Gene Electrotransfer via Conductivity-Clamped Electric Field Focusing Pivots Sensori-Motor DNA Therapeutics: "A Spoonful of Sugar Helps the Medicine Go Down".

Viral vectors and lipofection-based gene therapies have dispersion-dependent transduction/transfection profiles that thwart precise targeting. The study describes the development of focused close-field gene electrotransfer (GET) technology, refining spatial control of gene expression. Integration of fluidics for precise delivery of "naked" plasmid deoxyribonucleic acid (DNA) in sucrose carrier within the focused electric field enables negative biasing of near-field conductivity ("conductivity-clamping"-CC), increasing the efficiency of plasma membrane molecular translocation. This enables titratable gene delivery with unprecedently low charge transfer. The clinic-ready bionics-derived CC-GET device achieved neurotrophin-encoding miniplasmid DNA delivery to the cochlea to promote auditory nerve regeneration; validated in deafened guinea pig and cat models, leading to improved central auditory tuning with bionics-based hearing. The performance of CC-GET is evaluated in the brain, an organ problematic for pulsed electric field-based plasmid DNA delivery, due to high required currents causing Joule-heating and damaging electroporation. Here CC-GET enables safe precision targeting of gene expression. In the guinea pig, reporter expression is enabled in physiologically critical brainstem regions, and in the striatum (globus pallidus region) delivery of a red-shifted channelrhodopsin and a genetically-encoded Ca2+ sensor, achieved photoactivated neuromodulation relevant to the treatment of Parkinson's Disease and other focal brain disorders.

Therapeutics for hearing preservation and improvement of patient outcomes in cochlear implantation-Progress and possibilities.

The emergence of therapeutics targeted at hearing loss holds great promise in the development of novel treatments for this heterogenous condition. Whilst such therapeutics are largely designed to be efficacious in and of themselves, the possibility of combination with devices, namely cochlear implants, could result in much more effective treatment options. Here, we review the otoprotective molecules currently in clinical development, as well as generic steroids, discussing mechanisms of action and mode of delivery to the perilymph of the cochlea. Presenting both preclinical and clinical data, we explore the challenges these molecules face in reaching the inner ear. Furthermore, we consider the role of the cochlear implant as a drug delivery platform along with the ability of these drugs to preserve residual hearing and improve outcomes in implant recipients.

Platinum dissolution and tissue response following long-term electrical stimulation at high charge densities.

Objective. Established guidelines for safe levels of electrical stimulation for neural prostheses are based on a limited range of the stimulus parameters used clinically. Recent studies have reported particulate platinum (Pt) associated with long-term clinical use of these devices, highlighting the need for more carefully defined safety limits. We previously reported no adverse effects of Pt corrosion products in the cochleae of guinea pigs following 4 weeks of electrical stimulation using charge densities far greater than the published safe limits for cochlear implants. The present study examines the histopathological effects of Pt within the cochlea following continuous stimulation at a charge density well above the defined safe limits for periods up to 6 months.Approach. Six cats were bilaterally implanted with Pt electrode arrays and unilaterally stimulated using charge balanced current pulses at a charge density of 267µC cm-2phase-1using a tripolar electrode configuration. Electrochemical measurements were made throughout the implant duration and evoked potentials recorded at the outset and on completion of the stimulation program. Cochleae were examined histologically for particulate Pt, tissue response, and auditory nerve survival; electrodes were examined for surface corrosion; and cochlea, brain, kidney, and liver tissue analysed for trace levels of Pt.Main results. Chronic stimulation resulted in both a significant increase in tissue response and particulate Pt within the tissue capsule surrounding the electrode array compared with implanted, unstimulated control cochleae. Importantly, there was no stimulus-induced loss of auditory neurons (ANs) or increase in evoked potential thresholds. Stimulated electrodes were significantly more corroded compared with unstimulated electrodes. Trace analysis revealed Pt in both stimulated and control cochleae although significantly greater levels were detected within stimulated cochleae. There was no evidence of Pt in brain or liver; however, trace levels of Pt were recorded in the kidneys of two animals. Finally, increased charge storage capacity and charge injection limit reflected the more extensive electrode corrosion associated with stimulated electrodes.Significance. Long-term electrical stimulation of Pt electrodes at a charge density well above existing safety limits and nearly an order of magnitude higher than levels used clinically, does not adversely affect the AN population or reduce neural function, despite a stimulus-induced tissue response and the accumulation of Pt corrosion product. The mechanism resulting in Pt within the unstimulated cochlea is unclear, while the level of Pt observed systemically following stimulation at these very high charge densities does not appear to be of clinical significance.

Subthreshold Electrical Stimulation for Controlling Protein-Mediated Impedance Increases in Platinum Cochlear Electrode.

OBJECTIVE: This study evaluated subthreshold biphasic stimulation pulses as a strategy to stabilize electrode impedance via control of protein adsorption. Following implantation, cochlear electrodes undergo impedance fluctuations thought to be caused by protein adsorption and/or inflammatory responses. Impedance increases can impact device power consumption, safe charge injection limits, and long-term stability of electrodes. METHODS: Protein-mediated changes in polarization impedance (Zp) were measured by voltage transient responses to biphasic current pulses and electrochemical impedance spectroscopy, with and without protein solutions. Four subthreshold stimulation regimes were studied to assess their effects on protein adsorption and impedance; (1) symmetric charge-balanced pulses delivered continuously, (2) at 10% duty cycle, (3) at 1% duty cycle, and (4) an asymmetric charge balanced pulse delivered continuously with a cathodic phase twice as long as the anodic phase. RESULTS: The Zp of electrodes incubated in protein solutions without stimulation for 2 h increased by between ∼28% and ∼55%. Subthreshold stimulation reduced the rate at which impedance increased following exposure to all protein solutions. Decreases in Zp were dependent on the type of protein solution and the stimulation regime. Subthreshold stimulation pulses were more effective when delivered continuously compared to 1% and 10% duty cycles. CONCLUSION: These results support the potential of subthreshold stimulation pulses to mitigate protein-mediated increase in impedance. SIGNIFICANCE: This research highlights the potential of clinically translatable stimulation pulses to mitigate perilymph protein adsorption on cochlear electrodes, a key phenomenon precursor of the inflammatory response.

Chronic intracochlear electrical stimulation at high charge densities: reducing platinum dissolution.

OBJECTIVE: Cochleae of long-term cochlear implant users have shown evidence of particulate platinum (Pt) corroded from the surface of Pt electrodes. The pathophysiological effect of Pt within the cochlea has not been extensively investigated. We previously evaluated the effects of Pt corrosion at high charge densities and reported negligible pathophysiological impact. The present study extends this work by examining techniques that may reduce Pt corrosion. APPROACH: Deafened guinea pigs were continuously stimulated for 28 d using biphasic current pulses at extreme charge densities using: (i) electrode shorting; (ii) electrode shorting with capacitive coupling (CC); or (iii) electrode shorting with alternating leading phase (AP). On completion of stimulation, cochleae were examined for corrosion product, tissue response, auditory nerve (AN) survival and trace levels of Pt; and electrodes examined for surface corrosion. MAIN RESULTS: Pt corrosion was evident at ≥200 µC cm-2 phase-1; the amount dependent on charge density (p< 0.01) and charge recovery technique (p < 0.01); reduced corrosion was apparent using CC. Tissue response increased with charge density (p< 0.007); cochleae stimulated at ≥200 µC cm-2 phase-1 exhibited a vigorous response including a focal region of necrosis and macrophages. Notably, tissue response was not dependent on the charge recovery technique (p = 0.56). Despite stimulation at high charge densities resulting in significant levels of Pt corrosion, there was no stimulus induced loss of ANs. SIGNIFICANCE: Significant increases in tissue response and Pt corrosion were observed following stimulation at high charge densities. Charge recovery using CC, and to a lesser extent AP, reduced the amount of Pt corrosion but not the tissue response. Stimulation at change densities an order of magnitude higher than those used when programming cochlear implant recipients in the clinic, produced a vigorous tissue response and corrosion products without evidence of neural loss.

Protecting against electrode insertion trauma using dexamethasone.

OBJECTIVE: To compare the benefits of a dexamethasone-eluting array for hearing preservation and cochlear histopathology in low trauma (soft-surgery) and high trauma models of cochlear implant surgery. METHODS: Adult guinea pigs were implanted with an intra-cochlear array using two different surgical procedures: either a soft-surgery approach or following generation of electrode insertion trauma (high trauma). Two methods of dexamethasone delivery were evaluated: elution from an electrode array alone, and elution from a cochlear implant electrode array in combination with a pre-operative systemic injection. All electrode arrays were implanted for a period of 4 weeks. Outcome measures at 4 weeks post-implantation included auditory brainstem response (ABR) thresholds, histological analysis of spiral ganglion neuron density, fibrotic tissue, new bone growth, and cochlear damage. RESULTS: Animals exposed to high surgical trauma showed greater hearing loss than those in the low trauma model, irrespective of the presence of dexamethasone. Whilst the area of intra-cochlear fibrotic tissue growth post-implantation was also independent of dexamethasone administration, new bone growth was significantly reduced in its presence. Our high trauma model effectively obliterated the organ of Corti and significantly reduced spiral ganglion neuron densities in the lower basal turn. This trauma-induced reduction in spiral ganglion neuron survival decreased with the inclusion of a dexamethasone-eluting array. A pre-operative systemic injection of dexamethasone did not significantly improve any outcome measures beyond those provided with a dexamethasone-eluting array alone. CONCLUSION: Dexamethasone-eluting intra-cochlear arrays may inhibit osteoneogenesis, and reduce spiral ganglion neuron loss following traumatic cochlear implantation.

Chronic intracochlear electrical stimulation at high charge densities results in platinum dissolution but not neural loss or functional changes in vivo.

OBJECTIVE: Although there are useful guidelines defining the boundary between damaging and non-damaging electrical stimulation they were derived from acute studies using large surface area electrodes in direct contact with cortical neurons. These parameters are a small subset of the parameters used by neural stimulators. More recently, histological examination of cochleae from patients that were long-term cochlear implant users have shown evidence of particulate platinum (Pt). The pathophysiological effect of Pt within the cochlea is unknown. We examined the response of the cochlea to stimulus levels beyond those regarded as safe, and to evaluate the pathophysiological response of the cochlea following chronic stimulation at charge densities designed to induce Pt corrosion in vivo. APPROACH: 19 guinea pigs were systemically deafened and implanted with a cochlear electrode array containing eight Pt electrodes of 0.05, 0.075 or 0.2 mm2 area. Animals were electrically stimulated continuously for 28 d using charge balanced current pulses at charge densities of 400, 267 or 100 µC/cm2/phase. Electrically-evoked auditory brainstem responses (EABRs) were recorded to monitor neural function. On completion of stimulation electrodes were examined using scanning electron microscopy (SEM) and cochleae examined histology. Finally, analysis of Pt was measured using energy dispersive x-ray spectroscopy (EDS) and inductively coupled plasma mass spectrometry (ICP-MS). MAIN RESULTS: Compared with unstimulated control electrodes and electrodes stimulated at 100 µC/cm2/phase, stimulation at 267 or 400 µC/cm2/phase resulted in significant Pt corrosion. Cochleae stimulated at these high charge densities contained particulate Pt. The extent of the foreign body response depended on the level of stimulation; cochleae stimulated at 267 or 400 µC/cm2/phase exhibited an extensive tissue response that included a focal region of necrosis close to the electrode. Despite chronic stimulation at high charge densities there was no loss of auditory neurons (ANs) in stimulated cochleae compared with their contralateral controls. Indeed, we report a statistically significant increase in AN density proximal to electrodes stimulated at 267 or 400 µC/cm2/phase. Finally, there was no evidence of a reduction in AN function associated with chronic stimulation at 100, 267 or 400 µC/cm2/phase as evidenced by stable EABR thresholds over the stimulation program. SIGNIFICANCE: Chronic electrical stimulation of Pt electrodes at 267 or 400 µC/cm2/phase evoked a vigorous tissue response and produced Pt corrosion products that were located close to the electrode. Despite these changes at the electrode/tissue interface there was no evidence of neural loss or a reduction in neural function.

Neurotrophin gene augmentation by electrotransfer to improve cochlear implant hearing outcomes.

This Review outlines the development of DNA-based therapeutics for treatment of hearing loss, and in particular, considers the potential to utilize the properties of recombinant neurotrophins to improve cochlear auditory (spiral ganglion) neuron survival and repair. This potential to reduce spiral ganglion neuron death and indeed re-grow the auditory nerve fibres has been the subject of considerable pre-clinical evaluation over decades with the view of improving the neural interface with cochlear implants. This provides the context for discussion about the development of a novel means of using cochlear implant electrode arrays for gene electrotransfer. Mesenchymal cells which line the cochlear perilymphatic compartment can be selectively transfected with (naked) plasmid DNA using array - based gene electrotransfer, termed 'close-field electroporation'. This technology is able to drive expression of brain derived neurotrophic factor (BDNF) in the deafened guinea pig model, causing re-growth of the spiral ganglion peripheral neurites towards the mesenchymla cells, and hence into close proximity with cochlear implant electrodes within scala tympani. This was associated with functional enhancement of the cochlear implant neural interface (lower neural recruitment thresholds and expanded dynamic range, measured using electrically - evoked auditory brainstem responses). The basis for the efficiency of close-field electroporation arises from the compression of the electric field in proximity to the ganged cochlear implant electrodes. The regions close to the array with highest field strength corresponded closely to the distribution of bioreporter cells (adherent human embryonic kidney (HEK293)) expressing green fluorescent reporter protein (GFP) following gene electrotransfer. The optimization of the gene electrotransfer parameters using this cell-based model correlated closely with in vitro and in vivo cochlear gene delivery outcomes. The migration of the cochlear implant electrode array-based gene electrotransfer platform towards a clinical trial for neurotrophin-based enhancement of cochlear implants is supported by availability of a novel regulatory compliant mini-plasmid DNA backbone (pFAR4; plasmid Free of Antibiotic Resistance v.4) which could be used to package a 'humanized' neurotrophin expression cassette. A reporter cassette packaged into pFAR4 produced prominent GFP expression in the guinea pig basal turn perilymphatic scalae. More broadly, close-field gene electrotransfer may lend itself to a spectrum of potential DNA therapeutics applications benefitting from titratable, localised, delivery of naked DNA, for gene augmentation, targeted gene regulation, or gene substitution strategies.

Comparing perilymph proteomes across species.

OBJECTIVES/HYPOTHESIS: Biological components of perilymph affect the electrical performance of cochlear implants. Understanding the perilymph composition of common animal models will improve the understanding of this impact and improve the interpretation of results from animal studies and how it relates to humans. STUDY DESIGN: Analysis and comparison of the proteomes of human, guinea pig, and cat perilymph. METHODS: Multiple perilymph samples from both guinea pigs and cats were analysed via liquid chromatography with tandem mass spectrometry. Proteins were identified using the Mascot database. Human data were obtained from a published dataset. Proteins identified were refined to form a proteome for each species. RESULTS: Over 200 different proteins were found per species. There were 81, 39, and 64 proteins in the final human, guinea pig, and cat proteomes, respectively. Twenty-one proteins were common to all three species. Fifty-two percent of the cat proteome was found in the human proteome, and 31% of the guinea pig was common to human. The cat proteome had similar complexity to the human proteome in three protein classes, whereas the guinea pig had a similar complexity in two. The presence of albumin was significantly higher in human perilymph than in the other two species. Immunoglobulins were more abundant in the human than in the cat proteome. CONCLUSIONS: Perilymph proteomes were compared across three species. The degree of crossover of proteins of both guinea pig and cat with human indicate that these animals suitable models for the human cochlea, albeit the cat perilymph is a closer match. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E47-E52, 2018.

Evaluation of focused multipolar stimulation for cochlear implants: a preclinical safety study.

OBJECTIVE: Cochlear implants (CIs) have a limited number of independent stimulation channels due to the highly conductive nature of the fluid-filled cochlea. Attempts to develop highly focused stimulation to improve speech perception in CI users includes the use of simultaneous stimulation via multiple current sources. Focused multipolar (FMP) stimulation is an example of this approach and has been shown to reduce interaction between stimulating channels. However, compared with conventional biphasic current pulses generated from a single current source, FMP is a complex stimulus that includes extended periods of stimulation before charge recovery is achieved, raising questions on whether chronic stimulation with this strategy is safe. The present study evaluated the long-term safety of intracochlear stimulation using FMP in a preclinical animal model of profound deafness. APPROACH: Six cats were bilaterally implanted with scala tympani electrode arrays two months after deafening, and received continuous unilateral FMP stimulation at levels that evoked a behavioural response for periods of up to 182 d. Electrode impedance, electrically-evoked compound action potentials (ECAPs) and auditory brainstem responses (EABRs) were monitored periodically over the course of the stimulation program from both the stimulated and contralateral control cochleae. On completion of the stimulation program cochleae were examined histologically and the electrode arrays were evaluated for evidence of platinum (Pt) corrosion. MAIN RESULTS: There was no significant difference in electrode impedance between control and chronically stimulated electrodes following long-term FMP stimulation. Moreover, there was no significant difference between ECAP and EABR thresholds evoked from control or stimulated cochleae at either the onset of stimulation or at completion of the stimulation program. Chronic FMP stimulation had no effect on spiral ganglion neuron (SGN) survival when compared with unstimulated control cochleae. Long-term implantation typically evoked a mild foreign body reaction proximal to the electrode array; however stimulated cochleae exhibited a small but statistically significant increase in the tissue response. Finally, there was no evidence of Pt corrosion following long-term FMP stimulation; stimulated electrodes exhibited the same surface features as the unstimulated control electrodes. SIGNIFICANCE: Chronic intracochlear FMP stimulation at levels used in the present study did not adversely affect electrically-evoked neural thresholds or SGN survival but evoked a small, benign increase in inflammatory response compared to control ears. Moreover chronic FMP stimulation does not affect the surface of Pt electrodes at suprathreshold stimulus levels. These findings support the safe clinical application of an FMP stimulation strategy.

Understanding the cochlear implant environment by mapping perilymph proteomes from different species.

Cochlear implants operate within a bony channel of the cochlea, bathed in a fluid known as the perilymph. The perilymph is a complex fluid containing ions and proteins, which are known to actively interact with metallic electrodes. To improve our understanding of how cochlear implant performance varies in preclinical in vivo studies in comparison to human trials and patient outcomes, the protein composition (or perilymph proteome) is needed. Samples of perilymph were gathered from feline and guinea pig subjects and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) to produce proteomes and compare against the recently published human proteome. Over 64% of the proteins in the guinea pig proteome were found to be common to the human proteome. The proportions of apolipoproteins, enzymes and immunoglobulins showed little variation between the two proteomes, with other classes showing similarity. This establishes a good basis for comparison of results. The results for the feline profile showed less similarity with the human proteome and would not provide a quality comparison. This work highlights the suitability of the guinea pig to model the biological environment of the human cochlear and the need to carefully select models of the biological environment of a cochlear implant to more adequately translate in vitro and in vivo studies to the clinic.

Meningitis and a safe dexamethasone-eluting intracochlear electrode array.

OBJECTIVES: To evaluate the potential risk of pneumococcal meningitis associated with the use of a dexamethasone-eluting intracochlear electrode array as compared with a control array. METHODS: In two phases, adult Hooded-Wistar rats were implanted via the middle ear with an intracochlear array and were inoculated with Streptococcus pneumoniae 5 days post-surgery. Phase I created a dosing curve by implanting five groups (n = 6) with a control array, then inoculating 5 days later with different numbers of S. pneumoniae: 0 CFU, 10(3) CFU, 10(4) CFU, 10(4) CFU repeated, or 10(5) CFU (colony forming units). A target infection rate of 20% was aimed for and 10(4) CFU was the closest to this target with 33% infection rate. In phase II, we implanted two groups (n = 10), one with a dexamethasone-eluting array, the other a control array, and both groups were inoculated with 10(4) CFU of S. pneumoniae 5 days post-surgery. RESULTS: The dexamethasone-eluting array group had a 40% infection rate; the control array group had a 60% infection rate. This difference was not statistically significant with a P value of ≥0.5. CONCLUSION: The use of a dexamethasone-eluting intracochlear electrode array did not increase the risk of meningitis in rats when inoculated with S. pneumoniae via the middle ear 5 days following implantation.

Development of a safe dexamethasone-eluting electrode array for cochlear implantation.

OBJECTIVES: Cochlear implantation can result in trauma leading to increased tissue response and loss of residual hearing. A single intratympanic application of the corticosteroid dexamethasone is sometimes used clinically during surgery to combat the potential effect of trauma on residual hearing. This project looked at the safety and efficacy of dexamethasone eluted from an intracochlear array in vivo. METHODS: Three trials were conducted using normal hearing adult guinea pigs implanted with successive iterations of dexamethasone-eluting (DX1, DX2, and DX3) or non-eluting (control) intracochlear electrode arrays. The experimental period for each animal was 90 days during which hearing tests were performed at multiple time points. RESULTS: There was no significant difference between matched control array and dexamethasone array groups in terms of spiral ganglion neuron density, organ of Corti condition, or fibrosis and ossification. A cochleostomy seal was present in all implanted cochleae. There were no differences in the degree of hearing threshold shifts between DX1 and DX3 and their respective control arrays. Cochleae implanted with DX2 arrays showed less hearing loss and marginally better spiral ganglion neuron survival than their control array counterparts. Post-explant inspection of the DX2 and DX3 arrays revealed a difference in pore density following dexamethasone elution. CONCLUSION: The dexamethasone doses used were safe in the guinea pig cochlea. Dexamethasone did not inhibit formation of a cochleostomy seal. The level of hearing protection afforded by dexamethasone eluting from an intracochlear array may depend upon the degree of elution and level of trauma inflicted.