Viral vector tropism for supporting cells in the developing murine cochlea

Gene-based therapeutics are being developed as novel treatments for genetic hearing loss. One roadblock to effective gene therapy is the identification of vectors which will safely deliver therapeutics to targeted cells. The cellular heterogeneity that exists within the cochlea makes viral tropism a vital consideration for effective inner ear gene therapy. There are compelling reasons to identify a viral vector with tropism for organ of Corti supporting cells. Supporting cells are the primary expression site of connexin 26 gap junction proteins that are mutated in the most common form of congenital genetic deafness (DFNB1). Supporting cells are also primary targets for inducing hair cell regeneration. Since many genetic forms of deafness are congenital it is necessary to administer gene transfer-based therapeutics prior to the onset of significant hearing loss. We have used transuterine microinjection of the fetal murine otocyst to investigate viral tropism in the developing inner ear. For the first time we have characterized viral tropism for supporting cells following in utero delivery to their progenitors. We report the inner ear tropism and potential ototoxicity of three previously untested vectors: early-generation adenovirus (Ad5.CMV.GFP), advanced-generation adenovirus (Adf.11D) and bovine adeno-associated virus (BAAV.CMV.GFP). Adenovirus showed robust tropism for organ of Corti supporting cells throughout the cochlea but induced increased ABR thresholds indicating ototoxicity. BAAV also showed tropism for organ of Corti supporting cells, with preferential transduction toward the cochlear apex. Additionally, BAAV readily transduced spiral ganglion neurons. Importantly, the BAAV-injected ears exhibited normal hearing at 5 weeks of age when compared to non-injected ears. Our results support the use of BAAV for safe and efficient targeting of supporting cell progenitors in the developing murine inner ear.     

A glucocorticoid reduces adverse effects of adenovirus vectors in the cochlea

Gene transfer using a recombinant adenovirus is a powerful tool for research and clinical applications, but its cytotoxicity and immune response limit its use, especially when repeated application of the vector is necessary. This study investigated the effects of dexamethasone (DEX)-induced immunosuppression on the outcome of adenovirus gene transfer in guinea pig inner ears. Animals received DEX for 29 days. Their inner ear was inoculated with 5 micro l of adenovirus vector twice, on days 5 and 26. Auditory brainstem response was measured on days 1, 8 and 29. The animals were sacrificed on day 29, and reporter gene expression was evaluated. In control animals that received no DEX, postinoculation threshold shifts and lesions in the organ of Corti were observed and reporter gene expression was absent. In contrast, DEX-treated ears were largely protected, and transduction of inner ear cells was readily apparent. These data demonstrate that immunosuppressive treatment can reduce the negative consequences of repeated adenovirus-mediated gene therapy.     

Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants

A method for long term culture of utricular macula explants is demonstrated to be stable and reproducible over a period of 28 days in vitro (DIV). This culture system for four-day-old rat utricular maculae is potentially suitable for studies of hair cell loss, repair and regeneration processes as they occur in post-natal mammalian inner ear sensory epithelia. The cellular events that occur within utricular macula hair cell epithelia during 28 days of culture are documented from serial sections. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) and the assistance of morphometric computer software. Ultrastructural observations were made with transmission electron microscopy (TEM) for describing the changes in the fine detailed morphological characteristics that occurred in the explants related to time in vitro. After 2 DIV the density of HCs was 77%, at 21 DIV it was 69%, and at 28 DIV it was 52% of HCs present at explantation. Between 2 DIV and 28 DIV there was a 1.7% decrease of the vestibular macula HC density per DIV. The corresponding decrease of SC density within the utricular explants was less than 1% per DIV. The overall morphology of the epithelia, i.e. relationship of HCs to SCs, was well preserved during the first two weeks in culture. After this time a slight deterioration of the epithelia was observed and although type I and type II HCs were identified by TEM observations, these two HC types could no longer be distinguished from one another by LM observations. In preparations cultured for 21 DIV, SC nuclei were located more apical and further away from the basal membrane compared to their position in macula explants fixed immediately after dissection. The loss of cells that occurred was probably due to expulsion from the apical (i.e. luminal) surface of the sensory epithelia, but no lesions of the apical lining or ruptures of the basal membrane were observed. There were no significant changes in the volume of the vestibular HC comprising macular epithelium during the observation period of 28 DIV.     

Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.     

骨形态发生蛋白4在内耳发育及在毛细胞与螺旋神经节细胞再生中的研究进展

听力损失是人类最常见的感觉缺陷,主要涉及到毛细胞(HCs)和螺旋神经节细胞(SGCs)的损失等。在内耳发育过程中,骨形态发生蛋白4(BMP4)的表达具有时序性和特异性,并且通过调控分泌蛋白Tsukushin(TSK)、性别决定相关基因簇2蛋白(SOX2)等细胞因子,与WNT和SHH等信号通路之间相互作用,参与耳泡的诱导、前庭和耳蜗等器官的形成、HCs和SGCs等细胞的分化过程。此外,近些年在哺乳动物与非哺乳动物的耳蜗外植体中,发现BMP4在HCs和SGCs的再生中起重要作用。综述BMP4调控内耳发育、诱导HCs和SGCs再生的作用,以及相关的研究进展,以期为HCs和SGCs再生相关机制的阐明奠定基础,为听力损失的治疗带来新的思路与策略。     

A novel combination of drug therapy to protect residual hearing post cochlear implant surgery

Conclusions A cocktail combining NAC, Mannitol, and Dexamethasone may be used to prevent loss of residual hearing post-implantation. There is a window of opportunity to treat the cochlea before the onset of cell death in HCs. Objective Inner ear trauma caused by cochlear implant electrode insertion trauma (EIT) initiates multiple molecular mechanisms in hair cells (HCs) or support cells (SCs), resulting in initiation of programmed cell death within the damaged tissues of the cochlea, which leads to loss of residual hearing. In earlier studies L-N-acetylcysteine (L-NAC), Mannitol, and dexamethasone have been shown independently to protect the HCs loss against different types of inner ear trauma. These three molecules have different otoprotective effects. The goal of this preliminary study is to test the efficacy of a combination of these molecules to enhance the otoprotection of HCs against EIT. Methods OC explants were dissected from P-3 rats and placed in serum-free media. Explants were divided into control and experimental groups. Control group: (1) untreated controls; (2) EIT. Experimental group: (1) EIT?+?L-NAC (5, 2, or 1?mM); (2) EIT?+?Mannitol (100, 50, or 10?mM); (3) EIT?+?Dex (20, 10, or 5?μg/mL); (4) EIT?+?L-NAC?+?Mannitol?+?Dex. After EIT was caused in an in-vitro model of CI, explants were cultured in media containing L-NAC alone, Mannitol alone, or Dex alone at decreasing concentrations. Concentrations of L-NAC, Mannitol, and Dex that showed 50% protection of hair cell loss individually were used as a combination in experimental group 4. Results There was an increase of total hair cell (THC) loss in the EIT OC explants when compared with control group HC counts or the tri-therapy cochlea. This study defined the dosage of L-NAC, Mannitol, and Dex for the survival of 50% protection of hair cells in vitro. Their combination provided close to 96% protection, demonstrating an additive effect.     

Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear

Several classes of viral vectors including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus and vaccinia virus have been reported to infect cells of the inner ears of mammals and may be useful for protein manipulation and therapeutic purposes. We have screened a few of these for use as vectors to mediate gene transfer into the sensory hair cells of organotypic cultures from the neonatal mouse cochlea and utricle. Recombinant, replication-deficient adenovirus has emerged as a useful vector for several reasons: ease of vector generation at high titer; efficient hair cell specific infection; robust expression of reporter genes and minimal toxicity. Previously, we characterized adenovirus infected hair cells using a vector that carried the gene for green fluorescent protein (GFP). We screened GFP-positive cells electrophysiologically and found that although hair cells survive adenoviral vector infection, their mechanosensitivity was compromised. Until recently this has limited the scope of adenovirus application to the problems of inner ear physiology and pathophysiology. However, a modified adenoviral vector, now available, has been reported to have reduced ototoxicity in vivo. The modifications include the deletion of the adenoviral genes E1, E3, the viral polymerase, and the preterminal protein. We are currently working to characterize viral-mediated gene transfer into hair cells of the cultured mouse utricle using this new modified adenoviral vector. We have found that hair cells infected with the modified vector have intact hair bundles and robust mechanotransduction.     

Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.     

聋病基因研究新策略——基因敲除鼠听功能和内耳形态的系统研究

虽然对听功能进行的研究已有进展，但目前对感音神经性聋的诊治仍是一大难题。聋病基因的发现使得我们可以从不同的层面揭示听觉功能的分子奥秘。但是，从功能基因组学角度看，仅仅发现和克隆聋病基因还远不是我们想达到的目标．需要进一步深入研究基因的功能基因敲除是近年来发展和成熟起来的一项生物学新技术。通过在小鼠胚胎干细胞基因组水平的同源重组，造成目的基因的缺失突变，可以了解基因失活后对发育、生长、衰老以及器官、组织或细胞结构功能的影响，从而既可确切地从整体水平研究基出功能．又可建立疾病的动物模型。通过对内耳彤态和听功能的系统研究，体现了功能基闲组学的研究方向，并且可以建立基因缺陷致聋的动物模型。我们对SMAD4、SMAD5基因剔除小鼠进行了大量的听功能和内耳形态研究，发现基因缺陷导致小鼠严重听力障碍，并且内耳听觉器官包括毛细胞、支持细胞和螺旋神经节等出现不同程度的损害。可以认为，我们已经建立了一个基因缺陷导致聋病的动物模型。作为听功能基因研究新的平台，它将对听觉基因功能和聋病的分子机制研究有重要的意义，以此作为聋病基因研究的新策略，从而为最终的聋病基因治疗提供理论上的实验依据。     

Advances in inner ear gene therapy: exploring cochlear protection and regeneration

PURPOSE OF REVIEW: To review the application of gene therapy in the inner ear. Gene delivery to the inner ear was first reported in 1996. Since then the field has progressed on multiple fronts. RECENT DEVELOPMENTS: More diverse and sophisticated vectors are improving the efficiency of delivery to the inner ear. Research is transitioning from the delivery of marker genes to the delivery of therapeutic genes in animal models of inner ear disease. Three distinct areas of research are developing: (1) delivery of genes for protection of spiral ganglion neurons with potential application in cochlear implantation, (2) delivery of genes for protection of hair cells and hearing preservation in degenerative diseases and cochlear insults and (3) the use of gene therapy to transform cells from one phenotype to another and replace lost cells, potentially restoring lost function. SUMMARY: Currently, no specific drugs are targeted at inner ear disease. The use of gene therapy in the inner ear is being applied in animal models of ototoxicity and ischemia reperfusion injury. Gene therapy can protect the inner ear from damage and even restore function through the regeneration of hair cells.     

Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma.

BACKGROUND: Adenovirus-mediated gene therapy has been used for squamous cell carcinoma of the head and neck (SCCHN), but the in vivo efficacy has been limited by a lack of tissue specificity and low infection efficiency. We are interested in improving cancer gene therapy strategies using targeted adenovirus vectors. OBJECTIVE: To determine if the infection efficiency of adenovirus-mediated gene transfer to SCCHN cells could be enhanced by retargeting to the epidermal growth factor receptor (EGFR), which is known to be overexpressed in these tumors. DESIGN: Epidermal growth factor receptor retargeting in SCCHN cells was accomplished with a bispecific antibody that recognized the knob domain of adenovirus as well as EGFR. Using this retargeting schema, we compared the infection efficiency and specificity of unmodified and EGFR-retargeted adenovirus. RESULTS: Squamous cell carcinoma of the head and neck cell lines were shown to be infected by adenovirus with low efficiency, which is likely because of the low level of adenovirus receptor expressed in the SCCHN cells. Epidermal growth factor receptor retargeting markedly enhanced transduction in both SCCHN cell lines and primary tumor tissue, as indicated by the elevated levels of reporter gene expression. Furthermore, retargeting enhanced infection of tumor tissue compared with normal tissue from the same patient. CONCLUSIONS: Epidermal growth factor receptor retargeting enhanced adenovirus infection of SCCHN cells and, in doing so, augments the potency of the vector. This modification makes the vector potentially more valuable in the clinical setting.     

Collagen type II in the otic extracellular matrix. Effect on inner ear development

Immunocytochemistry was used to demonstrate type II collagen distribution during normal development of the mouse inner ear and in two malformed inner ears. Patterns of inner ear abnormalities and type II collagen distribution were compared between the malformed labyrinth of a mouse mutation (disproportionate micromelia, Dmm) and otic explants exposed to the teratogenic action of an L-proline analog, L-azetidine-2-carboxylic acid (LACA). The results suggest that type II collagen is an important constituent of the developing inner ear's extracellular matrix. Disruptions of the spatial and temporal pattern of collagen type II can adversely affect morphogenesis of the inner ear. A common mechanism of action is postulated for the causation of both the genetic and teratogen-induced inner ear malformations (i.e. disruption of the secretion of collagens to the otic extracellular matrix).     

MECOM promotes supporting cell proliferation and differentiation in cochlea

Permanent damage to hair cells (HCs) is the leading cause of sensory deafness. Supporting cells (SCs) are essential in the restoration of hearing in mammals because they can proliferate and differentiate to HCs. MDS1 and EVI1 complex locus (MECOM) is vital in early development and cell differentiation and regulates the TGF-β signaling pathway to adapt to pathophysiological events, such as hematopoietic proliferation, differentiation and cells death. In addition, MECOM plays an essential role in neurogenesis and craniofacial development. However, the role of MECOM in the development of cochlea and its way to regulate related signaling are not fully understood. To address this problem, this study examined the expression of MECOM during the development of cochlea and observed a significant increase of MECOM at the key point of auditory epithelial morphogenesis, indicating that MECOM may have a vital function in the formation of cochlea and regeneration of HCs. Meanwhile, we tried to explore the possible effect and potential mechanism of MECOM in SC proliferation and HC regeneration. Findings from this study indicate that overexpression of MECOM markedly increases the proliferation of SCs in the inner ear, and the expression of Smad3 and Cdkn2b related to TGF signaling is significantly down-regulated, corresponding to the overexpression of MECOM. Collectively, these data may provide an explanation of the vital function of MECOM in SC proliferation and trans-differentiation into HCs, as well as its regulation. The interaction between MECOM, Wnt, Notch and the TGF-β signaling may provide a feasible approach to induce the regeneration of HCs.     

Canalostomy is an ideal surgery route for inner ear gene delivery in big animal model

Abstract

Background: Inner gene therapy offers great promises as a potential treatment for hearing loss.

Aims/objectives: One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss.

Material and methods: Surgery was performed only in the right ear of each Bama miniature pig, and the left ear served as a control. The gene delivery to inner ear via round window membrane (RWM) and posterior semicircular canal (PSC) approach was performed with the viral vector AAV1-CMV-GFP.

Results: The gene delivery through RWM and the PSC (canalostomy) is able to perfuse the inner ear.

Conclusions and significance: The easy anatomic identification of the PSC, as to RWM, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery in big animal model.     

Mechanisms of programmed cell death signaling in hair cells and support cells post-electrode insertion trauma

Conclusion: Programmed cell death (PCD) initially starts in the support cells (SCs) after electrode insertion trauma (EIT), followed by PCD in hair cells (HCs). Activation of caspase-3 was observed only in SCs. Protecting both SCs and HCs with selective otoprotective drugs at an early stage post implantation may help to preserve residual hearing. Objectives: Cochlear implant EIT can initiate sensory cell losses via necrosis and PCD within the organ of Corti, which can lead to a loss of residual hearing. PCD appears to be a major factor in HC loss post-EIT. The current study aimed to: (1) determine the onset of PCD in both SCs and HCs within the traumatized organ of Corti; and (2) identify the molecular mechanisms active within the HCs and SCs that are undergoing PCD. Methods: Adult guinea pigs were assigned to one of two groups: (1) EIT and (2) unoperated contralateral ears as controls. Immunostaining of dissected organ of Corti surface preparations for phosphorylated-Jun, cleaved caspase-3, and 4-hydroxy-2,3-nonenal (HNE) were performed at 6, 12, and 24 h post-EIT and for contralateral control ears. Results: At 6 h post-EIT the SCs immunolabeled for the presence of phosphorylated-Jun and activated caspase-3. Phosphorylated p-Jun labeling was observed at 12 h in both the HCs and SCs of middle and basal cochlear turns. Cleaved caspase-3 was not observed in HCs of any cochlear turn at up to 24 h post-EIT. Lipid peroxidation (HNE immunostaining) was first observed at 12 h post-EIT in both the HCs and SCs of the basal turn, and reached the apical turn by 24 h post-EIT.     

A Model of Spontaneous Chronic Otitis Media with Effusion in Mice with ENU Induced Mutations

Background Chronic otitis media (COM) is a significant clinical problem. Understanding the mechanisms of COM is critical for its control and treatment. However, little is known of the processes leading to COM as a result of lack of animal models of N-ethyl-N-nitrosourea (ENU) induced mutations in otitis media with effusion (OME). Methods Otoscopy and auditory brain response(ABR) evaluation were carried out under sedation in Nmf391 nmf/nmf mice of 2, 4, 6 and 8 months of age. The mice were killed for study of middle and inner ear pathology. Results Tympanic membrane visualization and ABR thresholds in 1- to 8-month-old Nmf391 nmf/nmf mice showed spontaneous OME and inner ear diseases in approximately 100% of the animals.The significant elevation of ABR thresholds suggested a sensorineural component in hearing loss in addition to the conductive loss. Middle and inner ear histology showed various degrees of outer hair cells loss and middle ear inflammation in all the mice, but no inflammation cells in the inner ear. The ABR threshold at 32 kHz was significantly elevated. Conclusions This study shows histopathologic changes in the Nmf391 nmf/nmf mouse model of COM with effusion that have not been reported in human COM. This ENU induced mutation model of COM will be valuable for the characterization of middle ear inflammation and inner ear disease processes that are induced by middle ear infections. We propose that COM with effusion in this ENU induced mutation model is the cause of the cochlea hair cells damage.     

Helper-dependent adenovirus-mediated gene transfer into the adult mouse cochlea.

BACKGROUND: Gene therapy may provide a way to restore cochlear function to deaf patients. The most successful techniques for cochlear gene therapy have been injection of early-generation adenoviral vectors into scala media in guinea pigs. However, it is important to be able to perform gene therapy research in mice because there is wide availability of transgenic strains with hereditary hearing loss. PURPOSE: We demonstrate our technique for delivery of a third-generation adenoviral vector, helper-dependent adenovirus (HDAd), to the adult mouse cochlea. METHODS: Mice were injected with an HDAd that contained a reporter gene for either beta-galactosidase or green fluorescent protein into scala media. After 4 days, the cochleae were harvested for analyses. Auditory brainstem response monitoring of cochlear function was performed before making a cochleostomy, after making a cochleostomy, and before killing the animal. RESULTS: Beta-galactosidase was identified in the spiral ligament, the organ of Corti, and spiral ganglion cells by light microscopy. Green fluorescent protein epifluorescence was assessed in whole-mount organ of Corti preparations using confocal microscopy. This demonstrated transduction of inner hair cells, outer hair cells, and supporting cells. Paraffin-embedded cross sections similarly revealed gene transduction within the organ of Corti. Threshold shifts of 39.8 +/- 5.4 and 37.7 +/- 5.5 dB were observed in mice injected with HDAd or control buffer, respectively. CONCLUSION: The technique of scala media HDAd injection reliably infects the adult mouse cochlea, including cells within the organ of Corti, although the procedure itself adversely affects hearing.     

Gene transfer into supporting cells of the organ of Corti

To utilize the rapidly accumulating genetic information for developing new therapeutic technologies for inner ear disease, it is necessary to design technologies for expressing transgenes in the inner ear, especially in the organ of Corti. We examined the outcome of an adenovirus gene transfer into the organ of Corti via the scala media in guinea pigs. The transgene insert is the bacterial lacZ gene driven by a cytomegalovirus promoter. We demonstrate that the inoculation is detrimental to the hair cells that surround the site of inoculation, but the supporting cells in the organ of Corti survive and retain the ability to express the reporter transgene beta-gal. The ability to deliver transgenes that are expressed in the supporting cells is an important step in the development of clinically applicable treatments that involve hair cell regeneration.     

Gene transfer in human vestibular epithelia and the prospects for inner ear gene therapy.

Transfer of exogenous genetic material into the mammalian inner ear using viral vectors has been characterized over the last decade. A number of different viral vectors have been shown to transfect the varying cell types of the nonprimate mammalian inner ear. Several routes of delivery have been identified for introduction of vectors into the inner ear while minimizing injury to existing structures and at the same time ensuring widespread distribution of the agent throughout the cochlea and the rest of the inner ear.These studies raise the possibility that gene transfer may be developed as a potential strategy for treating inner ear dysfunction in humans. Furthermore, a recent report showing successful transfection of excised human vestibular epithelia offers proof of principle that viral gene transfer is a viable strategy for introduction and expression of exogenous genetic material to restore function to the inner ear. Human vestibular epithelia were harvested from patients undergoing labyrinthectomy, either for intractable Ménière's disease or vestibular schwannoma resection, and cultured for as long as 5 days. In those experiments, recombinant, multiply-deleted, replication-deficient adenoviral vectors were used to transfect and express a reporter gene as well as the functionally relevant gene, wild-type KCNQ4, a potassium channel gene that when mutated causes the autosomal dominant HL DFNA2.Here, we review the current state of viral-mediated gene transfer in the inner ear and discuss different viral vectors, routes of delivery, and potential applications of gene therapy. Emphasis is placed on experiments demonstrating viral transfection of human inner ear tissue and implications of these findings and for the future of gene therapy in the human inner ear.