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Efficient gene transfer to the mouse inner ear is achieved with a synthetic adeno-associated viral vector. Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities 1 , 2 . Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector 3 , for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Hearing loss is the most common sensory impairment in humans and currently disables 466 million people across the world. Congenital deafness affects at least 1 in 500 newborns, and over 50% are hereditary in nature. To date, existing pharmacologic therapies for genetic and acquired etiologies of deafness are severely limited. With the advent of modern sequencing technologies, there is a vast compendium of growing genetic alterations that underlie human hearing loss, which can be targeted by therapeutics such as gene therapy. Recently, there has been tremendous progress in the development of gene therapy vectors to treat sensorineural hearing loss (SNHL) in animal models . Nevertheless, significant hurdles remain before such technologies can be translated toward clinical use. These include addressing the blood-labyrinth barrier, engineering more specific and effective delivery vehicles, improving surgical access, and validating novel targets. In this review, we both highlight recent progress and outline challenges associated with gene therapy for human SNHL.

Background Vestibular schwannoma (VS) is an intracranial tumor arising from the Schwann cells of the vestibular nerve and is an important cause of sensorineural hearing loss (SNHL) in humans. The mechanisms underlying this SNHL are incompletely understood and currently, there are no drugs FDA approved specifically for VS. This knowledge gap significantly limits the development of effective treatments aimed at preventing, stabilizing, or reversing VS-induced SNHL. Methods To identify effector molecules involved in VS-induced SNHL, we analyzed 47 immune-related factors secreted by tumor tissue in over 50 patients with sporadic VS and studied their correlation with preoperative hearing ability and tumor size. The most promising effector molecules were validated in vivo in an anatomically accurate mouse model of VS, and in vitro with mouse fibroblasts (L929) and auditory cell lines representing pro-sensory precursors of hair cells (UB-OC1) and auditory neuroblasts (US-VOT-N33). Results We demonstrated that VS-induced SNHL was linked to increased secretion of TNF-α, IL-2R, CD163, eotaxin, and HGF, while larger tumor size was associated with higher levels of TNF-α, TNF-R2, IL-1α, IFN-α, MIP-1β, and IL-21 secretion. We identified heterogeneity among VS tumors in their capacity to secrete TNF-α. Tumors with high levels of TNF-α secretion released cytokines and chemokines that significantly correlated with poor hearing (TWEAK and eotaxin) or better hearing (LIF, GRO-α, MIP-1α, MIP-3α, and IL-1α). Among these, TWEAK was notably abundant, with levels exceeding those in normal nerve tissue, elevated in patients with non-serviceable hearing and strongly linked to poor hearing in patients with TNF-α high-secreting tumors. In vivo, we demonstrated that VS-secreted factors reach the inner ear, with elevated TNF-α and TWEAK in the perilymph and blood of tumor-bearing mice with impaired hearing. In vitro, TWEAK amplified TNF-α -mediated cytotoxicity in TNF-α sensitive cells (L929) and auditory cell lines (UB-OC1 and US-VOT-N33) at tumor-secreted concentrations. Conclusion This study provides compelling evidence that VS-secreted TNF-α and TWEAK act synergistically to drive tumor-induced SNHL. Targeting the TNF-α/TWEAK axis presents a promising new avenue for preventing VS-induced SNHL.

The energy of the electrochemical potential in the guinea pig cochlea is harvested and used to power a wireless transmitter. Endocochlear potential (EP) is a battery-like electrochemical gradient found in and actively maintained by the inner ear 1 , 2 . Here we demonstrate that the mammalian EP can be used as a power source for electronic devices. We achieved this by designing an anatomically sized, ultra-low quiescent-power energy harvester chip integrated with a wireless sensor capable of monitoring the EP itself. Although other forms of in vivo energy harvesting have been described in lower organisms 3 , 4 , 5 , and thermoelectric 6 , piezoelectric 7 and biofuel 8 , 9 devices are promising for mammalian applications, there have been few, if any, in vivo demonstrations in the vicinity of the ear, eye and brain. In this work, the chip extracted a minimum of 1.12 nW from the EP of a guinea pig for up to 5 h, enabling a 2.4 GHz radio to transmit measurement of the EP every 40–360 s. With future optimization of electrode design, we envision using the biologic battery in the inner ear to power chemical and molecular sensors, or drug-delivery actuators for diagnosis and therapy of hearing loss and other disorders.

The vast majority of hearing loss, the most common sensory impairment, and vertigo, which commonly causes falls, both reflect underlying dysfunction of inner ear cells. Perilymph sampling can thus provide molecular cues to hearing and balance disorders. While such \"liquid biopsy\" of the inner ear is not yet in routine clinical practice, previous studies have uncovered alterations in perilymph in patients with certain types of hearing loss. However, the proteome of perilymph from patients with intact hearing has been unknown. Furthermore, no complete characterization of perilymph from patients with vestibular dysfunction has been reported. Here, using liquid-chromatography with tandem mass spectrometry, we analyzed samples of normal perilymph collected from three patients with skull base meningiomas and intact hearing. We identified 228 proteins that were common across the samples, establishing a greatly expanded proteome of the previously inferred normal human perilymph. Further comparison to perilymph obtained from three patients with vestibular dysfunction with drop attacks due to Meniere's disease showed 38 proteins with significantly differential abundance. The abundance of four protein candidates with previously unknown roles in inner ear biology was validated in murine cochleae by immunohistochemistry and in situ hybridization: AACT, HGFAC, EFEMP1, and TGFBI. Together, these results motivate future work in characterizing the normal human perilymph and identifying biomarkers of inner ear disease.

Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects’ degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs.

Moderate acoustic overexposure in adult rodents is known to cause acute loss of synapses on sensory inner hair cells (IHCs) and delayed degeneration of the auditory nerve, despite the completely reversible temporary threshold shift (TTS) and morphologically intact hair cells. Our objective was to determine whether a cochlear synaptopathy followed by neuropathy occurs after noise exposure in pubescence, and to define neuropathic versus non-neuropathic noise levels for pubescent mice. While exposing 6 week old CBA/CaJ mice to 8-16 kHz bandpass noise for 2 hrs, we defined 97 dB sound pressure level (SPL) as the threshold for this particular type of neuropathic exposure associated with TTS, and 94 dB SPL as the highest non-neuropathic noise level associated with TTS. Exposure to 100 dB SPL caused permanent threshold shift although exposure of 16 week old mice to the same noise is reported to cause only TTS. Amplitude of wave I of the auditory brainstem response, which reflects the summed activity of the cochlear nerve, was complemented by synaptic ribbon counts in IHCs using confocal microscopy, and by stereological counts of peripheral axons and cell bodies of the cochlear nerve from 24 hours to 16 months post exposure. Mice exposed to neuropathic noise demonstrated immediate cochlear synaptopathy by 24 hours post exposure, and delayed neurodegeneration characterized by axonal retraction at 8 months, and spiral ganglion cell loss at 8-16 months post exposure. Although the damage was initially limited to the cochlear base, it progressed to also involve the cochlear apex by 8 months post exposure. Our data demonstrate a fine line between neuropathic and non-neuropathic noise levels associated with TTS in the pubescent cochlea.

Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7–14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders. Gene therapy using Adeno-associated viral vectors (AAV) rescues hearing and balance deficits in mouse models of human disorders. Here, the authors show that AAVAnc80L65 allows efficient cochlear gene transfer in nonhuman primates, and motivate future studies to evaluate gene therapy for hearing and balance disorders.