Alan Horsager

Virally delivered Channelrhodopsin-2 Safely and Effectively Restores Visual Function in Multiple Mouse Models of Blindness

Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Factors Affecting Perceptual Thresholds in Epiretinal Prostheses

PURPOSE The goal was to evaluate how perceptual thresholds are related to electrode impedance, electrode size, the distance of electrodes from the retinal surface, and retinal thickness in six subjects blind as a result of retinitis pigmentosa, who received epiretinal prostheses implanted monocularly as part of a U.S. Food and Drug Administration (FDA)-approved clinical trial. METHODS The implant consisted of an extraocular unit containing electronics for wireless data, power recovery, and generation of stimulus current, and an intraocular unit containing 16 platinum stimulating electrodes (260- or 520-microm diameter) arranged in a 4 x 4 pattern. The electrode array was held onto the retina by a small tack. Stimulation was controlled by a computer-based external system that allowed independent control over each electrode. Perceptual thresholds (the current necessary to see a percept on 79% of trials) and impedance were measured for each electrode on a biweekly basis. The distance of electrodes from the retinal surface and retinal thickness were measured by optical coherence tomography on a less regular basis. RESULTS Stimulation thresholds for detecting phosphenes correlated with the distance of the electrodes from the retinal surface, but not with electrode size, electrode impedance, or retinal thickness. CONCLUSIONS Maintaining close proximity between the electrode array and the retinal surface is critical in developing a successful retinal implant. With the development of chronic electrode arrays that are stable and flush on the retinal surface, it is likely that the influence of other factors such as electrode size, retinal degeneration, and subject age will become more apparent. (ClinicalTrials.gov number, NCT00279500.).

Brightness as a function of current amplitude in human retinal electrical stimulation.

PURPOSE With the goal of eventually restoring functional vision in patients with retinal degenerative diseases, USC/Second Sight Medical Products, Inc. chronically implanted blind human subjects with a prototype epiretinal prosthesis consisting of a 4 x 4 array of 16 stimulating electrodes. To accurately represent a visual scene, a visual prosthesis must convey luminance information across a range of brightness levels. To achieve this, the brightness of phosphenes produced by an individual electrode should scale appropriately with luminance, and the same luminance should produce equivalently bright phosphenes across the entire electrode array. The goal was to examine how apparent brightness changes as a function of stimulation intensity across electrodes. METHODS As described in previous studies, electrical stimulation of intact cells of the neural retina using this prosthetic device reliably elicits visual percepts in human subjects blinded by retinitis pigmentosa. Here, apparent brightness for a range of electrical amplitudes was measured using both subjective magnitude rating and brightness-matching procedures in chronically implanted human subjects. RESULTS It was found that apparent brightness can be described as a power function of stimulation intensity. The same model can also predict brightness matching across electrodes. CONCLUSIONS These results suggest that a relatively simple model for scaling current across electrodes may be capable of producing equivalently bright phosphenes across an entire array. (ClinicalTrials.gov number, NCT00279500.).

Predicting Visual Sensitivity in Retinal Prosthesis Patients

PURPOSE With the long-term goal of restoring functional vision in patients with retinal degenerative diseases, the eyes of blind human subjects were implanted chronically with epiretinal prostheses consisting of two-dimensional electrode arrays that directly stimulated cells of the neural retina. METHODS Psychophysical techniques were used to measure the brightness of electrically generated percepts on single electrodes using a variety of electrical stimulation patterns. RESULTS It was possible to predict the sensitivity of the human visual system to a wide variety of retinal electrical stimulation patterns using a simple and biologically plausible model. CONCLUSIONS This is the first study to demonstrate that, on the single-electrode level, retinal electrical stimulation in humans can produce visual qualia that are predictable using a quantitative model, a prerequisite for a successful retinal prosthesis. (ClinicalTrials.gov number, NCT00279500.).

Spatiotemporal Interactions in Retinal Prosthesis Subjects

PURPOSE Vision loss due to retinitis pigmentosa affects an estimated 15 million people worldwide. Through collaboration between Second Sight Medical Products, Inc., and the Doheny Eye Institute, six blind human subjects underwent implantation with epiretinal 4 x 4 electrode arrays designed to directly stimulate the remaining cells of the retina, with the goal of restoring functional vision by applying spatiotemporal patterns of stimulation. To better understand spatiotemporal interactions between electrodes during synchronous and asynchronous stimulation, the authors investigated how percepts changed as a function of pulse timing across the electrodes. METHODS Pulse trains (20, 40, 80, and 160 Hz) were presented on groups of electrodes with 800, 1600, or 2400 microm center-to-center separation. Stimulation was either synchronous (pulses were presented simultaneously across electrodes) or asynchronous (pulses were phase shifted). Using a same-different discrimination task, the authors were able to evaluate how the perceptual quality of the stimuli changed as a function of phase shifts across multiple electrodes. RESULTS Even after controlling for electric field interactions, subjects could discriminate between spatiotemporal pulse train patterns based on differences of phase across electrodes as small as 3 ms. These findings suggest that the quality of the percept is affected not only by electric field interactions but also by spatiotemporal interactions at the neural level. CONCLUSIONS During multielectrode stimulation, interactions between electrodes have a significant influence on the quality of the percept. Understanding how these spatiotemporal interactions at the neural level influence percepts during multielectrode stimulation is fundamental to the successful design of a retinal prosthesis.

Frequency and amplitude modulation have different effects on the percepts elicited by retinal stimulation.

PURPOSE In an effort to restore functional form vision, epiretinal prostheses that elicit percepts by directly stimulating remaining retinal circuitry were implanted in human subjects with advanced retinitis pigmentosa RP). In this study, manipulating pulse train frequency and amplitude had different effects on the size and brightness of phosphene appearance. METHODS Experiments were performed on a single subject with severe RP (implanted with a 16-channel epiretinal prosthesis in 2004) on nine individual electrodes. Psychophysical techniques were used to measure both the brightness and size of phosphenes when the biphasic pulse train was varied by either modulating the current amplitude (with constant frequency) or the stimulating frequency (with constant current amplitude). RESULTS Increasing stimulation frequency always increased brightness, while having a smaller effect on the size of elicited phosphenes. In contrast, increasing stimulation amplitude generally increased both the size and brightness of phosphenes. These experimental findings can be explained by using a simple computational model based on previous psychophysical work and the expected spatial spread of current from a disc electrode. CONCLUSIONS Given that amplitude and frequency have separable effects on percept size, these findings suggest that frequency modulation improves the encoding of a wide range of brightness levels without a loss of spatial resolution. Future retinal prosthesis designs could benefit from having the flexibility to manipulate pulse train amplitude and frequency independently (clinicaltrials.gov number, NCT00279500).

Aberrant sperm DNA methylation predicts male fertility status and embryo quality

OBJECTIVE To evaluate whether male fertility status and/or embryo quality during in vitro fertilization (IVF) therapy can be predicted based on genomewide sperm deoxyribonucleic acid (DNA) methylation patterns. DESIGN Retrospective cohort study. SETTING University-based fertility center. PATIENT(S) Participants were 127 men undergoing IVF treatment (where any major female factor cause of infertility had been ruled out), and 54 normozoospermic, fertile men. The IVF patients were stratified into 2 groups: patients who had generally good embryogenesis and a positive pregnancy (n = 55), and patients with generally poor embryogenesis (n = 72; 42 positive and 30 negative pregnancies) after IVF. INTERVENTION(S) Genomewide sperm DNA methylation analysis was performed to measure methylation at >485,000 sites across the genome. MAIN OUTCOME MEASURE(S) A comparison was made of DNA methylation patterns of IVF patients vs. normozoospermic, fertile men. RESULT(S) Predictive models proved to be highly accurate in classifying male fertility status (fertile or infertile), with 82% sensitivity, and 99% positive predictive value. Hierarchic clustering identified clusters enriched for IVF patient samples and for poor-quality-embryo samples. Models built to identify samples within these groups, from neat samples, achieved positive predictive value ≥ 94% while identifying >one fifth of all IVF patient and poor-quality-embryo samples in each case. Using density gradient prepared samples, the same approach recovered 46% of poor-quality-embryo samples with no false positives. CONCLUSION(S) Sperm DNA methylation patterns differ significantly and consistently for infertile vs. fertile, normozoospermic men. In addition, DNA methylation patterns may be predictive of embryo quality during IVF.

Drug and gene delivery to the back of the eye: from bench to bedside.

The ARVO 2012 Summer Eye Research Conference (SERC 2012) on “Drug and Gene Delivery to the Back of the Eye: From Bench to Bedside” was held June 15 and 16, 2012, at the University of Colorado Anschutz Medical Campus in Aurora, Colorado. The SERC provided a diverse group of approximately 150 scientists and physicians representing industry and academia from 14 countries with a unique opportunity to explore the latest approaches to drug and gene delivery to the posterior segment of the eye. Unlike the 2009 SERC meeting, which focused on novel drug delivery platforms while elucidating the anatomic barriers to reach the posterior segment,1 the most recent meeting explored strategies for bypassing ocular barriers using novel materials, nanoparticulate delivery systems, and gene therapy. It brought together experts in both ophthalmology and tangentially related areas to discuss the application and inherent technical challenges for translating experimental results from the laboratory bench to dependable medical therapies at the bedside and, where possible, it exemplified findings in ocular models with methods and results gleaned from disciplines outside of ophthalmology. The present review of the SERC provides investigators with tools to navigate these nascent approaches by exploring strategies from key laboratory investigations, drug development specialists, and clinical trials. The 2-day conference comprised the following six sessions: (1) barriers to drug delivery and transporter-guided drug design; (2) drug/gene delivery systems and cell therapies for the eye; (3) pharmacokinetics (PK), pharmacodynamics, and alternative routes of drug delivery; (4) nanotechnology for diagnosis and treatment of posterior eye disease; (5) translation of gene delivery for posterior eye disease; and (6) clinical trials. Rather than being a deliberate summary of each presentation, this review describes the common themes expressed during the six sessions.

Temporal Interactions during Paired-Electrode Stimulation in Two Retinal Prosthesis Subjects

PURPOSE Since 2002, six blind patients have undergone implantation of an epiretinal 4 × 4 electrode array designed to directly stimulate the remaining cells of the retina after severe photoreceptor degeneration due to retinitis pigmentosa. This study was conducted to investigate how the brightness of percepts is affected by pulse timing across electrodes in two of these patients. METHODS Subjects compared the perceived brightness of a standard stimulus (synchronous pulse trains presented across pairs of electrodes) to the perceived brightness of a test stimulus (pulse trains across the electrode pair phase shifted by 0.075, 0.375, 1.8, or 9 ms). The current amplitude necessary for each phase-shifted test stimulus to match the brightness of the standard was determined. RESULTS Depending on the electrode pair, interactions between electrodes were either facilitatory (the perceived brightness produced by stimulating the pair of electrodes was greater than that produced by stimulating either electrode alone) or suppressive (the perceived brightness produced by stimulating the pair of electrodes was less than that produced by stimulating either electrode alone). The amount of interaction between electrodes decreased as a function of increased separation both in time (the phase-shift between pulse trains) and space (center-to-center distance between the electrode pair). CONCLUSIONS For visual prostheses to represent visual scenes that are changing in both space and time requires the development of spatiotemporal models describing the effects of stimulation across multiple electrodes. During multielectrode stimulation, interactions between electrodes have a significant influence on subjective brightness that includes both facilitatory and suppressive effects, and these interactions can be described with a simple computational model. (ClinicalTrials.gov number, NCT00279500.).

The Perceptual Effects of Chronic Retinal Stimulation

Can functional vision be restored in blind human subjects using a microelectronic retinal prosthesis? The initial indications suggest that, yes, it is possible. However, the visual experience of these subjects is nothing like a digital scoreboard-like movie, with each electrode acting as an independent pixel. The work described here in this chapter suggests that there are interactions between pulses and across electrodes, at the electrical, retinal, or even cortical level that influence the quality of the percept. In particular, this work addresses the question, “how does the percept change as a function of pulse timing on single and multiple electrodes”? The motivation for the work described here is that these interactions must be understood and predictable if we are to develop a functional tool for blind human patients. In this chapter, we review work evaluating perceptual effects using chronic electric stimulation in three different implantable systems.