Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate.

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.

Genetic Medicine for Hearing Loss: OTOF as Exemplar.

Millions of people worldwide have disabling hearing loss because one of their genes generates an incorrect version of some specific protein the ear requires for hearing. In many of these cases, delivering the correct version of the gene to a specific target cell within the inner ear has the potential to restore cochlear function to enable high-acuity physiologic hearing. Purpose: In this review, we outline our strategy for the development of genetic medicines with the potential to treat hearing loss. We will use the example of otoferlin gene (OTOF)-mediated hearing loss, a sensorineural hearing loss due to autosomal recessive mutations of the OTOF gene.

Measurement of Ototoxicity Following Intracochlear Bisphosphonate Delivery.

HYPOTHESIS: Assessing the maximum safe dose for local bisphosphonate delivery to the cochlea enables efficient delivery without ototoxicity. BACKGROUND: Otosclerosis is a disease of abnormal bone metabolism affecting the otic capsule, which can cause conductive hearing loss. Larger otosclerotic lesions involving the cochlear endosteum and spiral ligament can result in sensorineural hearing loss. Bisphosphonates are used to treat patients with metabolic bone diseases, including otosclerosis. Local delivery is the most efficient way of delivery to the cochlea while avoiding systemic side effects. To attain intracochlear bisphosphonate delivery without ototoxicity, the maximum safe dose of bisphosphonates requires definition. In the present study, we tested increasing concentrations of zoledronate, a third-generation bisphosphonate in an intracochlear delivery system. We measured ototoxicity by monitoring distortion product otoacoustic emissions and compound action potentials. METHODS: Artificial perilymph and increasing molar concentrations of zoledronate were administered to the cochlea in guinea pigs via a cochleostomy. Hearing was measured at multiple time points. A fluorescently labeled zoledronate derivative (6-FAM-ZOL) was coadministered as an internal control for drug delivery. Specimens embedded in the resin blocks were ground to a mid-modiolar section and fluorescent photomicrographs were taken. RESULTS: No significant shift in hearing was observed in animals treated either with artificial perilymph or with 4% of the human systemic zoledronate dose. However, compound action potentials thresholds increased during infusion of 8% of the human systemic zoledronate dose, improved 4 hours later, and then increased again 4 weeks later. Using fluorescent photomicrography, intracochlear bisphosphonate delivery up to the apical cochlear turn was confirmed by visualizing 6-FAM-ZOL. CONCLUSION: These findings provide reference values for intracochlear bisphosphonate delivery in the treatment of cochlear otosclerosis and describe a useful method for tracking cochlear drug delivery.

Intracochlear Drug Delivery Through the Oval Window in Fresh Cadaveric Human Temporal Bones.

HYPOTHESIS: Drug delivered to the oval window can diffuse to the apex of the human cochlea. BACKGROUND: We have previously demonstrated that zoledronate, a nitrogen-containing bisphosphonate, can arrest the sensorineural hearing loss in cochlear otosclerosis. We have also shown that, in animals, delivery of bisphosphonate into the cochlea can dramatically increase delivery efficiency. Intracochlear drug delivery has the potential to increase local concentration of drug while decreasing the risk of systemic toxicity. In the present study, a fluorescently labeled bisphosphonate compound (6-FAM-ZOL) was introduced into the human cochlea through the oval window and its distribution within the temporal bone was quantified. METHODS: In three fresh human temporal bones, we introduced 6-FAM-ZOL via the oval window. We compared these specimens to control specimens treated with artificial perilymph alone. Specimens were then processed, embedded into methyl methacrylate, and ground to the mid-modiolar axis. We quantified the fluorescence in confocal images. RESULTS: We found 6-FAM-ZOL to be distributed up to the apical cochlear turn. In specimens treated with 6-FAM-ZOL, we identified a strong baso-apical gradient of fluorescent signal along the lateral cochlear wall and the modiolus both in the scala vestibuli and in the scala tympani. CONCLUSION: Bisphosphonate introduced via the oval window in the human cochlea can be delivered up to the apical cochlear turn. Interscalar communication is likely to play an important role in determining patterns of drug delivery in the inner ear.

Microfabricated reciprocating micropump for intracochlear drug delivery with integrated drug/fluid storage and electronically controlled dosing.

The anatomical and pharmacological inaccessibility of the inner ear is a major challenge in drug-based treatment of auditory disorders. This also makes pharmacokinetic characterization of new drugs with systemic delivery challenging, because efficacy is coupled with how efficiently a drug can reach its target. Direct delivery of drugs to cochlear fluids bypasses pharmacokinetic barriers and helps to minimize systemic toxicity, but anatomical barriers make administration of multiple doses difficult without an automated delivery system. Such a system may be required for hair-cell regeneration treatments, which will likely require timed delivery of several drugs. To address these challenges, we have developed a micropump for controlled, automated inner-ear drug delivery with the ultimate goal of producing a long-term implantable/wearable delivery system. The current pump is designed to be used with a head mount for guinea pigs in preclinical drug characterization experiments. In this system, we have addressed several microfluidic challenges, including maintaining controlled delivery at safe, low flow rates and delivering drug without increasing the volume of fluid in the cochlea. By integrating a drug reservoir and all fluidic components into the microfluidic structure of the pump, we have made the drug delivery system robust compared to previous systems that utilized separate, tubing-connected components. In this study, we characterized the pump's unique infuse-withdraw and on-demand dosing capabilities on the bench and in guinea pig animal models. For the animal experiments, we used DNQX, a glutamate receptor antagonist, as a physiological indicator of drug delivery. DNQX suppresses compound action potentials (CAPs), so we were able to infer the distribution and spreading of the DNQX over time by measuring the changes in CAPs in response to stimuli at several characteristic frequencies.

Non-Ototoxic Local Delivery of Bisphosphonate to the Mammalian Cochlea.

HYPOTHESIS: Local delivery of bisphosphonates results in superior localization of these compounds for the treatment of cochlear otosclerosis, without ototoxicity. BACKGROUND: Otosclerosis is a common disorder of abnormal bone remodeling within the human otic capsule. It is a frequent cause of conductive hearing loss from stapes fixation. Large lesions that penetrate the cochlear endosteum and injure the spiral ligament result in sensorineural hearing loss. Nitrogen-containing bisphosphonates (e.g., zoledronate) are potent inhibitors of bone remodeling with proven efficacy in the treatment of metabolic bone diseases, including otosclerosis. Local delivery to the cochlea may allow for improved drug targeting, higher local concentrations, and the avoidance of systemic complications. In this study, we use a fluorescently labeled bisphosphonate compound (6-FAM-ZOL) to determine drug localization and concentration within the otic capsule. Various methods for delivery are compared. Ototoxicity is evaluated by auditory brainstem responses and distortion product otoacoustic emissions. METHODS: 6-FAM-ZOL was administered to guinea pigs via intraperitoneal injection, placement of alginate beads onto the round window membrane, or microfluidic pump infusion via a cochleostomy. Hearing was evaluated. Specimens were embedded into resin blocks, ground to a mid-modiolar section, and quantitatively imaged using fluorescence microscopy. RESULTS: There was a dose-dependent increase in fluorescent signal after systemic 6-FAM-ZOL treatment. Local delivery via the round window membrane or a cochleostomy increased delivery efficiency. No significant ototoxicity was observed after either systemic or local 6-FAM-ZOL delivery. CONCLUSION: These findings establish important preclinical parameters for the treatment of cochlear otosclerosis in humans.

Microfabricated infuse-withdraw micropump component for an integrated inner-ear drug-delivery platform.

One of the major challenges in treatment of auditory disorders is that many therapeutic compounds are toxic when delivered systemically. Local intracochlear delivery methods are becoming critical in emerging treatments and in drug discovery. Direct infusion via cochleostomy, in particular, is attractive from a pharmacokinetics standpoint, as there is potential for the kinetics of delivery to be well-controlled. Direct infusion is compatible with a large number of drug types, including large, complex molecules such as proteins and unstable molecules such as siRNA. In addition, hair-cell regeneration therapy will likely require long-term delivery of a timed series of agents. This presents unknown risks associated with increasing the volume of fluid within the cochlea and mechanical damage caused during delivery. There are three key requirements for an intracochlear drug delivery system: (1) a high degree of miniaturization (2) a method for pumping precise and small volumes of fluid into the cochlea in a highly controlled manner, and (3) a method for removing excess fluid from the limited cochlear fluid space. To that end, our group is developing a head-mounted microfluidics-based system for long-term intracochlear drug delivery. We utilize guinea pig animal models for development and demonstration of the device. Central to the system is an infuse-withdraw micropump component that, unlike previous micropump-based systems, has fully integrated drug and fluid storage compartments. Here we characterize the infuse-withdraw capabilities of our micropump, and show experimental results that demonstrate direct drug infusion via cochleostomy in animal models. We utilized DNQX, a glutamate receptor antagonist that suppresses CAPs, as a test drug. We monitored the frequency-dependent changes in auditory nerve CAPs during drug infusion, and observed CAP suppression consistent with the expected drug transport path based on the geometry and tonotopic organization of the cochlea.

Quantitative analysis of ribbons, vesicles, and cisterns at the cat inner hair cell synapse: correlations with spontaneous rate.

Cochlear hair cells form ribbon synapses with terminals of the cochlear nerve. To test the hypothesis that one function of the ribbon is to create synaptic vesicles from the cisternal structures that are abundant at the base of hair cells, we analyzed the distribution of vesicles and cisterns around ribbons from serial sections of inner hair cells in the cat, and compared data from low and high spontaneous rate (SR) synapses. Consistent with the hypothesis, we identified a "sphere of influence" of 350 nm around the ribbon, with fewer cisterns and many more synaptic vesicles. Although high- and low-SR ribbons tended to be longer and thinner than high-SR ribbons, the total volume of the two ribbon types was similar. There were almost as many vesicles docked at the active zone as attached to the ribbon. The major SR-related difference was that low-SR ribbons had more synaptic vesicles intimately associated with them. Our data suggest a trend in which low-SR synapses had more vesicles attached to the ribbon (51.3 vs. 42.8), more docked between the ribbon and the membrane (12 vs. 8.2), more docked at the active zone (56.9 vs. 44.2), and more vesicles within the "sphere of influence" (218 vs. 166). These data suggest that the structural differences between high- and low-SR synapses may be more a consequence, than a determinant, of the physiological differences.

Cochlear kainate receptors.

Synaptic transmission between the cochlear hair cell and its afferent fiber is mediated by glutamate receptors. While kainate receptors are known to be present in the spiral ganglion, little is known of their distribution or functional role. We have detected all five kainate receptor subunits in the mouse cochlea with quantitative RT-PCR and with immunohistochemistry. We observed kainate receptors on afferent terminals co-localized with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA: ) receptors at the afferent synapse. Individual terminals innervating a single hair cell varied in their ratios of AMPA: to kainate receptor immunoreactivity. Infusion of the mouse cochlea via the scala tympani with UBP296, a recently developed antagonist with high specificity for the GluK1 kainate receptor (compared to the AMPA: receptor), reduced the compound action potential and elevated auditory neural thresholds without affecting the distortion product otoacoustic emission thresholds. Thus, the pharmacological evidence suggests that kainate receptors may contribute to the response to transmitter released from the hair cell during acoustic stimulation. It is plausible that afferent transmission at this synapse is mediated by a mix of AMPA: and kainate receptors.

Kinetics of reciprocating drug delivery to the inner ear.

Reciprocating drug delivery is a means of delivering soluble drugs directly to closed fluid spaces in the body via a single cannula without an accompanying fluid volume change. It is ideally suited for drug delivery into small, sensitive and unique fluid spaces such as the cochlea. We characterized the pharmacokinetics of reciprocating drug delivery to the scala tympani within the cochlea by measuring the effects of changes in flow parameters on the distribution of drug throughout the length of the cochlea. Distribution was assessed by monitoring the effects of DNQX, a reversible glutamate receptor blocker, delivered directly to the inner ear of guinea pigs using reciprocating flow profiles. We then modeled the effects of those parameters on distribution using both an iterative curve-fitting approach and a computational fluid dynamic model. Our findings are consistent with the hypothesis that reciprocating delivery distributes the drug into a volume in the base of the cochlea, and suggest that the primary determinant of distribution throughout more distal regions of the cochlea is diffusion. Increases in flow rate distributed the drug into a larger volume that extended more apically. Over short time courses (less than 2h), the apical extension, though small, significantly enhanced apically directed delivery of drug. Over longer time courses (>5h) or greater distances (>3mm), maintenance of drug concentration in the basal scala tympani may prove more advantageous for extending apical delivery than increases in flow rate. These observations demonstrate that this reciprocating technology is capable of providing controlled delivery kinetics to the closed fluid space in the cochlea, and may be suitable for other applications such as localized brain and retinal delivery.

A corticosteroid-responsive transcription factor, promyelocytic leukemia zinc finger protein, mediates protection of the cochlea from acoustic trauma.

Animals can be induced to resist cochlear damage associated with acoustic trauma by exposure to a variety of "conditioning" stimuli, including restraint stress, moderate level sound, heat stress, hypoxia, and corticosteroids. Here we identify in mice a corticosteroid-responsive transcription factor, PLZF (promyelocytic leukemia zinc finger protein), which mediates conditioned protection of the cochlea from acoustic trauma. PLZF mRNA levels in the cochlea are increased following conditioning stimuli, including restraint stress, dexamethasone administration, and moderate-to-high level acoustic stimulation. Heterozygous mutant (luxoid.Zbtb16(LU)/J) mice deficient in PLZF have hearing and responses to acoustic trauma similar to their wild type littermates but are unable to generate conditioning-induced protection from acoustic trauma. PLZF immunoreactivity is present in the spiral ganglion, lateral wall of the cochlea, and organ of Corti, all targets for acoustic trauma. PLZF is also present in the brain and PLZF mRNA in brain is elevated following conditioning stimuli. The identification of a transcription factor that mediates conditioned protection from trauma provides a tool for understanding the protective action of corticosteroids, which are widely used in treating acute hearing loss, and has relevance to understanding the role of corticosteroids in trauma protection.

Functional roles of high-affinity glutamate transporters in cochlear afferent synaptic transmission in the mouse.

In the cochlea, afferent transmission between inner hair cells and auditory neurons is mediated by glutamate receptors. Glutamate transporters located near the synapse and in spiral ganglion neurons are thought to maintain low synaptic levels of glutamate. We analyzed three glutamate transporter blockers for their ability to alter the effects of glutamate, exogenously applied to the synapse via perfusion of the scala tympani of the mouse, and compared that action to their ability to alter the effects of intense acoustic stimulation. Threo-beta-benzyloxyaspartate (TBOA) is a broad-spectrum glutamate transporter antagonist, affecting all three transporters [glutamate/aspartate transporter (GLAST), glutamate transporter-1 (GLT1), and excitatory amino acid carrier 1 (EAAC1)]. l-serine-O-sulfate (SOS) blocks both GLAST and EAAC1 without effect on GLT1. Dihydrokainate (DHK) is selective for GLT1. Infusion of glutamate (10 microM for 220 min), TBOA (200 microM for 220 min), or SOS (100 microM for 180 min) alone did not alter auditory neural thresholds. When infused together with glutamate, TBOA and SOS produced significant neural threshold shifts, leaving otoacoustic emissions intact. In addition, both TBOA and SOS exacerbated noise-induced hearing loss by producing larger neural threshold shifts and delaying recovery. DHK did not alter glutamate- or noise-induced hearing loss. The evidence points to a major role for GLAST, both in protecting the synapse from exposure to excess extracellular glutamate and in attenuating hearing loss due to acoustic overstimulation.

Drug delivery for treatment of inner ear disease: current state of knowledge.

Delivery of medications to the inner ear has been an area of considerable growth in both the research and clinical realms during the past several decades. Systemic delivery of medication destined for treatment of the inner ear is the foundation on which newer delivery techniques have been developed. Because of systemic side effects, investigators and clinicians have begun developing and using techniques to deliver therapeutic agents locally. Alongside the now commonplace use of intratympanic gentamicin for Meniere's disease and the emerging use of intratympanic steroids for sudden sensorineural hearing loss, novel technologies, such as hydrogels and nanoparticles, are being explored. At the horizon of inner ear drug-delivery techniques, intracochlear devices that leverage recent advances in microsystems technology are being developed to apply medications directly into the inner ear. Potential uses for such devices include neurotrophic factor and steroid delivery with cochlear implantation, RNA interference technologies, and stem-cell therapy. The historical, current, and future delivery techniques and uses of drug delivery for treatment of inner ear disease serve as the basis for this review.

Development of a microfluidics-based intracochlear drug delivery device.

BACKGROUND: Direct delivery of drugs and other agents into the inner ear will be important for many emerging therapies, including the treatment of degenerative disorders and guiding regeneration. METHODS: We have taken a microfluidics/MEMS (MicroElectroMechanical Systems) technology approach to develop a fully implantable reciprocating inner-ear drug-delivery system capable of timed and sequenced delivery of agents directly into perilymph of the cochlea. Iterations of the device were tested in guinea pigs to determine the flow characteristics required for safe and effective delivery. For these tests, we used the glutamate receptor blocker DNQX, which alters auditory nerve responses but not cochlear distortion product otoacoustic emissions. RESULTS: We have demonstrated safe and effective delivery of agents into the scala tympani. Equilibration of the drug in the basal turn occurs rapidly (within tens of minutes) and is dependent on reciprocating flow parameters. CONCLUSION: We have described a prototype system for the direct delivery of drugs to the inner ear that has the potential to be a fully implantable means for safe and effective treatment of hearing loss and other diseases.

Regulated expression of surface AMPA receptors reduces excitotoxicity in auditory neurons.

Dynamic regulation of the expression of surface AMPA receptors (AMPARs) is a key mechanism to modulate synaptic strength and efficacy in the CNS and also to regulate auditory sensitivity. Here we address the role of surface AMPAR expression in excitotoxicity by blocking clathrin-mediated AMPAR endocytosis in auditory neurons. We used a membrane-permeable, dynamin-derived, myristoylated peptide (myr-Dyn) to inhibit surface AMPAR endocytosis induced by glutamate receptor agonists in culture and by noise exposure in vivo. Myr-Dyn infused into the mouse cochlea induced excitotoxic responses to acoustic stimuli that were normally not excitotoxic. These included vacuolization in the nerve terminals and spiral ganglion as well as irreversible auditory brain stem response threshold shifts. In cultured spiral ganglion neuronal cells, blockade of the reduction of surface AMPARs exacerbated neuronal death by incubation with N-methyl-d-aspartate and AMPA. This excitotoxic neuronal death could be prevented by calpeptin, a calpain-specific inhibitor. These results suggest that the reduction of surface AMPAR by endocytosis during excitatory stimulation plays an important role in limiting the excitotoxic damage to the neuron.

Proteomics analysis of perilymph and cerebrospinal fluid in mouse.

OBJECTIVES: Proteins in perilymph may alter the delivery profile of implantable intracochlear drug delivery systems through biofouling. Knowledge of protein composition will help anticipate interactions with delivered agents. STUDY DESIGN: Analysis of mouse perilymph. METHODS: Protein composition of perilymph and cerebrospinal fluid (CSF) was analyzed using a capillary liquid chromatography-mass spectrometry-based iTRAQ quantitative proteomics approach. We searched against a mouse subset of the Uniprot FASTA protein database. We sampled perilymph from the apex of the mouse cochlea to minimize CSF contamination. RESULTS: More than 50 explicit protein isoforms were identified with very high confidence. iTRAQ reporter ions allowed determination of relative molar amounts of proteins between perilymph and CSF. Protein in perilymph was almost three times more concentrated than in CSF. More than one-third of the proteins in perilymph comprised protease inhibitors, with serpins being the predominant group. Apolipoproteins constituted 16%. Fifteen percent of the proteins were enzymes. Albumin was the most abundant single protein (14%). Proteins with relatively high perilymph/CSF ratios included broad-spectrum protease inhibitors and apolipoproteins. DISCUSSION: Some proteins found in perilymph, such as albumin and HMW kininogen, have been implicated in biofouling through adsorption to device materials. The relatively large quantities of apolipoprotein and albumin may serve as a reservoir for acidic and lipophilic drugs. Alpha-2-glycoprotein can bind basic drugs. CONCLUSIONS: Perilymph is similar in protein composition to CSF, though amounts are 2.8 times higher. Protease inhibitors comprise the largest category of proteins.

Mastoid cavity dimensions and shape: method of measurement and virtual fitting of implantable devices.

Temporal bone implants can be used to electrically stimulate the auditory nerve, to amplify sound, to deliver drugs to the inner ear and potentially for other future applications. The implants require storage space and access to the middle or inner ears. The most acceptable space is the cavity created by a canal wall up mastoidectomy. Detailed knowledge of the available space for implantation and pathways to access the middle and inner ears is necessary for the design of implants and successful implantation. Based on temporal bone CT scans a method for three-dimensional reconstruction of a virtual canal wall up mastoidectomy space is described. Using Amira software the area to be removed during such surgery is marked on axial CT slices, and a three-dimensional model of that space is created. The average volume of 31 reconstructed models is 12.6 cm(3) with standard deviation of 3.69 cm(3), ranging from 7.97 to 23.25 cm(3). Critical distances were measured directly from the model and their averages were calculated: height 3.69 cm, depth 2.43 cm, length above the external auditory canal (EAC) 4.45 cm and length posterior to EAC 3.16 cm. These linear measurements did not correlate well with volume measurements. The shape of the models was variable to a significant extent making the prediction of successful implantation for a given design based on linear and volumetric measurement unreliable. Hence, to assure successful implantation, preoperative assessment should include a virtual fitting of an implant into the intended storage space. The above-mentioned three-dimensional models were exported from Amira to a Solidworks application where virtual fitting was performed. Our results are compared to other temporal bone implant virtual fitting studies. Virtual fitting has been suggested for other human applications.

Fabrication Methods and Performance of Low-Permeability Microfluidic Components for a Miniaturized Wearable Drug Delivery System.

In this paper, we describe low-permeability components of a microfluidic drug delivery system fabricated with versatile micromilling and lamination techniques. The fabrication process uses laminate sheets which are machined using XY milling tables commonly used in the printed-circuit industry. This adaptable platform for polymer microfluidics readily accommodates integration with silicon-based sensors, printed-circuit, and surface-mount technologies. We have used these methods to build components used in a wearable liquid-drug delivery system for in vivo studies. The design, fabrication, and performance of membrane-based fluidic capacitors and manual screw valves provide detailed examples of the capability and limitations of the fabrication method. We demonstrate fluidic capacitances ranging from 0.015 to 0.15 µL/kPa, screw valves with on/off flow ratios greater than 38 000, and a 45× reduction in the aqueous fluid loss rate to the ambient due to permeation through a silicone diaphragm layer.

Inner ear drug delivery for auditory applications.

Many inner ear disorders cannot be adequately treated by systemic drug delivery. A blood-cochlear barrier exists, similar physiologically to the blood-brain barrier, which limits the concentration and size of molecules able to leave the circulation and gain access to the cells of the inner ear. However, research in novel therapeutics and delivery systems has led to significant progress in the development of local methods of drug delivery to the inner ear. Intratympanic approaches, which deliver therapeutics to the middle ear, rely on permeation through tissue for access to the structures of the inner ear, whereas intracochlear methods are able to directly insert drugs into the inner ear. Innovative drug delivery systems to treat various inner ear ailments such as ototoxicity, sudden sensorineural hearing loss, autoimmune inner ear disease, and for preserving neurons and regenerating sensory cells are being explored.