Scott H. Greenwald

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.).

Measurements of the effect of surface slant on perceived lightness

When a planar object is rotated with respect to a directional light source, the reflected luminance changes. If surface lightness is to be a reliable guide to surface identity, observers must compensate for such changes. To the extent they do, observers are said to be lightness constant. We report data from a lightness matching task that assesses lightness constancy with respect to changes in object slant. On each trial, observers viewed an achromatic standard object and indicated the best match from a palette of 36 grayscale samples. The standard object and the palette were visible simultaneously within an experimental chamber. The chamber illumination was provided from above by a theater stage lamp. The standard objects were uniformly-painted flat cards. Different groups of naive observers made matches under two sets of instructions. In the Neutral Instructions, observers were asked to match the appearance of the standard and palette sample. In the Paint Instructions, observers were asked to choose the palette sample that was painted the same as the standard. Several broad conclusions may be drawn from the results. First, data for most observers were neither luminance matches nor lightness constant matches. Second, there were large and reliable individual differences. To characterize these, a constancy index was obtained for each observer by comparing how well the data were accounted for by both luminance matching and lightness constancy. The index could take on values between 0 (luminance matching) and 1 (lightness constancy). Individual observer indices ranged between 0.17 and 0.63 with mean 0.40 and median 0.40. An auxiliary slant-matching experiment rules out variation in perceived slant as the source of the individual variability. Third, the effect of instructions was small compared to the inter-observer variability. Implications of the data for models of lightness perception are discussed.

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.).

An Equivalent Illuminant Model for the Effect of Surface Slant on Perceived Lightness

In the companion study (C. Ripamonti et al., 2004), we present data that measure the effect of surface slant on perceived lightness. Observers are neither perfectly lightness constant nor luminance matchers, and there is considerable individual variation in performance. This work develops a parametric model that accounts for how each observers lightness matches vary as a function of surface slant. The model is derived from consideration of an inverse optics calculation that could achieve constancy. The inverse optics calculation begins with parameters that describe the illumination geometry. If these parameters match those of the physical scene, the calculation achieves constancy. Deviations in the models parameters from those of the scene predict deviations from constancy. We used numerical search to fit the model to each observers data. The model accounts for the diverse range of results seen in the experimental data in a unified manner, and examination of its parameters allows interpretation of the data that goes beyond what is possible with the raw data alone.

Mouse Models of NMNAT1-Leber Congenital Amaurosis (LCA9) Recapitulate Key Features of the Human Disease

The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.

S-opsin knockout mice with the endogenous M-opsin gene replaced by an L-opsin variant

Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease-causing mutations by replacing exon 2 through 6 of the mouse M-opsin gene with the corresponding cDNA for a human L-opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without coexpression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described.

Cone-isolating ON–OFF electroretinogram for studying chromatic pathways in the retina

The electroretinogram (ERG) provides information about outer retina function in both clinical and research applications. ERG components elicited by light increments and decrements can be separated using a long-flash paradigm in which periods of light ON and OFF are alternated. Here, the ON-OFF ERG is combined with a silent substitution technique to elicit responses from individual cone photoreceptor classes by modulating the intensities of three color lights between the two periods. The results focus on the short wavelength (S) cone pathways since they are vulnerable to disease and because there are many unanswered questions about S-cone ON and OFF circuitry.

Role of a Dual Splicing and Amino Acid Code in Myopia, Cone Dysfunction and Cone Dystrophy Associated with L/M Opsin Interchange Mutations

Purpose Human long (L) and middle (M) wavelength cone opsin genes are highly variable due to intermixing. Two L/M cone opsin interchange mutants, designated LIAVA and LVAVA, are associated with clinical diagnoses, including red-green color vision deficiency, blue cone monochromacy, cone degeneration, myopia, and Bornholm Eye Disease. Because the protein and splicing codes are carried by the same nucleotides, intermixing L and M genes can cause disease by affecting protein structure and splicing. Methods Genetically engineered mice were created to allow investigation of the consequences of altered protein structure alone, and the effects on cone morphology were examined using immunohistochemistry. In humans and mice, cone function was evaluated using the electroretinogram (ERG) under L/M- or short (S) wavelength cone isolating conditions. Effects of LIAVA and LVAVA genes on splicing were evaluated using a minigene assay. Results ERGs and histology in mice revealed protein toxicity for the LVAVA but not for the LIAVA opsin. Minigene assays showed that the dominant messenger RNA (mRNA) was aberrantly spliced for both variants; however, the LVAVA gene produced a small but significant amount of full-length mRNA and LVAVA subjects had correspondingly reduced ERG amplitudes. In contrast, the LIAVA subject had no L/M cone ERG. Conclusions Dramatic differences in phenotype can result from seemingly minor differences in genotype through divergent effects on the dual amino acid and splicing codes. Translational Relevance The mechanism by which individual mutations contribute to clinical phenotypes provides valuable information for diagnosis and prognosis of vision disorders associated with L/M interchange mutations, and it informs strategies for developing therapies.

Allele-specific editing of rhodopsin P23H knock-in mice broadens therapeutic potential of CRISPR/Cas for dominant genetic diseases

No effective treatment exists for dominant inherited diseases. Here, we present a CRISPR/Cas9 genome editing strategy that combines multiple innovations to achieve specific and efficient disruption of the pathogenic allele with a single-nucleotide mutation (Rho-P23H) in a mouse model of dominant retinitis pigmentosa. Our study demonstrates the possibility of a spacer-mediated allele-specific editing approach, which may be applicable to a broad range of dominant disorders in which the mutant allele is not discriminable by placement of a PAM sequence.Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR/Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs (PAMs) that can lead to allele restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single guide RNAs (tru-sgRNAs) to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa (RP). We found that approximately 45% of the mutant P23H allele was edited at DNA level, and that the relative RNA expression of wild-type Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in the outer nuclear layer was significantly delayed in treated regions of the Rho-P23H retinas at five weeks of age. Our proof-of-concept study, therefore, outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patient. Our study also extends allele-specific editing strategies beyond discrimination within the PAM sites, with potentially broad applicability to other dominant diseases.