Boyuan Yan,Suresh Viswanathan,Scott E. Brodie,Wen-Tao Deng,Kirsten E. Coleman,William W. Hauswirth,Sheila Nirenberg
A clinically viable approach to restoring visual function using optogenetic gene therapy
optogenetic gene therapy,retinal prosthesis,preclinical study,retinitis pigmentosa,vision restoration
Optogenetic gene therapies offer a promising strategy for restoring vision to patients with retinal degenerative diseases, such as retinitis pigmentosa (RP). Several clinical trials have begun in this area using different vectors and optogenetic proteins (Clinical Identifiers: NCT02556736, NCT03326336, NCT04945772, and NCT04278131). Here we present preclinical efficacy and safety data for the NCT04278131 trial, which uses an AAV2 vector and Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using electroretinograms (ERGs). Safety was assessed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), electroretinograms (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed.
A clinically viable approach to restoring visual function using optogenetic gene therapy

Optogenetic gene therapies offer a promising strategy for restoring vision to patients with retinal degenerative diseases, such as retinitis pigmentosa (RP). Several clinical trials have begun in this area using different vectors and optogenetic proteins (Clinical Identifiers: NCT02556736, NCT03326336, NCT04945772, and NCT04278131). Here we present preclinical efficacy and safety data for the NCT04278131 trial, which uses an AAV2 vector and Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using electroretinograms (ERGs). Safety was assessed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), electroretinograms (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed.

Retinitis pigmentosa (RP) is a large group of inherited retinal disorders in which progressive degeneration of photoreceptors leads to vision loss. The clinical manifestations of affected individuals present first as night blindness, followed by reduction of peripheral vision and, eventually, loss of central vision. Current treatments for patients with RP are limited. There is some evidence to suggest that vitamin A and fish oil supplements may slow vision loss in some patients with early disease, but they are not able to reverse the disease. For a subset of patients whose retinal degeneration is caused by a mutation in the RPE65 gene, targeted gene therapy is now possible,,, and is currently being used to treat patients (reviewed in Maguire et al.). However, because RP is a genetically heterogeneous disease, with more than 100 different genes or loci that lead to the common endpoint of vision loss (https://web.sph.uth.edu/retnet/sum-dis.htm#B-diseases), this particular gene therapy does not apply to the majority of RP patients. New gene therapies that employ optogenetics, however, are opening up new options for patients with RP. With optogenetics, it is possible to treat the disease in a way that is independent of the underlying gene defect, allowing a much broader range of patients to obtain benefit. Briefly, one of the main versions of the approach is as follows: an optogenetic protein, such as a channelrhodopsin (ChR), is delivered to the retina’s output cells, the ganglion cells,,,,,, although bipolar cells have also been utilized.,,, The ChR is then light-activated, which causes the ganglion cells to fire and send neural signals through the optic nerve to the brain. Different investigators are using different strategies to perform the light activation, with some incorporating externally worn goggles that contain an embedded light-delivery device (reviewed in Barrett et al.).,, Nirenberg and Pandarinath take the approach a step further, using a light-delivery device that sends signals in the retina’s neural code, causing the ganglion cell firing to closely mimic that of the normal retina. This combination of optogenetic gene therapy and neurally coded stimulation has the potential to offer significantly better and more detailed vision restoration to patients with advanced stage blindness due to RP. The initial optogenetic therapies utilized channelrhodopsin-2 (ChR2) as the optogenetic protein,,,, as this was one of the first optogenetic proteins discovered (reviewed in Mattis et al.). While highly promising as a concept, this particular protein had limitations as a therapy because it (1) requires very bright light to activate it, and (2) its excitation wavelength is short (near blue, with peak excitation at 460 nm), both of which increase its risk of producing phototoxic effects. In the past decade, more optogenetic proteins,,, and light-activated photoswitches (Gaub et al.; reviewed in Tochitsky et al.; Berry et al.) have been developed, providing an increasing arsenal of tools to move the field forward. As a result, there are now several clinical trials using optogenetics as a therapeutic: ClinicalTrial.gov identifier: NCT02556736 (Retrosense/Allergan/AbbVie), ClinicalTrial.gov identifier: NCT03326336 (GenSight Biologics), ClinicalTrial.gov identifier: NCT04945772 (Nanoscope Therapeutics Inc.), and ClinicalTrial.gov identifier: NCT04278131 (Bionic Sight Inc.). In this paper, we present the main preclinical studies underlying NCT04278131, the Bionic Sight trial. The vector used in this trial, referred to as BS01 (AAV2-CAG-ChronosFP-WPRE), utilizes Chronos as the optogenetic protein. It was chosen because Chronos was reported to be >10-fold more sensitive to light than ChR2, and has a longer excitation wavelength (peak excitation is 500 nm), which together bring a significant reduction in potential phototoxicity., In the studies shown here and in NCT04278131, Chronos is fused to green fluorescent protein (GFP) to produce a protein called ChronosFP. Here we present data in animal models on both the efficacy and safety of ChronosFP-expressing vectors. These data can later be compared with the results of the clinical trial. Reporting preclinical data is highly valuable, as it provides insight into what measures in animal studies were important and had predictive value when the treatment was subsequently applied to humans.

To test the efficacy, photopic electroretinograms (ERGs) were performed in adult rd1 mice, a widely used model for retinal degenerative disease,,,,: rd1 has an earlier onset of retinal degeneration than the rd10 model. Briefly, the photopic ERG response in normal animals is composed of three components: the a-wave, which corresponds to photoreceptor signaling; the b-wave, which corresponds to bipolar cell signaling; and the photopic negative response (PhNR), which corresponds to ganglion cell signaling. In rd1 animals, which lose photoreceptor outer segments by 8 weeks of age,, none of these components are present. If these animals are engineered to express an optogenetic protein in their ganglion cells, the prediction is that a PhNR-like wave would emerge, reflecting the newly created optogenetic activity of the ganglion cells. We thus conducted ERGs on BS01-treated eyes to test for the presence of a newly created PhNR-like wave. Seven rd1 mice received BS01 by intravitreal injection into one eye at a dose of 5 × 108 vg/eye, which is comparable to a dose of 5 × 1011 vg/eye in humans,, (see also Table S1). This dose was chosen as a starting point, aiming to be comparable to one of the first intravitreal doses of an AAV-type vector in human trials that was available at the time (NCT02416622); this is also comparable to studies in animal models., Outcomes were measured at 10 weeks post injection, when the ChronosFP expression was expected to have peaked. The untreated eye of each animal served as the control. The results showed that six of the seven animals produced clear and reliable light-evoked ERG responses (PhNR-like waves) in their treated eyes; this is in contrast to the ERGs produced by the untreated eyes of the same rd1 animals, which showed flat ERG responses (Figure 1). The results were significant at both a group level (p < 0.001, unpaired t test comparing the magnitude of the ERG signal from treated eyes with those from the untreated eyes), and at an individual level (p < 0.004, paired t test, comparing, for each animal, its treated eye with its untreated counterpart). The ChronosFP-induced PhNR has a short latency; this occurs because the ganglion cells are being activated directly, rather than through the photoreceptor-to-bipolar cells pathway.

Following the experiments shown in Figure 1, the effectiveness of BS01 over a broader range of doses was explored, from 10-fold lower (5 × 107 vg/eye) to 2-fold higher (1 × 109 vg/eye). Figure 2 shows the results over this 20-fold range; all dose levels were statistically significantly different from the control group (p < 0.01, Student’s t test, comparing each dose group with the untreated group), and, as expected, the amplitudes of the responses increased with increasing vector dose. Given the robust PhNR-like responses at the higher vector doses, we also explored whether the intensity of the light stimulation could be reduced and still produce a light response. Specifically, using the two highest doses, we assessed how far the light level could be dropped while still maintaining a PhNR-like amplitude that was significantly above the control level. The results showed that the light level required for a vector dose of 1 × 109 vg/eye could be reduced by approximately a factor of 10 (Figure 3). For the next highest dose, 5 × 108 vg/eye, the light level could be reduced by a factor of 6 (p < 0.01, Student’s t test). The effects on lower vector doses were not tested, since there was little room for adjustment. These results served to narrow the range of light levels needed for testing in a clinical trial, reducing the burden of exploration with patients. We emphasize also that the controls in this study (see Figure 2), which were blind, untreated eyes, always received the maximal light intensity (0.06 mW/mm2). Thus, without the vector treatment, blind eyes showed no response even to the brightest light in this regimen, whereas with BS01 treatment, the eyes responded with robust ERG signals even when light levels were dropped substantially. We also note that light levels can likely be dropped further when perceptual studies are performed with human subjects, since ERG responses are well known to be less sensitive than perceptual ones.

The ERG responses in rd1 mice demonstrated that BS01 is effective in producing light responses in blind animals. To assess the vector’s safety, studies were performed in rats, nonhuman primates, and mice, using several tests, including immunohistochemical analyses and cell counts (rats), ERGs (nonhuman primates), and ocular tolerance/toxicology assays (mice).

Similar to the rd1 mouse line and human patients with RP, S334ter-3 rats have an inherited retinal degenerative disease that leads to severe loss of photoreceptors. Two sets of experiments were performed. The first assessed the safety of the BS01 vector, the second assessed the safety of the BS01 vector combined with the light stimulation needed to activate it. For the first set, three groups of animals were used: a low-dose group that received 6.8 × 108 vg/eye, which corresponds to 6.7 × 107 vg/eye in mice, a high-dose group that received 2.7 × 109 vg/eye, which corresponds to 2.6 × 108 vg/eye in mice, and a control group that contained both vehicle-treated eyes and untreated eyes. Note that there is a wide range of vitreous volumes that have been reported for rat eyes (13–54 μL). To be conservative with respect to safety, we assumed the largest volume for the rat vitreous (54 μL) when converting to the mouse equivalent dose (mouse vitreous is 5.3 μL). Thus, the doses used here may be considerably higher (as much as four times higher) and, therefore, provide an even stronger assessment of the toxicity of BS01 to ganglion cells. For reference, Table S1 provides dose equivalents for different species, including mouse, rat, nonhuman primate, and human. The assay performed was to count the number of cells in the ganglion cell layer. If treatment with BS01 was detrimental to the targeted cells, one would expect a loss of cells in the ganglion cell layer of the treated retinas compared with controls. Ganglion cells were labeled with two markers, one that labels ganglion cells in general (Brn3) and one that labels the ChronosFP-expressing cells specifically (an antibody to GFP). Cell counts were performed 5 months after BS01 injection to allow for cell death, if it occurred, as well as removal of cellular debris. Figure 4 shows the results: there was no statistically significant difference in retinal ganglion cell counts between the low-dose group and the control group 5 months after treatment (p > 0.1, Student’s t test) or between the high-dose group and the control group (p > 0.5, Student’s t test). The second set of experiments assessed the safety of the vector plus light stimulation. These were divided into two parts. The first focused on testing for the presence of ChronosFP-expressing cells. Since these cells had been made light-sensitive by expressing ChronosFP in them, there was a possibility that light stimulation would damage them, limiting the value of a therapy that required light stimulation. To assess the safety of light-activating ChronosFP-expressing cells, we treated two groups of four animals with BS01, both at 2.7 × 109 vg/eye. The animals in one group received light stimulation similar to the exposure expected in a clinical trial with an optogenetic vector (12 2-h sessions of direct stimulation over a period of 8 weeks at 0.1 mW/mm2 [see materials and methods]), while the other group of animals received no light stimulation. The retinas were processed 5 months after the light stimulation. The results showed no difference in the number of ChronosFP-expressing cells in the two groups, indicating no loss of ChronosFP-expressing cells as a result of the light exposure (Figures 5A and 5B) (p > 0.7, Student’s t test). The second part of the study focused on photoreceptors, assessing whether the light stimulation needed to drive ChronosFP in the ganglion cells caused damage to the naturally light-absorbing cells in the retina, the photoreceptors. For these experiments, wild-type (WT), rather than S334ter-3, animals, were used since adult S334ter-3 rats no longer have a photoreceptor layer as a result of their retinal degenerative disease., Six WT animals were assessed: In each, one eye received BS01 plus light treatment (12 2.5-h sessions over a period of 6 weeks at 0.1 mW/mm2), and the other eye received no treatment. The BS01 dose was 8.4 × 10 9 vg/eye, which corresponds to 8.2 × 108 vg/eye in mice. The retinas were removed 6 months post-injection. The results (Figures 5C and 5D) showed no loss of photoreceptors (cones) in the BS01-plus light treated retinas compared with the untreated retinas (p > 0.4, Student’s t test). These results are consistent with previous results on light stimulation for optogenetic therapies reported in Yan et al. showing no loss to the photoreceptor layer, which contains both rods and cones, as measured by retinal thickness (outer nuclear layer thickness) and using the same wavelength and light level.

The study in rats assessed tissue integrity using immunohistochemistry and cell count assays. To examine safety electrophysiologically and in a species similar to humans, ERGs were performed on cynomolgus macaques. Three animals (six eyes total) were assessed 7 months after vector administration by intravitreal injection. One animal (both eyes) received BS01 and two animals (both eyes) received a variant that used the same transgene (ChronosFP) but was packaged using a different AAV2 capsid (the AAV2tYF capsid). The doses fell within the range used in the efficacy study in Figure 2; doses were 3.7 × 1010 vg/eye and 1.2 × 1011 vg/eye, which is equivalent to 1 × 108 vg/eye and 3.2 × 108 vg/eye in mice (see also Table S1). Four animals (eight eyes total) were untreated and served as controls. To assess whether ERG responses were adversely affected in treated vs. untreated eyes, we focused on the three standard photopic ERG components: the a- and b-waves, which reflect photoreceptor and bipolar cell responses, respectively, and the PhNR wave, which reflects ganglion cell responses. Seven months after vector injection, intensity/response data were fit to a generalized Naka-Rushton function to derive the saturated amplitude for each wave, the Vmax, following reference. As shown in Figure 6, there was no statistically significant reduction in Vmax observed for any of the three ERG components as a result of the treatment (p > 0.2, for all waves, comparing the Vmax values in the treated group with those in the untreated group). The ERG safety experiments shown in Figure 6 show results at a macroscale, i.e., whole retina electrophysiology. In this section, we provide additional safety evaluation at the single-cell level. Multi-electrode array (MEA) recordings from excised retinas of cynomolgus macaques that were previously injected with ChronosFP-expressing vectors were evaluated for receptive field size and mean firing rate. The retinas from six eyes (three animals) were treated with an array of ChronosFP-expressing vectors; for these experiments, the capsid was the AAV2 variant AAV2tYF, the promoters were CAMKII, hCACNA1G, and mNefL1.6, and the dose range was from 2.2 × 1011 vg/eye to 7.6 × 1012 vg/eye (equivalent to 5.8 × 108 vg/eye to 2 × 1010 vg/eye in mouse). The results showed that the distributions of receptive field sizes and firing rates from the ChronosFP-treated group were not statistically significantly different from those from the untreated group (Figure 7) (p > 0.2, Kolmogorov-Smirnov test), that is, retinal ganglion cells sampled from eyes that had been injected intravitreally with a ChronosFP-expressing vector showed receptive field sizes and firing rates that were very similar to those from untreated retinas, with the stimuli used to assess firing rates drawn from natural scenes (e.g., trees, landscapes, people walking).

Last, we assessed the safety of BS01 in terms of local tolerance. A total of 120 rd1 mice were divided into three groups (40 animals per group): two dose groups spanning a factor of 10 in dose level (4.25 × 108 vg/eye and 4.25 × 109 vg/eye) and a vehicle-alone group. Injections were performed intravitreally to the right eye, and the left eye remained untreated. Each group had two time points, week 4 and week 12 (20 animals at each time point), when animals were euthanized. Transgene expression in the injected eyes was verified (Figure 8, Table S2). ChronosFP expression, measured by GFP-immunolabeling (magenta), was detected in nerve fiber layer, inner plexiform layer, optic disc, and the extending axons in all animals. Ophthalmic examinations were performed on weeks 2, 4, 8, and 12 post-injection, and the findings are summarized in Table S3. A slit lamp was used to assess anterior segments including cornea, iris, and lens. An indirect ophthalmoscope was used to assess the posterior segments including vitreous chambers and retinas. Previous phenotypic characterizations of rd1 mice have shown that their eyes have vessel attenuation and pigment patches at an early age., Consistent with this, these phenotypes were present at similar frequencies in both the BS01 and vehicle-injected eyes in our study, suggesting that they were not caused by the vector. In hematoxylin and eosin-stained retinal sections, BS01-related microscopic findings included minimal to slight mononuclear cell infiltrates in the vitreous, where minimal is the lowest level in a 5-level classification (Table S4). By terminal euthanization (week 12), only the lowest (minimal level) mononuclear cell infiltrates were present.

Optogenetic gene therapies offer a potential strategy for restoring sight to patients with retinal degenerative diseases. In this study, we assessed the efficacy and safety of a potential therapeutic AAV2 vector that uses Chronos as the optogenetic protein. Efficacy was assessed in mice in a dose-dependent manner using ERGs, and safety was evaluated in several species using several tests, including immunohistochemical analyses and cell counts (rats), ERGs (nonhuman primates), and ocular toxicology assays (mice). The results showed that Chronos-expressing vectors were efficacious over a broad range of vector doses and stimulating light intensities, and were well tolerated: no test article-related findings were observed in the anatomical and electrophysiological assays performed. These results paved the way for the ongoing clinical trial, ClinicalTrial.gov. NCT04278131.

While optogenetic gene therapy applied to retinal ganglion cells holds great promise for sending visual signals to the brain, we do not expect it to serve as a complete treatment for patients with advanced stage blindness. This is because the retina is an image processing tissue, and optogenetic therapy needs to be combined with a device that performs this processing for the patients. Briefly, when images enter a normal retina, they activate the photoreceptors, which convert the images into electrical signals. The signals are then passed through the retinal circuity, which extracts features from the images (the building blocks for visual perceptions) and converts the features into a code. The coded signals are then sent from the retinal ganglion cells to the brain. When optogenetic therapy is combined with a neural coding device, it opens the door to producing meaningful perceptions (e.g., of faces, objects, and landscapes), as described and demonstrated using the mouse retina in Nirenberg and Pandarinath. Chronos is a particularly good fit to interface with a neural coding device, because of its fast kinetics. The temporal resolution driven by a channelrhodopsin is largely limited by the off-kinetics of its photocurrent (i.e., the kinetics of current decay after the cessation of light). As one of the fastest channelrhodopsins, Chronos has an off time of 3.6 ms, which is much faster than ChR2 (∼10 ms) and ChrimsonR (15.8 ms).

Light safety has been a concern for clinical applications of optogenetics, as early channelrhodopsins required bright light of relatively short wavelengths. However, this problem has been significantly reduced in two ways: (1) by the properties of Chronos: it has significantly higher light sensitivity and a longer peak absorption wavelength (500 nm), and (2) by using a device that delivers neurally coded stimuli. The latter helps because neuronal firing is well known to be sparse (cells fire on average only 5%–10% of the time),,,, so stimulation that follows the neural code keeps the accumulated light exposure low, even when very bright pulses, such as those used in Figures 2 and 3 (0.06 mW/mm2), are used. A previous study assessed exposure limits for various stimulation protocols, following the limits provided by the American National Standards Institute (ANSI 2014). In particular, the study took movies of natural scenes, encoded them into trains of light pulses following Nirenberg and Panderinath, and assessed the exposure the light produced relative to the ANSI limits. Because of the sparse nature of the neurally coded representation of the movies, the exposure fell well below the ANSI limit, even using 0.06 mW/mm2 for the pulses, running the movies for 8 h, and including the newer (2014) restriction by ANSI, the Luminance Dose Restriction. Briefly, the Luminance Dose Restriction in the 2014 ANSI takes into account studies by Morgan et al., in nonhuman primates that showed retinal damage was occurring from exposures to 568 nm light at intensity levels below those reported in ANSI 2007. These experiments led to an addition to the damage spectrum. The damage spectrum of the Luminance Dose Restriction, denoted V(λ), where λ is wavelength, peaks at 555 nm and decreases toward both ends of the visual spectrum (e.g., V(555 nm) = 1.0, V(590 nm) = 0.76, V(500 nm) = 0.32). Yan et al. provides software for other investigators in the field to assess any stimulation protocol with respect to the ANSI 2014 limits.

Currently, there are four clinical trials using optogenetics as a potential therapy for RP (ClinicalTrial.gov identifier: NCT02556736, NCT03326336, NCT04278131, and NCT04945772). Among these, NCT02556736, NCT03326336, and NCT04278131 target retinal ganglion cells, and NCT04945772 targets ON bipolar cells. An advantage of targeting bipolar cells is the potential to utilize the remaining intrinsic retinal processing pathway. However, during retinal degeneration, the inner retina undergoes progressive remodeling, which makes this strategy less suitable for patients with advanced disease. For the studies targeting ganglion cells, several different optogenetic proteins are being used. The first clinical trial of an optogenetic therapy in humans (NCT02556736) began in 2015 using ChR2 as the optogenetic protein, based on the pioneering work of Zhuo-Hua Pan and his colleagues. Two years later, another clinical trial (NCT03326336) was started, using ChrimsonR (peak excitation: 590 nm), a red-shifted variant with faster kinetics, producing some positive results in one subject. Chronos has favorable qualities relative to Chrimson and ChR2 in both light sensitivity and kinetics: as shown in Klapoetke et al., it is 7-fold or more sensitive to light than Chrimson and more than 10-fold more sensitive than ChR2, according to the light levels required to achieve 100% spiking in cultured neurons; the off-time of Chronos is 3.6 ms compared with ChR2 (∼10 ms) and ChrimsonR (15.8 ms). The faster off-time offers the ability to follow stimuli with rapid temporal variations, which is critical for devices delivering neurally coded visual input. With respect to light safety, while ChrimsonR was believed to have the highest margin of safety due to its red-shifted spectrum (peak excitation: 590 nm), this may no longer be the case, as indicated in ANSI 2014 (Luminance Dose Restriction in ANSI 2014) (see previous section about the updated damage spectrum from ANSI 2014, based on monkey studies).

In this paper, we referred to the ERG responses from Chronos-expressing retinas as “PhNR-like,” because the newly created wave comes from ganglion cells, as does the PhNR in WT animals. Note, though, that we expect the Chronos-produced wave to be different from the WT-produced one, because the former is produced by direct activation of the ganglion cells, as opposed to the latter, which rides on waves of signals created by other cells in the retinal pathway. As a result, the Chronos-produced signal is expected to occur with a short latency, which is what was observed and shown in Figures 1 and 2. With respect to the amplitude of the response, it is difficult to predict how it should compare to the amplitude in the WT, but we do note that in mice treated with the highest vector dose used in this study (see Figure 2) the amplitude reached the same approximate level as is observed in WT PhNR (∼20 μV for the saturated response, Liu et al.). Thus, in summary, while the BS01 treatment does not produce a completely normal PhNR, it does produce a significant ganglion cell response, which we referred to as “PhNR-like” and has the potential to provide visual signals to the brain and help visually impaired patients.

All vectors were prepared in a balanced salt solution with 0.014% Tween 20 and delivered to the eye by intravitreal injection. For rodents, animals were anesthetized with intraperitoneal ketamine/xylazine (72 mg/kg ketamine and 4 mg/kg xylazine for mice, and 80 mg/kg ketamine and 10 mg/kg xylazine for rats), and the pupil was dilated with an atropine sulfate ophthalmic solution (1%). Using a Hamilton syringe under a dissecting microscope, the needle was passed through the sclera into the vitreous cavity. The injected volume was 1 μL for mice and 4 μL for rats. For nonhuman primates, animals were anesthetized with a mixture of ketamine/dexmedetomidine (5–10 mg/kg ketamine and 0.01–0.02 mg/kg dexmedetomidine) and then maintained with inhaled isoflurane/oxygen mixture. Pupils were dilated with 1% atropine sulfate, 2.5% phenylephrine hydrochloride, applied topically. The vector was injected intravitreally using a 3/10 mL U-100 insulin syringe with a 30-Gauge needle. The injected volume was 80–100 μL. All animal experiments and procedures were performed according to the guidelines approved by the Institutional Animal Care and Use Committees.

For mice, animals were anesthetized with intraperitoneal ketamine/xylazine (72 mg/kg ketamine and 4 mg/kg xylazine), and the pupil was dilated with 1% atropine sulfate, 2.5% phenylephrine hydrochloride. To perform the ERG, a tungsten-wire electrode was placed on the corneal surface of the recorded eye and referenced to an electrode in the mouth. Visual stimuli were delivered with an LED stimulator with a 505-nm peak wavelength. The stimulator was placed 1.7 cm away from the cornea, subtending a visual angle of approximately 100°, with a peak intensity of 0.06 mW/mm2. The stimulation was delivered as pulsed light, periodic at 10 Hz, pulse width at 11.2 ms. Data collection was carried out with the Espion E3 ERG console (Diagnosys LLC, Lowell, MA). Mouse model was rd1, which has rapid retinal degeneration. As shown in Nirenberg and Pandarinath, photoreceptor outer segments disappear but the ganglion cell layer remains largely intact. The advantage to this model is that ERG responses recorded can be attributed to the transduced cells and not riding on signals passed from the photoreceptors. For nonhuman primates, animals were anesthetized with a mixture of ketamine (5–10 mg/kg)/dexmedetomidine (0.01–0.02 mg/kg). Pupils were dilated with topical agents (1% atropine sulfate, 2.5% phenylephrine hydrochloride). To perform the ERG, a tungsten-wire electrode was placed on the corneal surface of the eye being tested, and referenced to a needle electrode placed in the scalp. Visual stimuli were delivered with a mini-ganzfeld stimulator placed close to the recorded eye. For the Naka-Rushton fit, photopic ERG stimulation was used following Joshi and colleagues: 5-ms red light ganzfeld flashes ranging from 0.00625 to 1.6 cd s/m2 on a constant 7 cd/m2 blue background.

Light stimulation was performed in 12 2- to 2.5-h sessions over a period of 6–8 weeks. The light was at an intensity of 0.1 mW/mm2 (a peak wavelength of 505 nm). The light was delivered in pulses with a pulse width of 5 ms, as it would be if neurally coded stimuli were used., The stimulus subtending a visual angle of approximately 60° covered a large area of central retina, 4 mm diameter. In each session, animals were anesthetized with isoflurane (99.9%) to a depth that minimized eye movements. Each animal was placed on its left side, with its right eye illuminated by the stimulus. The pupil was dilated with an atropine sulfate ophthalmic solution (1%) and the eye was kept wet with artificial tears applied regularly (every 7 min). The left eye was left untreated. Between sessions, the animals were exposed to normal room light with standard day/night cycles, as is standard in a rodent animal housing facility. Two to 4 weeks after the sessions were completed, the animals were euthanized and the retinas removed for examination.

For wholemount retinas, eyes were removed and fixed in 4% paraformaldehyde in PBS for 30 min. Retinas were dissected and fixed overnight in 4% paraformaldehyde. Autofluorescence was quenched with 1% sodium borohydride in PBS for 5 minuets. The retinas were permeabilized and blocked with 5% normal donkey serum (NDS), 1% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100 for 1 h. The retinas were then labeled with rabbit anti-GFP Alexa Fluor 555 1:200 (Invitrogen-Molecular Probes, Life Technologies, Carlsbad, CA) overnight in 5% NDS, 1% BSA in PBS or with fluorescein peanut agglutinin (FITC PNA) 1:500 in 2% BSA (Vector Laboratories, Burlington, CA) in PBS for 15 min. Then, the retinas were washed five times in PBS and incubated for 1.5 h at room temperature with the Alexa Fluor 647 donkey anti-goat immunoglobulin IgG 1:100 (Invitrogen-Molecular Probes, Life Technologies, Carlsbad, CA). The retinas were thoroughly washed in PBS and mounted. When processed as sections, eyes were fixed in 4% paraformaldehyde. After 1 h, cornea and lens were removed without disturbing the retina. The retinas were further fixed for an additional 2–3 h at room temperature. The eyecups were rinsed with PBS and cryoprotected by 30% sucrose/PBS for 4 h at room temperature, then embedded in cryostat compound (Tissue TEK OCT, Sakura Finetek USA, Inc., Torrance, CA) and frozen at −80°C. Retinas were sectioned perpendicularly from dorsal to ventral at 12-μm thickness. For immunohistochemistry, retinal sections were rinsed in PBS and incubated in 0.3% Triton X-100 in PBS for 15 min, then blocked in 5% BSA in PBS for 1 h at room temperature. Sections were then incubated with anti-Brn3a (1:500, Santa Cruz, sc-31984) and anti-GFP (1:200 dilution, Life Technologies, A11122) at room temperature overnight. They were washed with PBS three times, followed by incubating with IgG secondary antibodies tagged with Alexa 594 and Alexa 488 (1:500 dilution, Molecular Probes, Eugene OR) at room temperature for 2 h, then washed with PBS. Sections were mounted with Vectashield Mounting Medium for Fluorescence (Vector Lab, H-10400, Burlingame, CA) and coverslipped.

Electrophysiological recordings were obtained in vitro from isolated retinas. Briefly, the anterior portion of the eye and vitreous were removed immediately after enucleation, and the eyecup was placed in Ringer’s solution and stored in darkness for at least 20 min before dissection. Under dim red light illumination, pieces of retina 1.5–3.0 mm in diameter were cut from central regions and placed onto a MEA for recording. The Ringer’s solution was bubbled with 95% O2 and 5% CO2 and maintained at 35–36°C, pH 7.4. The stimulation and recording of retinal ganglion cells was performed as in Nirenberg and Pandarinath. Spike waveforms were recorded using a Plexon Instruments Multichannel Neuronal Acquisition Processor (Dallas, TX). A standard spike sorting method was used to identify individual cells as in Nirenberg and Pandarinath.

Before injections, an ocular examination was performed. If ocular findings were present, the animal was excluded from the study. Ocular examinations were also performed on weeks 2, 4, 8, and 12 post-injection by a licensed veterinary ophthalmologist. A slit lamp was used to assess anterior segments including cornea, iris, and lens. An indirect ophthalmoscope was used to assess the posterior segments including vitreous chambers and retinas. All observations were made in a masked fashion. There were two euthanizaton time points: week 4 and week 12. At scheduled necropsies, eyes of animals were collected in 4% paraformaldehyde. Following sufficient time in fixation, the tissues were trimmed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and slides were examined. Immunohistochemical stains for GFP were also examined to verify the transgene expression.

The authors declare that the data supporting the findings of this study are available within the paper and its supplemental information files. Otherwise, data generated and analyzed during the current study are available from the corresponding author on reasonable request.