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CHARGE syndrome is a multiple anomaly developmental disorder characterized by a variety of sensory deficits, including sensorineural hearing loss of unknown etiology. Most cases of CHARGE are caused by heterozygous pathogenic variants in CHD7, the gene encoding Chromodomain DNA-binding Protein 7 (CHD7), a chromatin remodeler important for the development of neurons and glial cells. Previous studies in the Chd7 Gt/+ mouse model of CHARGE syndrome showed substantial neuron loss in the early stages of the developing inner ear that are compensated for by mid-gestation. In this study, we sought to determine if early developmental delays caused by Chd7 haploinsufficiency affect neurons, glial cells, and inner hair cell innervation in the mature cochlea. Analysis of auditory brainstem response recordings in Chd7 Gt/+ adult animals showed elevated thresholds at 4 kHz and 16 kHz, but no differences in ABR Wave I peak latency or amplitude compared to wild type controls. Proportions of neurons in the Chd7 Gt/+ adult spiral ganglion and densities of nerve projections from the spiral ganglion to the organ of Corti were not significantly different from wild type controls. Inner hair cell synapse formation also appeared unaffected in mature Chd7 Gt/+ cochleae. However, histological analysis of adult Chd7 Gt/+ cochleae revealed diminished satellite glial cells and hypermyelinated Type I spiral ganglion axons. We characterized the expression of CHD7 in developing inner ear glia and found CHD7 to be expressed during a tight window of inner ear development at the Schwann cell precursor stage at E9.5. While cochlear neurons appear to differentiate normally in the setting of Chd7 haploinsufficiency, our results suggest an important role for CHD7 in glial cells in the inner ear. This study highlights the dynamic nature of CHD7 activity during inner ear development in mice and contributes to understanding CHARGE syndrome pathology., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest SG is on the advisory boards for the Cystic Fibrosis Foundation, Applied Genetic Technologies Corporation and Roche. He also receives research support without financial remuneration from Med-El Corporation and is a consultant for Cochlear Corporation and Sirocco Therapeutics. YR is on the advisory board of Applied Genetic Technologies Corporation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hair cells (HCs) in the cochlea are responsible for transducing mechanical sound energy into neural impulses which lead to the perception of sound. Loss of these sensory cells is the most common cause of sensorineural hearing loss, and spontaneous HC regeneration does not occur in mature mammals. Among the future potential treatment modalities is gene therapy, which is defined as the administration of either DNAs or RNAs as active pharmaceutical ingredients for inducing a clinically-beneficial response. Gene therapy is being envisioned and evaluated as a potential tool for addressing a number of human inner ear disorders. This paper is a hybrid Review and Research Paper, including unpublished data and a review of HC regeneration studies in live animal models. Current gene therapeutic approaches for replacing lost HC populations have been aimed at converting supporting cells surviving within the neuro-epithelium to new HCs by inducing upregulation of bHLH transcription factors such as Atoh1 or reciprocal silencing of Notch signaling with siRNAs, to tip the balance of transcriptional regulation toward a HC fate. Development of one or more of these techniques may yield a path to effective restoration of inner ear form and function. This review also describes other approaches and molecular targets that may prove efficacious and provides perspectives on future clinical challenges and opportunities for gene therapy to become a valuable weapon for the long-anticipated realization of this regenerative treatment., Competing Interests: Declaration of competing interest YR is on the advisory board of Applied Genetic Technologies Corporation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The inner ear contains the sensory organs for hearing and balance. Both hearing and balance are commonly affected in individuals with CHARGE syndrome (CS), an autosomal dominant condition caused by heterozygous pathogenic variants in the CHD7 gene. Semicircular canal dysplasia or aplasia is the single most prevalent feature in individuals with CHARGE leading to deficient gross motor skills and ambulation. Identification of CHD7 as the major gene affected in CHARGE has enabled acceleration of research in this field. Great progress has been made in understanding the role of CHD7 in the development and function of the inner ear, as well as in related organs such as the middle ear and auditory and vestibular neural pathways. The goals of current research on CHD7 and CS are to (a) improve our understanding of the pathology caused by CHD7 pathogenic variants and (b) to provide better tools for prognosis and treatment. Current studies utilize cells and whole animals, from flies to mammals. The mouse is an excellent model for exploring mechanisms of Chd7 function in the ear, given the evolutionary conservation of ear structure, function, Chd7 expression, and similarity of mutant phenotypes between mice and humans. Newly recognized developmental functions for mouse Chd7 are shedding light on how abnormalities in CHD7 might lead to CS symptoms in humans. Here we review known human inner ear phenotypes associated with CHD7 pathogenic variants and CS, summarize progress toward diagnosis and treatment of inner ear-related pathologies, and explore new avenues for treatment based on basic science discoveries., (© 2017 Wiley Periodicals, Inc.)

microRNAs (miRNAs) are regulators of differentiation and development of inner ear cells. Mutations in miRNAs lead to deafness in humans and mice. Among inner ear pathologies, inflammation may lead to structural and neuronal defects and eventually to hearing loss and vestibular dysfunction. While the genetic factors of these pathways have not been defined, autoimmunity participates in these processes. We report that inflammatory stimuli in the inner ear induce activation of the innate immune system via miR-224 and pentraxin 3 (Ptx3). miR-224 is a transcriptional target of nuclear factor κB, a key mediator of innate immunity. Ptx3 is a regulator of the immune response. It is released in response to inflammation and regulated by nuclear factor κB. We show that miR-224 and Ptx3 are expressed in the inner ear and we demonstrate that miR-224 targets Ptx3. As a model of the innate immune response, we injected lipopolysaccharide into the scala tympani of mouse inner ears. This resulted in changes in the levels of miR-224 and Ptx3, in addition to activation of the complement system, as measured by immune cell infiltration and activated C3. This suggests that while miR-224 regulates Ptx3 under normal conditions, upon inflammation, both are recruited to offer a front line of defense in acting as responders to inflammation in the inner ear. miR-224 diminishes the innate immune response by down-regulating Ptx3 expression, while Ptx3 stimulates the innate immune response. An understanding of the molecular components of the inflammatory pathway may help develop therapeutics for reducing inflammation associated with inner ear injury., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

CHARGE syndrome is a multiple congenital anomaly disorder that leads to life-threatening birth defects, such as choanal atresia and cardiac malformations as well as multiple sensory impairments, that affect hearing, vision, olfaction and balance. CHARGE is caused by heterozygous mutations in CHD7, which encodes an ATP-dependent chromatin remodeling enzyme. Identification of the mechanisms underlying neurological and sensory defects in CHARGE is a first step toward developing treatments for CHARGE individuals. Here, we used mouse models of Chd7 deficiency to explore the function of CHD7 in the development of the subventricular zone (SVZ) neural stem cell niche and inner ear, structures that are important for olfactory bulb neurogenesis and hearing and balance, respectively. We found that loss of Chd7 results in cell-autonomous proliferative, neurogenic and self-renewal defects in the perinatal and mature mouse SVZ stem cell niche. Modulation of retinoic acid (RA) signaling prevented in vivo inner ear and in vitro neural stem cell defects caused by Chd7 deficiency. Our findings demonstrate critical, cooperative roles for RA and CHD7 in SVZ neural stem cell function and inner ear development, suggesting that altered RA signaling may be an effective method for treating Chd7 deficiency.

Animal studies on inner ear development, repair and regeneration provide understanding of molecular pathways that can be harnessed for treating inner ear disease. Use of transgenic mouse technology, in particular, has contributed knowledge of genes that regulate development of hair cells and innervation, and of molecular players that can induce regeneration, but this technology is not applicable for human treatment, for practical and ethical reasons. Therefore other means for influencing gene expression in the inner ear are needed. We describe several gene vectors useful for inner ear gene therapy and the practical aspects of introducing these vectors into the ear. We then review the progress toward using gene transfer for therapies in both auditory and balance systems, and discuss the technological milestones needed to advance to clinical application of these methods., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Severe to profound deafness is most often secondary to a loss of or injury to cochlear mechanosensory cells, and there is often an associated loss of the peripheral auditory neural structures, specifically the spiral ganglion neurons and peripheral auditory fibers. Cochlear implantation is currently our best hearing rehabilitation strategy for severe to profound deafness. These implants work by directly electrically stimulating the remnant auditory neural structures within the deafened cochlea. When administered to the deafened cochlea in animal models, neurotrophins, specifically brain derived neurotrophic factor and neurotrophin-3, have been shown to dramatically improve spiral ganglion neuron survival and stimulate peripheral auditory fiber regrowth. In animal models, neurotrophins administered in combination with cochlear implantation has resulted in significant improvements in the electrophysiological and psychophysical measures of outcome. While further research must be done before these therapies can be applied clinically, neurotrophin therapies for the inner ear show great promise in enhancing CI outcomes and the treatment of hearing loss., (Copyright © 2012 Wiley Periodicals, Inc.)

The membrane glycoprotein CTL2/SLC44A2 is expressed by supporting cells in the inner ear and has been identified as a target of antibodies that may induce auto-immune hearing loss. To determine if CTL2/SLC44A2 also has roles in inner ear development and to distinguish between isoform-specific roles, we assessed age-related changes in expression of CTL2/SLC44A2 isoforms and protein in the developing murine inner ear. We determined that both isoform p1 and isoform p2 (named for the upstream p1 and proximal p2 promoters that control alternate exons 1a and 1b) were robustly expressed as early as E14 and persisted during embryonic development, but after birth the p1 isoform fell to barely detectable levels while isoform p2 levels were maintained. This trend continued and became even more apparent later in post-natal development and remained in mature ears until at least 6 weeks of age. In aged (18 mo old) mice, the level of isoform p1 transcripts rose again to levels similar to the p2 isoform like that seen early in development. At the earliest stage examined, CTL2/SLC44A2 protein was expressed in both immature supporting cells and immature sensory cells, but after birth expression in the sensory cells declined in both the utricle and cochlea and by day P1 expression of CTL2/SLC44A2 was restricted to supporting cells. The changes we observed in isoform distribution are indicative of differential developmental roles and age related changes between the two isoforms of CTL2/SLC44A2 in the inner ear., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Inner ear neurogenesis is positively regulated by the pro-neural bHLH transcription factors Ngn1 and NeuroD, but the factors that act upstream of this regulation are not well understood. Recent evidence in mouse and Drosophila suggests that neural development depends on proper chromatin remodeling, both for maintenance of neural stem cells and for proper neuronal differentiation. Here, we show that CHD7, an ATP-dependent chromatin remodeling enzyme mutated in human CHARGE syndrome, is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis. Conditional deletion of Chd7 in the developing otocyst using Foxg1-Cre resulted in cochlear hypoplasia and complete absence of the semicircular canals and cristae. Conditional knockout and null otocysts also had reductions in vestibulo-cochlear ganglion size and neuron number in combination with reduced expression of Ngn1, Otx2 and Fgf10, concurrent with expansion of the neural fate suppressor Tbx1 and reduced cellular proliferation. Heterozygosity for Chd7 mutations had no major effects on expression of otic patterning genes or on cell survival, but resulted in decreased proliferation within the neurogenic domain. These data indicate that epigenetic regulation of gene expression by CHD7 must be tightly coordinated for proper development of inner ear neuroblasts.

Unlabelled: Health care professionals tending to patients with inner ear disease face inquiries about therapy options, including treatments that are being developed for future use but not yet available. The devastating outcome of sensorineural hearing loss, combined with the permanent nature of the symptoms, make these inquiries demanding and frequent. The vast information accessible online and the publicity for breakthroughs in research add to patient requests for access to advanced and innovative therapies, even before these are available for clinical use. This can sometimes be taxing on the health care provider who is in contact with the patients. Here we aim to equip the provider with information about some of the progress made for protective and reparative approaches for treating inner ears., Learning Outcomes: (1) Readers will be able to explain why hearing loss is irreversible and common, (2) readers will be able to explain the importance of protective measures and the progress made in discovery and design of novel biological protective molecules, (3) readers will be able to describe reparative approaches currently under investigation (such as tissue engineering), the main difficulties in the design of such therapies and the major hurdles that remain for making novel technologies clinically viable, and (4) readers will be able to explain to their patients some of the progress in developing new treatments without making the promise of imminent clinical use. With this information, readers will be able to guide patients to make better choices for their treatment and to guide students toward research in this exciting field., (Copyright 2010 Elsevier Inc. All rights reserved.)

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells., (Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

During normal development, cells divide, then differentiate to adopt their individual form and function in an organism. Under most circumstances, mature cells cannot transdifferentiate, changing their fate to adopt a different form and function. Because differentiated cells cannot usually divide, the repair of injuries as well as regeneration largely depends on the activation of stem cell reserves. The mature cochlea is an exception among epithelial cell layers in that it lacks stem cells. Consequently, the sensory hair cells that receive sound information cannot be replaced, and their loss results in permanent hearing impairment. The lack of a spontaneous cell replacement mechanism in the organ of Corti, the mammalian auditory sensory epithelium, has led researchers to investigate circumstances in which transdifferentiation does occur. The hope is that this information can be used to design therapies to replace lost hair cells and restore impaired hearing in humans.

Bone Morphogenetic Protein 4 (BMP4) is a member of the TGF-beta superfamily and is known to be important for the normal development of many tissues and organs, including the inner ear. Bmp4 homozygous null mice die as embryos, but Bmp4 heterozygous null (Bmp4(+/-)) mice are viable and some adults exhibit a circling phenotype, suggestive of an inner ear defect. To understand the role of BMP4 in inner ear development and function, we have begun to study C57BL/6 Bmp4(+/-) mice. Quantitative testing of the vestibulo-collic reflex, which helps maintain head stability, demonstrated that Bmp4(+/-) mice that exhibit circling behavior have a poor response in the yaw axis, consistent with semicircular canal dysfunction. Although the hair cells of the ampullae were grossly normal, the stereocilia were greatly reduced in number. Auditory brainstem responses showed that Bmp4(+/-) mice have elevated hearing thresholds and immunohistochemical staining demonstrated decreased numbers of neuronal processes in the organ of Corti. Thus Bmp4(+/-) mice have structural and functional deficits in the inner ear.

It is possible to manipulate gene expression in cochlear tissue, but technical issues have made this challenging in the mature in vivo inner ear. Generally, the most common reasons for such manipulations involve basic science or therapeutic quests. Examples of experimental studies are those designed to elucidate the role of a specific gene or a gene expression cascade or to understand the function of a particular cell type. Therapeutic goals may include replacing a defective gene or enhancing tissue protection, repair, or regeneration. This review summarizes the main technical approaches that are viable options for in vivo manipulation of gene expression in the mature inner ear, as well as major research and clinical issues likely to benefit from such genetic manipulations.

The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.

Inner ear disease due to hair cell loss is common, and no restorative treatments for the balance and hearing impairment are currently available. To develop clinical means for enhancing protection and regeneration in the inner ear, it is necessary to understand the molecular basis for hereditary and acquired deafness and vestibular disorders. One approach is to identify and characterize genes that regulate protection or repair in other systems. For that purpose, we have used the differential display assay and compared gene expression between normal and acoustically traumatized inner ears of chicks. Several chick cDNAs that were identified are considered as candidates for roles in the reparative process that follows trauma in the basilar papilla. The mammalian vestibular epithelium has a limited regenerative capability. To identify genes that may participate in the regenerative response, we have used gene arrays profiling, comparing normal to drug-traumatized vestibular epithelia. We identified several genes that are differentially expressed in traumatized vestibular epithelium, including several insulin-like growth factor-I binding proteins. To use this molecular knowledge for enhancing protection and repair in the organ of Corti, it is necessary to overexpress the genes of choice in the inner ear. Using viral-mediated gene transfer, we overexpressed transgenic glial cell line-derived neurotrophic factor and demonstrated a robust protective effect against acoustic and ototoxic inner ear trauma. Future identification of the genes that are important for protection and regeneration, along with improved gene transfer technology, will allow the use of gene therapy for treating hereditary and environmental inner ear disease., (Copyright 2002 S. Karger AG, Basel)