with hearing and balance deficits due to mutations in Tmc1 and closely related Tmc2. Here we report restoration of function in inner and outer hair cells, enhanced hair cell survival, restoration of cochlear and vestibular function, restoration of neural responses in auditory cortex and recovery of behavioral responses to auditory and vestibular stimulation. Secondarily, we find that inner ear Tmc gene therapy restores breeding efficiency, litter survival and normal growth rates in mouse models of genetic inner ear dysfunction. Although challenges remain, the data suggest that Tmc gene therapy may be well suited for further development and perhaps translation to clinical application.

De novo variants in GREB1L are associated with non-syndromic inner ear malformations and deafness.

Congenital inner ear malformations affecting both the osseous and membranous labyrinth can have a devastating impact on hearing and language development. With the exception of an enlarged vestibular aqueduct, non-syndromic inner ear malformations are rare, and their underlying molecular biology has thus far remained understudied. To identify molecular factors that might be important in the developing inner ear, we adopted a family-based trio exome sequencing approach in young unrelated subjects with severe inner ear malformations. We identified two previously unreported de novo loss-of-function variants in GREB1L [c.4368G>T;p.(Glu1410fs) and c.982C>T;p.(Arg328*)] in two affected subjects with absent cochleae and eighth cranial nerve malformations. The cochlear aplasia in these affected subjects suggests that a developmental arrest or problem at a very early stage of inner ear development exists, e.g., during the otic pit formation. Craniofacial Greb1l RNA expression peaks in mice during this time frame (E8.5). It also peaks in the developing inner ear during E13-E16, after which it decreases in adulthood. The crucial function of Greb1l in craniofacial development is also evidenced in knockout mice, which develop severe craniofacial abnormalities. In addition, we show that Greb1l-/- zebrafish exhibit a loss of abnormal sensory epithelia innervation. An important role for Greb1l in sensory epithelia innervation development is supported by the eighth cranial nerve deficiencies seen in both affected subjects. In conclusion, we demonstrate that GREB1L is a key player in early inner ear and eighth cranial nerve development. Abnormalities in cochleovestibular anatomy can provide challenges for cochlear implantation. Combining a molecular diagnosis with imaging techniques might aid the development of individually tailored therapeutic interventions in the future.

Outlook and future of inner ear therapy.

Drug delivery to the inner ear is an ideal method to treat a wide variety of otologic conditions. A broad range of potential applications is just beginning to be explored. New approaches combine principles of inner ear pharmacokinetics with emerging technologies of drug delivery including novel delivery systems, drug-device combinations, and new categories of drugs. Strategies include cell-specific targeting, manipulation of gene expression, local activation following systemic delivery, and use of stem cells, viral vectors, and gene editing systems. Translation of these therapies to the clinic remains challenging given the potential risks of intracochlear and intralabyrinthine trauma, our limited understanding of the etiologies of particular inner ear disorders, and paucity of accurate diagnostic tools at the cellular level. This review provides an overview of future methods, delivery systems, disease targets, and clinical considerations required for translation to clinical medicine.

Delivery of Adeno-Associated Virus Vectors in Adult Mammalian Inner-Ear Cell Subtypes Without Auditory Dysfunction.

Hearing loss, including genetic hearing loss, is one of the most common forms of sensory deficits in humans with limited options of treatment. Adeno-associated virus (AAV)-mediated gene transfer has been shown to recover auditory functions effectively in mouse models of genetic deafness when delivered at neonatal stages. However, the mouse cochlea is still developing at those time points, whereas in humans, the newborn inner ears are already fully mature. For effective gene therapy to treat genetic deafness, it is necessary to determine whether AAV-mediated therapy can be equally effective in the fully mature mouse inner ear without causing damage to the inner ear. This study tested several AAV serotypes by canalostomy in adult mice. It is shown that most AAVs transduce the sensory inner hair cells efficiently, but are less efficient at transducing outer hair cells. A subset of AAVs also transduces non-sensory cochlear cell types. Neither the surgical procedure of canalostomy nor the AAV serotypes damage hair cells or impair normal hearing. The studies indicate that canalostomy can be a viable route for safe and efficient gene delivery, and they expand the repertoire of AAVs to target diverse cell types in the adult inner ear.

Superior Canal Dehiscence Syndrome Affecting 3 Families.

Importance: Superior canal dehiscence syndrome (SCDS) is an increasingly recognized cause of hearing loss and vestibular symptoms, but the etiology of this condition remains unknown. Objective: To describe 7 cases of SCDS across 3 families. Design, Setting, and Participants: This retrospective case series included 7 patients from 3 different families treated at a neurotology clinic at a tertiary academic medical center from 2010 to 2014. Patients were referred by other otolaryngologists or were self-referred. Each patient demonstrated unilateral or bilateral SCDS or near dehiscence. Interventions: Clinical evaluation involved body mass index calculation, audiometry, cervical vestibular evoked myogenic potential testing, electrocochleography, and multiplanar computed tomographic (CT) scan of the temporal bones. Zygosity testing was performed on twin siblings. Main Outcomes and Measures: The diagnosis of SCDS was made if bone was absent over the superior semicircular canal on 2 consecutive CT images, in addition to 1 physiologic sign consistent with labyrinthine dehiscence. Near dehiscence was defined as absent bone on only 1 CT image but with symptoms and at least 1 physiologic sign of labyrinthine dehiscence. Results: A total of 7 patients (5 female and 2 male; age range, 8-49 years) from 3 families underwent evaluation. Family A consisted of 3 adult first-degree relatives, of whom 2 were diagnosed with SCDS and 1 with near dehiscence. Family B included a mother and her child, both of whom were diagnosed with unilateral SCDS. Family C consisted of adult monozygotic twins, each of whom was diagnosed with unilateral SCDS. For all cases, dehiscence was located at the arcuate eminence. Obesity alone did not explain the occurrence of SCDS because 5 of the 7 cases had a body mass index (calculated as weight in kilograms divided by height in meters squared) less than 30.0. Conclusions and Relevance: Superior canal dehiscence syndrome is a rare, often unrecognized condition. This report of 3 multiplex families with SCDS provides evidence in support of a potential genetic contribution to the etiology. Symptomatic first-degree relatives of patients diagnosed with SCDS should be offered evaluation to improve detection of this disorder.

Zebrafish models of human eye and inner ear diseases.

Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies.

CHD7 mutations and CHARGE syndrome in semicircular canal dysplasia.

OBJECTIVE: To determine whether patients with semicircular canal dysplasia have mutations in CHD7. BACKGROUND: CHARGE syndrome is a nonrandom clustering of congenital anomalies, including ocular coloboma, heart defects, choanal atresia or stenosis, retarded growth and development, genital hypoplasia, and inner and outer ear anomalies including deafness. Semicircular canal dysplasia has been included as a major diagnostic criterion for CHARGE syndrome. Mutations in the gene CHD7 on chromosome 8q12.1 are a major cause of CHARGE syndrome, but the extent to which patients with semicircular canal dysplasia have CHD7 mutations is not fully understood. STUDY DESIGN: Cross-sectional analysis of CHD7 in 12 patients with semicircular canal dysplasia and variable clinical features of CHARGE syndrome. RESULTS: We identified 6 CHD7 mutations, 5 of which occurred in patients who fulfilled Verloes' diagnostic criteria for typical CHARGE syndrome, and three of which were previously unreported. Of the 3 remaining CHD7 mutation-positive patients, one had atypical CHARGE by diagnostic criteria. Four MRI records were available, which revealed 2 patients with cochlear nerve aplasia and 1 patient with Chiari 1 malformation. CONCLUSION: These data provide additional evidence that CHD7 mutations are a significant cause of semicircular canal atresia in children with full or partial CHARGE syndrome.

Familial superior canal dehiscence syndrome.

IMPORTANCE: The etiology of superior canal dehiscence (SCD) involving the arcuate eminence is not completely understood, but genetic factors may play a role. One hypothesis is that patients are born with a defect of the superior canal, and an acute event (such as head trauma) or progressive loss of bone (eg, due to dural pulsations) may result in the onset of SCD symptoms. Familial SCD has only been briefly mentioned in the literature to date. OBSERVATIONS: We report 3 families that each had 2 members with SCD syndrome. We found that first-degree relatives presented with similar complaints and that temporal bone computed tomography scans between relatives showed very similar skull base topography and anatomic SCD defects. CONCLUSIONS AND RELEVANCE: The presence of symptomatic SCD among first-degree relatives and similar skull base topography suggests that genetics may play a role in the etiology of SCD.

Inner ear symptoms and disease: pathophysiological understanding and therapeutic options.

In recent years, huge advances have taken place in understanding of inner ear pathophysiology causing sensorineural hearing loss, tinnitus, and vertigo. Advances in understanding comprise biochemical and physiological research of stimulus perception and conduction, inner ear homeostasis, and hereditary diseases with underlying genetics. This review describes and tabulates the various causes of inner ear disease and defines inner ear and non-inner ear causes of hearing loss, tinnitus, and vertigo. The aim of this review was to comprehensively breakdown this field of otorhinolaryngology for specialists and non-specialists and to discuss current therapeutic options in distinct diseases and promising research for future therapies, especially pharmaceutic, genetic, or stem cell therapy.

A causative link between inner ear defects and long-term striatal dysfunction.

There is a high prevalence of behavioral disorders that feature hyperactivity in individuals with severe inner ear dysfunction. What remains unknown is whether inner ear dysfunction can alter the brain to promote pathological behavior. Using molecular and behavioral assessments of mice that carry null or tissue-specific mutations of Slc12a2, we found that inner ear dysfunction causes motor hyperactivity by increasing in the nucleus accumbens the levels of phosphorylated adenosine 3',5'-monophosphate response element-binding protein (pCREB) and phosphorylated extracellular signal-regulated kinase (pERK), key mediators of neurotransmitter signaling and plasticity. Hyperactivity was remedied by local administration of the pERK inhibitor SL327. These findings reveal that a sensory impairment, such as inner ear dysfunction, can induce specific molecular changes in the brain that cause maladaptive behaviors, such as hyperactivity, that have been traditionally considered exclusively of cerebral origin.

Noddy, a mouse harboring a missense mutation in protocadherin-15, reveals the impact of disrupting a critical interaction site between tip-link cadherins in inner ear hair cells.

In hair cells of the inner ear, sound or head movement increases tension in fine filaments termed tip links, which in turn convey force to mechanosensitive ion channels to open them. Tip links are formed by a tetramer of two cadherin proteins: protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which have 11 and 27 extracellular cadherin (EC) repeats, respectively. Mutations in either protein cause inner ear disorders in mice and humans. We showed recently that these two cadherins bind tip-to-tip in a "handshake" mode that involves the EC1 and EC2 repeats of both proteins. However, a paucity of appropriate animal models has slowed our understanding both of the interaction and of how mutations of residues within the predicted interface compromise tip link integrity. Here, we present noddy, a new mouse model for hereditary deafness. Identified in a forward genetic screen, noddy homozygotes lack inner ear function. Mapping and sequencing showed that noddy mutant mice harbor an isoleucine-to-asparagine (I108N) mutation in the EC1 repeat of PCDH15. Residue I108 interacts with CDH23 EC2 in the handshake and its mutation impairs the interaction in vitro. The noddy mutation allowed us to determine the consequences of blocking the handshake in vivo: tip link formation and bundle morphology are disrupted, and mechanotransduction channels fail to remain open at rest. These results offer new insights into the interaction between PCDH15 and CDH23 and help explain the etiology of human deafness linked to mutations in the tip-link interface.

Gene transfer in inner ear cells: a challenging race.

Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders.