Hearing and hair cells are protected by adenoviral gene therapy with TGF-β1 and GDNF

Glial cell line-derived neurotrophic factor (GDNF) overexpression in the inner ear can protect hair cells against degeneration induced by aminoglycoside ototoxicity. The protective efficiency of GDNF increases when it is combined with co-factors such as transforming growth factor β1 (TGF-β1), a ubiquitous cytokine. The aim of this study was to determine whether TGF-β1 receptors are expressed in the inner ear and whether a cocktail of GDNF and TGF-β1 transgenes provides enhanced protection of the inner ear against ototoxic trauma. Using RT-PCR analysis, we determined that both TGF-β1 receptors, type 1 and 2 are present in rat cochlea. We co-inoculated two adenoviral vectors, one encoding human TGF-β1 gene (Ad.TGF-β1) and the other encoding human GDNF gene (Ad.GDNF) into guinea pig cochleae 4 days prior to injecting an ototoxic dose of aminoglycosides. Inoculated ears had better hearing and fewer missing inner hair cells after exposure to the aminoglycoside ototoxicity, as compared with controls and ears treated only with Ad.GDNF. Cochleae with TGF-β1 overexpression exhibited fibrosis in the scala tympani regardless of the presence of GDNF. Our results suggest that the adenovirus-mediated overexpression of GDNF and TGF-β1 can be used in combination to protect cochlear hair cells and hearing from ototoxic trauma.     

Hair cell protection from aminoglycoside ototoxicity by adenovirus-mediated overexpression of glial cell line-derived neurotrophic factor

Aminoglycosides are commonly used antimicrobial drugs that often have ototoxic side effects. The ototoxicity often involves permanent loss of cochlear hair cells (HCs). Neurotrophic factors have been shown to protect a variety of tissues, including HCs, from toxic trauma. To determine if glial cell line-derived neurotrophic factor (GDNF) can protect cochlear HCs from trauma, we inoculated an adenoviral vector encoding the human GDNF gene into guinea pig cochleae via the round window membrane 4 days prior to injection of aminoglycosides. Control groups showed little or no negative influence of the viral inoculation on cochlear structure and function. In contrast, ears that were inoculated with the GDNF vector had better hearing and fewer missing HCs after exposure to the ototoxins, as compared with controls. Our results demonstrate the feasibility of gene therapy for cochlear application and suggest that virus-mediated overexpression of GDNF may be developed as a valuable prevention against trauma-induced HC death.     

Sound preconditioning therapy inhibits ototoxic hearing loss in mice

Therapeutic drugs with ototoxic side effects cause significant hearing loss for thousands of patients annually. Two major classes of ototoxic drugs are cisplatin and the aminoglycoside antibiotics, both of which are toxic to mechanosensory hair cells, the receptor cells of the inner ear. A critical need exists for therapies that protect the inner ear without inhibiting the therapeutic efficacy of these drugs. The induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death and hearing loss. We hypothesized that exposure to sound that is titrated to stress the inner ear without causing permanent damage would induce HSPs in the cochlea and inhibit ototoxic drug–induced hearing loss. We developed a sound exposure protocol that induces HSPs without causing permanent hearing loss. We used this protocol in conjunction with a newly developed mouse model of cisplatin ototoxicity and found that preconditioning mouse inner ears with sound has a robust protective effect against cisplatin-induced hearing loss and hair cell death. Sound therapy also provided protection against aminoglycoside-induced hearing loss. These data indicate that sound preconditioning protects against both classes of ototoxic drugs, and they suggest that sound therapy holds promise for preventing hearing loss in patients receiving these drugs.     

Adenovirus-mediated overexpression of a gene prevents hearing loss and progressive inner hair cell loss after transient cochlear ischemia in gerbils

The use of adenoviral vectors has recently provided a novel strategy for direct gene transfer into the cochlea. In this study, we assessed the utility of an adenoviral vector expressing glial-cell-derived neurotrophic factor (GDNF) in ischemia-reperfusion injury of the gerbil cochlea. The vector was injected through the round window 4 days before ischemic insult. The distribution of a reporter transgene was confirmed throughout the cochlea from the basal to the apical turn and Western blot analysis indicated significant upregulation of GDNF protein 11 days following virus inoculation. Hearing ability was assessed by sequentially recording compound action potentials (CAP), and the degree of hair cell loss in the organ of Corti was evaluated in specimens stained with rhodamine-phalloidin and Hoechst 33342. On the seventh day of ischemia, the CAP threshold shift and inner hair cell loss were remarkably suppressed in the Ad-GDNF group compared with the control group. These results suggest that adenovirus-mediated overexpression of GDNF is useful for protection against hair cell damage, which otherwise eventually occurs after transient ischemia of the cochlea.     

Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity

We previously demonstrated that an artificial protein, TAT-FNK, has antiapoptotic effects against cochlear hair cell (HC) damage caused by ototoxic agents when applied systemically. To examine the feasibility of topical protein therapy for inner ear disorders, we investigated whether gelatin sponge soaked with TAT-FNK and placed on the guinea pig round window membrane (RWM) could deliver the protein to the cochlea and attenuate aminoglycoside (AG)-induced cochlear damage in vivo. First, we found that the immunoreactivity of TAT-myc-FNK was distributed throughout the cochlea. The immunoreactivity was observed from 1-24 h after application. When Tat-FNK was applied 1 h before ototoxic insult (a combination of kanamycin sulfate and ethacrynic acid), auditory brainstem response threshold shifts and the extent of HC death were significantly attenuated. When cochlear organotypic cultures prepared from P5 rats were treated with kanamycin, TAT-FNK significantly reduced the extent of caspase-9 activation and HC death. These findings indicate that TAT-FNK topically applied on the RWM can enter the cochlea by diffusion and effectively prevent AG-induced apoptosis of cochlear HCs by suppressing the mitochondrial caspase-9 pathway.     

Current status and prospects of gene therapy for the inner ear

Inner ear diseases are common and often result in hearing disability. Sensorineural hearing loss is the main cause of hearing disability. So far, no effective treatment is available although some patients may benefit from a hearing aid equipped with a hearing amplifier or from cochlear implantation. Inner ear gene therapy has become an emerging field of study for the treatment of hearing disability. Numerous new discoveries and tremendous advances have been made in inner ear gene therapy including gene vectors, routes of administration, and therapeutic genes and targets. Gene therapy may become a treatment option for inner ear diseases in the near future. In this review, we summarize the current state of inner ear gene therapy including gene vectors, delivery routes, and therapeutic genes and targets by examining and analyzing publications on inner ear gene therapy from the literature and patent documents, and identify promising patents, novel techniques, and vital research projects. We also discuss the progress and prospects of inner ear gene therapy, the advances and shortcomings, with possible solutions in this field of research.     

Nephrotoxic and ototoxic agents

It is well established that many drugs, such as the aminoglycoside antibiotics and the chemotherapeutic drug cisplatin, are capable of inducing both nephrotoxicity and ototoxicity. The factors that selectively predispose the kidney and inner ear to the toxic effects of these agents as well as the mechanism by which damage is produced are not well defined. The two organs differ greatly in their exposure to these toxic agents. The kidney has an abundant vascular supply and tends to selectively concentrate a number of drugs within the renal cortex or medulla, often to toxic levels. The vascular supply of the inner ear is not as extensive. In addition, the stria vascularis of the cochlea may act as a functional regulator of drug entry into inner ear fluids. The absorption of drugs into perilymph and endolymph is poorly understood. Selective accumulation theories of drug accumulation in the inner ear must be questioned because of the results of recent pharmacokinetic studies, which give contrary data. Drug-induced ototoxicity and nephrotoxicity can be explained on a cellular level. Studies using radiolabeled gentamicin suggest that binding mechanisms of the drug to the plasma membrane of the outer hair cells of the cochlea and vestibular apparatus and to the brush border receptors of the renal proximal convoluted tubules are similar. This suggests the same receptor sites for aminoglycosides occur in otic and renal organs. Calcium channels are implicated because of the reversibility of aminoglycoside-induced changes in the cochlear microphonic by calcium and other divalent cations. Calcium channel blockers, such as verapamil, reduce the nephrotoxicity of a number of drugs that are also ototoxic. Studies are needed to assess potential prevention of ototoxicity by use of these same calcium channel blocking agents. Aminoglycosides concentrate within the lysosomes of renal proximal tubular cells. Possibly, they also may concentrate in lysosomes within the cells of cochlear and vestibular structures. Nephrotoxic heavy metals concentrate within proximal tubular cells and, some, such as lead or bismuth, specifically concentrate within intracytoplasmic or intranuclear inclusion bodies. Studies are necessary to determine if the same metals accumulate within the cochlear and vestibular cells, inclusion bodies, or both. These questions and others must be answered before it can be determined why many nephrotoxic drugs and agents are also ototoxic.     

Exosomes derived from mouse inner ear stem cells attenuate gentamicin‐induced ototoxicity in vitro through the miR‐182‐5p/FOXO3 axis

Gentamicin‐induced cochlear hair cell ototoxicity, such as oxidative stress and apoptosis, could be attenuated by mouse inner ear stem cells (IESCs). However, it is still unclear whether such protective effects could be mediated by exosomes derived from IESCs (IESCs‐ex). In the present study, HEI‐OC1 cells were exposed to gentamicin (2 mM) to establish an ototoxicity model and further treated with exosomes isolated from miR‐182‐5p transferred or non‐transferred IESCs. IESCs‐ex improved HEI‐OC1 cell viability, as assayed by the 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyltetrazolium bromide method, and alleviated the oxidative stress response induced by the gentamicin treatment, as confirmed by measuring the malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase levels. IESCs‐ex increased relative miR‐182‐5p expression and decreased FOXO3 expression in the gentamicin‐exposed HEI‐OC1 cells. Furthermore, exosomes derived from miR‐182‐5p mimics that were pre‐treated with IESCs could increase miR‐182‐5p and Bcl‐2 expressions and decrease FOXO3 and Bax expressions in gentamicin‐exposed HEI‐OC1 cells. All of these results indicate that IESCs‐ex could attenuate gentamicin‐induced HEI‐OC1 cell apoptosis and oxidative stress through the miR‐182‐5p/FOXO3 axis.     

Transduction of the contralateral ear after adenovirus-mediated cochlear gene transfer

Cochlear gene transfer is a promising new approach for inner ear therapy. Previous studies have demonstrated hair cell protection with cochlear gene transfer not only in the inoculated, but also in the uninoculated ear. To characterize the kinetics of viral spread, we investigated the extent of transgene expression in the contralateral (uninoculated) cochlea after unilateral adenoviral cochlear gene transfer. We used a lacZ reporter gene vector, and demonstrated spread of the adenovirus into the cerebrospinal fluid (CSF) after cochlear inoculation of 25 microl viral vector. Direct virus application into the CSF resulted in transduction of both cochleae, whereas virus inoculation into the bloodstream did not. The cochlear aqueduct was identified as the most likely route of virus spread to the contralateral cochlea. These data enhance our understanding of the kinetics of virus-mediated transgene expression in the inner ear, and assist in the development of clinical applications for inner ear gene therapy. Our results showed a functional communication between the CSF and the perilymphatic space of the inner ear, that is not only of importance for otological gene transfer, but also for CNS gene transfer. Gene Therapy (2000) 7, 377-383.     

Ototoxicity induced by gentamicin and furosemide

OBJECTIVE: To present a case of ototoxicity induced by furosemide and once-daily gentamicin therapy. CASE SUMMARY: A 60-year-old white woman presented to the hospital with community-acquired pneumonia and urinary tract infection. The antibiotic regimen included gentamicin and, after 5 doses, the patient reported profound bilateral hearing loss. A Pure Tone Audiogram suggested moderate to moderately severe sensorineural hearing loss bilaterally. The only risk factors present included her age, elevated temperature, and the use of furosemide. DISCUSSION: Several risk factors may predispose a patient to developing aminoglycoside ototoxicity: the 1555 chromosomal mutation, preexisting disorders of hearing and balance, hypovolemia, bacteremia, liver and renal dysfunction, and the simultaneous administration of other ototoxic medications. The cumulative dose and duration of aminoglycoside therapy are more important than serum concentrations. Administration of an aminoglycoside followed by furosemide may increase the risk of ototoxicity. The aminoglycoside interacts with the cell membranes in the inner ear, increasing their permeability. This theoretically allows the loop diuretic to penetrate into the cells in higher concentrations, causing more severe damage. CONCLUSIONS: Auditory toxicity occurred after only 5 days of gentamicin therapy and 1 dose of furosemide. An aminoglycoside followed by furosemide may increase the risk for ototoxicity. Clinicians need to be aware of the synergistic potential of ototoxic medications.     

A modified adenovirus can transfect cochlear hair cells in vivo without compromising cochlear function.

The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1(-), E3(-)) and replication-defective (E1(-), E3(-), pol(-)) adenovirus vectors were used to transduce the bacterial beta-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1(-), E3(-)) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1(-), E3(-), pol(-)) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and beta-galactosidase (beta-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1(-), E3(-), pol(-)) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.     

Comparative Ototoxicity of Amikacin and Gentamicin in Cats

The ototoxic potentials of two aminoglycoside antibiotics, amikacin and gentamicin, were compared in cats, using several otoxicity assessment techniques. Daily subcutaneous doses of 90 and 45 mg of amikacin per kg and 18 and 9 mg of gentamicin per kg (approximately six and three times the daily human dose) were administered to cats for extended periods of time until cochlear or vestibular dysfunction developed. Renal tissue damage and serum and perilymph antibiotic concentrations were also monitored. Amikacin selectively produced an impairment of cochlear function after an approximate cumulative dose of 3,600 mg/kg obtained after 41 days at 90 mg/kg per day or 78 days at 45 mg/kg per day, as determined by electrophysiological assessment. Gentamicin caused an impairment of vestibular function after an approximate cumulative dose of 700 mg/kg obtained after 42 days at 18 mg/kg per day or 68 days at 9 mg/kg per day, as determined by ataxia and impaired righting reflex. Gentamicin also moderately reduced electrophysiological cochlear responses and appeared to cause histological renal tissue change more frequently than did amikacin.     

A novel bovine virus efficiently transduces inner ear neuroepithelial cells.

Disruption of the cellular composition or arrangement of the sensory epithelia due to hair cell or supporting cell damage leads to hearing loss and vestibular dysfunctions. These peripheral hearing disorders make good targets for gene therapy; however, development requires efficient gene transfer methods for the inner ear. Here we characterized the cellular tropism of a novel adeno-associated bovine virus vector (BAAV) in cultured rat inner ear epithelia. To help identify transduced cells, we used beta-actin-GFP as a reporter gene. We found that BAAV efficiently transduced auditory and vestibular hair cells as well as all types of supporting cells with no apparent pathological effects. The number of transduced hair cells significantly increased in both a dose- and a time-dependent manner. Transduction was independent of the cells' maturation state and was observed in both P2 and P10 cultures. Interestingly, even after several days of incubation with BAAV, hair cells demonstrated varying progression of beta-actin-GFP incorporation into the stereocilia. This suggests that the onset of viral transduction can occur throughout the course of the experiment. Of the other tested AAVs, AAV2 and AAV5 transduced only a small percentage of inner and vestibular hair cells, respectively, whereas no transduction was detected with AAV4.     

Kinetics of gentamicin uptake and release in the rat. Comparison of inner ear tissues and fluids with other organs.

The kinetics of entry and release of gentamicin was investigated in fluids and tissues of the inner ear of the rat, as well as in renal cortex, and in organs that do not share susceptibility to the toxic effects of aminoglycosides. Various modes of administration were used to achieve different patterns of drug plasma concentrations. Electrophysiological and histological examinations were performed to correlate pharmacokinetics and ototoxicity. Results show that: the uptake of the drug by the inner ear tissues is dose dependent and manifests a rapid saturation kinetics with a concentration plateau of about 1 micrograms/mg of protein. The low ratio of the perilymph and endolymph to plasma concentrations argues against the concept of an accumulation of the drug in the inner ear over drug levels in plasma, which has been considered as the basic mechanism of ototoxicity. In renal cortex, the kinetics appears similar to that of the inner ear but the concentrations achieved are 10-fold higher than in cochlear tissues. In other organs (liver, heart, lung, and spleen), no saturation could be demonstrated within the duration of the experiment. Ototoxicity seems to be related to the penetration of the drug into compartment(s) from which the half-life of disappearance is extremely slow. Rapid uptake, early saturation, and long exposure of the tissues to the drug may account for the development of toxicity in inner ear and kidney.     

神经营养因子修复神经组织机制的研究:脑细胞生长肽对豚鼠耳蜗毛细胞内酸性磷酸酶的影响

背景脑细胞生长肽(cerebrocellular growth peptide,CCGP)对庆大霉素引起的耳蜗毛细胞内酸性磷酸酶(acid phosphatase,ACP)的变化是否有影响?对受损耳蜗组织是否有促进修复的作用?目的观察CCGP对庆大霉素引起的耳中毒豚鼠ACP的影响.设计随机对照研究.地点和材料实验地点泰山医学院听觉研究室.选用健康杂色豚鼠40只,对照组10只,肌肉注射生理盐水1 mL/(kg·d);庆大霉素组15只,肌肉注射硫酸庆大霉素80 mg/(kg·d);CCGP组15只,肌肉注射硫酸庆大霉素同庆大霉素组,并肌肉注射CCGP 1 mg/(kg·d).各组用药25d.方法用脑干听觉诱发电位(brainstem auditory evoked potential,BAEP)和组织化学方法检测动物听阈的变化和耳蜗毛细胞ACP显色变化.主要观察指标各组动物BAEP反应阈值和ACP显色变化.结果用药前BAEP反应阈值[dB(peSLP)]生理盐水组32.62±2.33,庆大霉素组31.87±2.63,CCGP组32.56±2.39.用药后BAEP反应阈值均有不同程度的升高,用药后25 d BAEP反应阈值生理盐水组32.81±2.48,庆大霉素组56.73±17.21,CCGP组42.87±9.95,庆大霉素组、CCGP组与生理盐水组比较,差异均有显著性意义(t=3.113,4.335,P均＜0.01),CCGP组与庆大霉素组比较,差异有显著性意义(t=2.700,P＜0.05).ACP显色变化生理盐水组毛细胞ACP染色呈棕褐色,毛细胞排列整齐.庆大霉素组ACP变化显著,毛细胞显色失明显;CCGP组ACP显色变化较轻,两组铺片显示有明显差别.结论CCGP能降低庆大霉素的耳毒性,减轻由于溶酶体的破坏溢出的ACP引起的毛细胞的损伤.     

Treatment of peripheral sensorineural hearing loss: gene therapy

Noise, chemicals and genetic defects are all common causes of irreversible hearing loss, which at present have no cure. Gene therapy may soon be utilized in both the protection and the treatment of these exogenous and endogenous sources of hearing loss. Gene therapy technology is rapidly developing and the inner ear is a particularly feasible model for gene therapy. This review outlines our current understanding of the mechanisms behind deafness and prospects for treatment, discusses the inner ear model in detail and reviews the efforts that have been made in inner ear gene therapy. Finally, the proposed next steps will be discussed. The viral mediated delivery of neurotrophins and antioxidants offers imminent promise in preventing and treating exogenous hearing loss and improving cochlear implant therapy.     

Cell-gene delivery of brain-derived neurotrophic factor to the mouse inner ear.

Sensorineural hearing loss is a common disability, but treatment options are currently limited to cochlear implants and hearing aids. Studies are therefore being conducted to provide alternative means of biological therapy, including gene therapy. Safe and effective methods of gene delivery to the cochlea need to be developed to facilitate the clinical application of these therapeutic treatments for hearing loss. In this study, we examined the potential of cell-gene therapy with nonviral vectors for delivery of therapeutic molecules into the cochlea. NIH3T3 cells were transfected with the brain-derived neurotrophic factor (Bdnf) gene using lipofection and then transplanted into the mouse inner ear. Immunohistochemistry and Western blotting demonstrated the survival of grafted cells in the cochlea for up to 4 weeks after transplantation. No significant hearing loss was induced by the transplantation procedure. A Bdnf-specific enzyme-linked immunosorbent assay revealed a significant increase in Bdnf production in the inner ear following transplantation of engineered cells. These findings indicate that cell-gene delivery with nonviral vectors may be applicable for the local, sustained delivery of therapeutic molecules into the cochlea.     

Adeno-associated virus-mediated gene delivery into the scala media of the normal and deafened adult mouse ear

Murine models are ideal for studying cochlear gene transfer, as many hearing loss-related mutations have been discovered and mapped within the mouse genome. However, because of the small size and delicate nature, the membranous labyrinth of the mouse is a challenging target for the delivery of viral vectors. To minimize injection trauma, we developed a procedure for the controlled release of adeno-associated viruses (AAVs) into the scala media of adult mice. This procedure poses minimal risk of injury to structures of the cochlea and middle ear, and allows for near-complete preservation of low and middle frequency hearing. In this study, transduction efficiency and cellular specificity of AAV vectors (serotypes 1, 2, 5, 6 and 8) were investigated in normal and drug-deafened ears. Using the cytomegalovirus promoter to drive gene expression, a variety of cell types were transduced successfully, including sensory hair cells and supporting cells, as well as cells in the auditory nerve and spiral ligament. Among all five serotypes, inner hair cells were the most effectively transduced cochlear cell type. All five serotypes of AAV vectors transduced cells of the auditory nerve, though serotype 8 was the most efficient vector for transduction. Our findings indicate that efficient AAV inoculation (via the scala media) can be performed in adult mouse ears, with hearing preservation a realistic goal. The procedure we describe may also have applications for intra-endolymphatic drug delivery in many mouse models of human deafness.     

Inner ear supporting cells protect hair cells by secreting HSP70

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70^–/– mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.