Kohei Kawamoto

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo

Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.

Aminoglycoside ototoxicity in adult CBA, C57BL and BALB mice and the Sprague–Dawley rat

The availability of genetic information, transgenic and knock-out animals make the mouse a primary model in biomedical research. Aminoglycoside ototoxicity, however, has rarely been studied in mature mice because they are considered highly resistant to the drugs. This study presents models for kanamycin ototoxicity in adult CBA/J, C57BL/6 and BALB/c mouse strains and a comparison to Sprague-Dawley rats. Five-week-old mice were injected subcutaneously twice daily with 400-900 mg kanamycin base/kg body weight for 15 days. Kanamycin induced dose-dependent auditory threshold shifts of up to 70 dB at 24 kHz as measured by auditory brain stem-evoked responses. Vestibular function was also affected in all strains. The functional deficits were accompanied by hair cell loss in both cochlear and vestibular neurosensory epithelia. Concomitant administration of the antioxidant 2,3-dihydroxybenzoate significantly attenuated the kanamycin-induced threshold shifts. In adult male Sprague-Dawley rats, doses of 1 x 500 mg or 2 x 300 mg kanamycin base/kg body weight/day x 14 days induced threshold shifts of approximately 50 dB at 20 kHz. These were accompanied by loss of outer hair cells. The order of susceptibility, BALB>CBA>C57, was not due to differences in the pharmacokinetics of kanamycin. It also did not correlate with the presence of Ahl/Ahl2 genes which predispose C57 and BALB strains, respectively, to accelerated age-related hearing loss. Pigmentation, however, paralleled this rank order suggesting an influence of melanin on cochlear antioxidant status.

Spiral ganglion neurons are protected from degeneration by GDNF gene therapy

Perceptual benefits from the cochlear prosthesis are related to the quantity and quality of the patients auditory nerve population. Multiple neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have been shown to have important roles in the survival of inner ear auditory neurons, including protection of deafferented spiral ganglion cells (SGCs). In this study, GDNF gene therapy was tested for its ability to enhance survival of SGCs after aminoglycoside/diuretic-induced insult that eliminated the inner hair cells. The GDNF transgene was delivered by adenoviral vectors. Similar vectors with a reporter gene (lacZ) insert served as controls. Four or seven days after bilateral deafening, 5 microl of an adenoviral suspension (Ad-GDNF or Ad-lacZ) or an artificial perilymph was injected into the left scala tympani of guinea pigs. Animals were sacrificed 28 days after deafening and their inner ears prepared for SGC counts. Adenoviral-mediated GDNF transgene expression enhanced SGC survival in the left (viral-treated) deafened ears. This observation suggests that GDNF is one of the survival factors in the inner ear and may help maintain the auditory neurons after insult. Application of GDNF and other survival factors via gene therapy has great potential for inducing survival of auditory neurons following hair cell loss.

Glial cell line-derived neurotrophic factor and chronic electrical stimulation prevent VIII cranial nerve degeneration following denervation

As with other cranial nerves and many CNS neurons, primary auditory neurons degenerate as a consequence of loss of input from their target cells, the inner hair cells (IHCs). Electrical stimulation (ES) of spiral ganglion cells (SGCs) has been shown to enhance their survival. Glial cell line‐derived neurotrophic factor (GDNF) has also been shown to increase survival of SGCs following IHC loss. In this study, the combined effects of the GDNF transgene delivered by adenoviral vectors (Ad‐GDNF) and ES were tested on SGCs after first eliminating the IHCs. Animal groups received Ad‐GDNF or ES or both. Ad‐GDNF was inoculated into the cochlea of guinea pigs after deafening, to overexpress human GDNF. ES‐treated animals were implanted with a cochlear implant electrode and chronically stimulated. A third group of animals received both Ad‐GDNF and ES (GDNF/ES). Electrically evoked auditory brainstem responses were recorded from ES‐treated animals at the start and end of the stimulation period. Animals were sacrificed 43 days after deafening and their ears prepared for evaluation of IHC survival and SGC counts. Treated ears exhibited significantly greater SGC survival than nontreated ears. The GDNF/ES combination provided significantly better preservation of SGC density than either treatment alone. Insofar as ES parameters were optimized for maximal protection (saturated effect), the further augmentation of the protection by GDNF suggests that the mechanisms of GDNF‐ and ES‐mediated SGC protection are, at least in part, independent. We suggest that GDNF/ES combined treatment in cochlear implant recipients will improve auditory perception. These findings may have implications for the prevention and treatment of other neurodegenerative processes. J. Comp. Neurol. 454:350–360, 2002.

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.

Adenovirus-Mediated Expression of Brain-Derived Neurotrophic Factor Protects Spiral Ganglion Neurons from Ototoxic Damage

Hair cell loss, the most common cause of deafness, is often associated with auditory nerve degeneration. Our goal was to determine the influence of combined ciliary-derived neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) gene therapy on the survival of spiral ganglion neurons (SGNs) after elimination of inner hair cells in the mature guinea pig ear. Seven days after bilateral deafening, a 5-µl suspension of CNTF and/or BDNF adenovirus vectors was injected into the left scala tympani through the round window. Animals were sacrificed 28 days after deafening, and their inner ears were prepared for SGN counts. The SGN counts revealed that BDNF alone and the combined CNTF and BDNF treatment significantly enhanced SGN survival. CNTF did not significantly enhance the protective effect of BDNF. These data present possible strategies for enhancing SGN survival in cochlear implant procedures.

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.

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 beta1 (TGF-beta1), a ubiquitous cytokine. The aim of this study was to determine whether TGF-beta1 receptors are expressed in the inner ear and whether a cocktail of GDNF and TGF-beta1 transgenes provides enhanced protection of the inner ear against ototoxic trauma. Using RT-PCR analysis, we determined that both TGF-beta1 receptors, type 1 and 2 are present in rat cochlea. We co-inoculated two adenoviral vectors, one encoding human TGF-beta1 gene (Ad.TGF-beta1) 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-beta1 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-beta1 can be used in combination to protect cochlear hair cells and hearing from ototoxic trauma.

Gene transfer into guinea pig cochlea using adeno-associated virus vectors

Several genes are candidates for treating inner ear diseases. For clinical applications, minimally invasive approaches to the inner ear are desirable along with minimal side‐effects.