Multi-Characteristic Opsin Therapy to Functionalize Retina, Attenuate Retinal Degeneration, and Restore Vision in Mouse Models of Retinitis Pigmentosa

Abstract

Purpose: Retinal degeneration 1 and 10 (rd1 and rd10) mice are useful animal models of retinitis pigmentosa (RP) with rapidly and slowly progressive pathologies, respectively. Our study aims were to determine the effect of adeno-associated viral vector 2 (AAV2)-delivered multi-characteristic opsin (MCO-010; under the control of a metabotropic glutamate receptor-6 promoter enhancer) on the morphological and functional characteristics of vision in both rd1 and rd10 mice.

Methods: Various retinal measures of MCO-010 transduction and electrophysiological, behavioral, and other routine blood analyses were performed in the rd1 and/or rd10 mice after intravitreal injection of 1 µL of MCO-010 or AAV2 vehicle. Functional tests included electroretinogram, visually evoked potential, and behavior assay (optomotor and water maze). Retinal thickness, intraocular pressure, and plasma cytokine levels were also determined.

Results: Following intravitreal MCO-010 injection, approximately 80% of bipolar cells were transduced in the retina, and no alterations in retinal thickness were observed at 4 months post-injection. However, retinal thickness significantly decreased in control mice. MCO-010 treatment increased head movements and induced faster navigation of mice to the platform in a water-maze test. The MCO-010 gene therapy helped preserve visually evoked electrical response in the retina and visual cortex. No ocular toxicity, immunotoxicity, or phototoxicity was observed in the MCO-010–treated mice, even under chronic intense light conditions.

Conclusions: Intravitreal MCO-010 was well tolerated in rd1 and rd10 mice models of RP, and it appeared to attenuate retinal photoreceptor degeneration based on retinal structure and functional outcome measures.

Translational Relevance: As reported here, optogenetic treatment of the inner retina attenuates further retinal degeneration in addition to photosensitizing higher order neurons, and this disease-modifying aspect should be evaluated in optogenetic clinical trials.

Introduction

Loss of outer retinal cells in diseases such as retinitis pigmentosa (RP) and advanced dry age-related macular degeneration (dAMD) causes severe visual impairment and currently does not have any approved treatment. Such disorders are characterized by degeneration of photoreceptors in the retina, which hinders visual ability due to non-photoactivation of the retina and the lack of transmission of signals to the visual cortex.^1–5 Furthermore, the degree of visual loss increases with aging,⁶ and this is a major concern for the ever-increasing elderly population in our society. The inability to replace lost photoreceptors in RP and dAMD necessitates the discovery and clinical utility of suitable alternative therapeutics that will provide clinically significant improvements in vision, retard or halt disease progression, and help patients function better in their visually dependent daily activities. Conventional gene replacement and gene editing therapies may have limited effectiveness in treating advanced stages of retinal degeneration, as these approaches rely on the presence of viable photoreceptors to rescue or transduce. However, in cases where photoreceptor loss is severe, there are insufficient target cells for these therapies to act upon. In contrast, optogenetic approaches^7–12 offer a promising alternative by functionalizing the remaining, non-photoreceptor retinal cells, such as bipolar cells or retinal ganglion cells, to become light sensitive. This strategy has gained significant interest within the ophthalmic community as a potential treatment option for advanced retinal degeneration, where traditional gene therapies may fall short. By conferring light sensitivity to the surviving retinal cells, optogenetic therapy aims to restore visual function downstream of the damaged photoreceptors. 

Recently, optogene therapies based on precise optical modulation^13–16 of nervous system components have shown promising results for the treatment of several neurological^17–24 and retinal disorders.^25,26 By photosensitizing higher order retinal neurons (e.g., ON bipolar cells) with ambient light-sensitive cation-channel proteins, delivered via safe viral vectors, we aimed to convert the recipient bipolar cells into artificial photoreceptors and thus restore vision. Unlike artificial implants, which are available to treat blindness under U.S. Food and Drug Administration Humanitarian Device Exemptions, invasive surgery is not required for optogenetics treatment. In addition, optogenetic therapy of retinal degenerative diseases is gene-mutation agnostic and does not require viable photoreceptor cells which degenerate in RP and other inherited retinal diseases. 

Our recent work showed significant improvement of visually guided behavior in retinal degeneration 10 (rd10) mouse models of RP upon activation of multi-characteristic opsin (MCO-010)-expressing cells by ambient light.²⁷ In the current study, for the first time to our knowledge, we demonstrated that intravitreally delivered MCO-010 attenuated further retinal degeneration and provided useful functional vision in two retinal degenerated mice models (rd10 and rd1). Attenuation of progressive retinal degeneration was observed in optical coherence tomography (OCT) and immunohistological analyses. The improvement of vision after MCO-010 treatment was evaluated by studying the performance of the recipient animals in a water-maze test system over a longer duration and spatial frequency-dependent optomotor responses. In addition, we report measurement of the light-activated responses at the retina and visual cortex levels using in vivo electrophysiology. Furthermore, we demonstrated that the MCO-010–enabled vision restoration therapy was well tolerated and safe in the rd1 and rd10 mice. 

Materials and Methods

Ethics Statement

All experimental procedures were conducted according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and a Nanoscope Technologies Institutional Animal Care and Use Committee–approved protocol. 

MCO-010 Mechanism of Action

MCO is a fusion protein of three mutated subunits (derived from Chlamydomonas reinhardtii,²⁸ Chlamydomonas noctigama,²⁹ and Discosoma³⁰) without any linkers, and it is the most bioengineered non-mammalian protein that has been heterologously expressed in human subjects.²⁷ Adeno-associated viral vector 2 (AAV2) containing MCO-010 transgene was manufactured at Charles River Laboratories (Rockville, MD). MCO opens when exposed to light, allowing the flow of cations into the cells which then depolarize the cells to transmit visually evoked signal to the brain via the optic nerve.³¹ 

MCO-010 Dose Calculation and Conversion

The animals received intravitreal injections of MCO-010 vector ranging from 1E13 to 1E10 vector genomes/mL based on quantitative polymerase chain reaction (qPCR) titration targeting inverted terminal repeat (ITR) sequences. For injection volume of 1 µL/eye, this corresponds to the range of 1E10 to 1E7 vector genomes (vg)/eye. Applying a conversion factor of 2.9 (ratio between ITR copies and MCO gene copies historically observed across production lots), the corresponding range of estimated doses injected per eye was 3.4E9 to 3.4E6 MCO gene copies (gc) per eye. For the vehicle control groups, which lacked the MCO transgene, doses are represented in terms of vector genomes per eye, meaning an injection of 1 µL from a 1E12-vg/mL solution would correspond to 1E9 vg/eye. 

Mouse Models with Retinal Degeneration

Retinal degenerated (rd10) mice (B6.CXB1-Pde6b^rd10/J) have a spontaneous missense point mutation in Pde6b (cGMP phosphodiesterase 6B, rod receptor, beta polypeptide), which leads to the later onset and slower retinal degeneration similar to human disease (RP). Approximately 12-week-old retinal degenerated mice (B6.CXB1-Pde6b^rd10/J; The Jackson Laboratory, Bar Harbor, ME) were used in the reported in vivo experiments. The rd1 mice have a retinal degeneration 1 mutation (Pde6b^rd1) and have early onset of photoreceptor degeneration, causing complete blindness by 3 to 4 weeks of age. Mice were maintained on a 12∶12 light:dark cycle. 

Randomization and Masking

Randomized block experimental design was implemented in order to ensure that different doses of MCO-010 or phosphate-buffered saline (PBS) were assigned to each experimental group with a known, equal probability of receiving a given treatment. Mice were selected randomly, and group number, cage number, and mice number were assigned prior to injection based on restricted randomization. The cages were housed in a random order on the shelves, and the injections were performed in a random order. To avoid bias, the video imaging–based experiments, including behavioral tests (water maze and optomotor), and the analyses, which included retina thickness measurements, were performed by individuals masked with respect to the treatments. After the randomized allocation of animals to the treatments, animals, samples, and treatments were coded until the data were analyzed. The objective measures of electrophysiology were performed by trained scientists and therefore were not masked. 

Intravitreal Injection of MCO-010 to rd10 Mouse Eye

Aseptic conditions were used for all surgical procedures, and surgical tools were sterilized in an autoclave. Each mouse was anesthetized, and a light topical ocular anesthetic (proparacaine HCl 0.5%) was instilled into the eye of the animal. The MCO-010 solution (∼1 µL) was injected by a sterilized 29-gauge needle of a Hamilton microliter syringe inserted through the sclera into the vitreous cavity (intravitreal injection). The MCO-010 solution was injected into both eyes. In the case of the control, AAV2 vehicle (∼1 µL) was injected into the eyes intravitreally by a sterilized 29-gauge needle. The cornea was kept moist with a balanced salt solution during the surgical procedure. 

Immunostaining

The MCO-010–injected and control eyes were fixed in a modified Davidson solution overnight and finally stored in 1× PBS. Next, the eyecup/retinal sections were prepared and subjected to 0.5% Triton X-100 (washing solution) three times. The nonspecific binding of antibodies was blocked by 4% serum for 60 minutes and washed with washing solution three times. The samples were incubated with primary antibodies (1:500 dilution), including mCherry Antibody (NBP1-96752; Novus Biologicals, Toronto, ON, Canada), PKCα Polyclonal Antibody (PA5-17551; Thermo Fisher Scientific, Waltham, MA), Caspase-3 Antibody (sc-7272; Santa Cruz Biotechnology, Dallas, TX), Phospho-CD45 (Ser1007) Polyclonal Antibody (PA5-38448; Thermo Fisher Scientific), and CtBP-2 (BDB612044; BD Biosciences, Franklin Lakes, NJ), overnight at 4°C. After the samples were washed with 0.5% Triton X-100 solution in 1× PBS three times, the secondary antibody (Thermo Fisher Scientific), either Goat Anti-Rabbit IgG (Alexa Fluor 488 nm) or Goat Anti-Mouse IgG (Alexa 594 nm), diluted 1:250 in washing solution, was loaded for 1 hour at room temperature. The samples were then stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:200 dilution). 

OCT Imaging

OCT imaging is a standard ophthalmic assessment tool that provides quantitative measurements of anterior segment and retinal structures instead of a subjective evaluation. Optical sectioning/imaging using spectral-domain OCT (SD-OCT) was performed to monitor any changes in ocular structures due to intravitreal injection of different doses of MCO-010 or vehicle control. We employed the NS-NEEL OCT-guided electrophysiology system (Nanoscope Instruments, Bedford, TX),³² with an imaging wavelength centered around 860 nm. Animals were anesthetized using a mixture of ketamine, xylazine, and acepromazine. For dilating the pupil, a drop of tropicamide was topically applied to the eye. B-scan and C-scan OCT images of the retina were acquired. SD-OCT images of the cornea, lens, and retina after intravitreal MCO-010 injection in rd1/rd10 mice were compared to the images before injection. ImageJ software (National Institutes of Health, Bethesda, MD) was used to analyze the SD-OCT images. The total thickness of the retina was measured by point measures (at five different locations excluding the optic nerve region). 

Intraocular Pressure Measurement in Intravitreally Injected Mice

For intraocular pressure (IOP) measurement, the mouse was placed on a wire cage and allowed ∼1 minute of resting time. The tonometer (iCare TONOVET Plus; Icare Finland, Vantaa, Finland) was loaded with a new probe allowing for self-calibration. The TONOVET was positioned parallel to the eye with a distance of about half an inch and aimed at the center of the cornea. Three trials of IOP measurement were carried out, each trial consisting of six readings. 

Optomotor Response After MCO-010 Injection

For optomotor behavior assessment, mice were placed on a platform (in the center of a drum) surrounded by rotating vertical stripes. The optomotor system was custom made, and the performance of the system was validated using blind and wild-type mice. The functional recovery of vision was evaluated via head-tracking responses.³³ The basic principle of this analysis is that, whenever a moving pattern is presented to an animal, the (light-sensing) animal will move its head as a transient corrective measure to maintain stable vision.^34,35 The advantage of this method is that it does not require any previous training of the animal. The animal’s head movements were recorded at different spatial frequencies (0.05–1 cycle per degree [cpd]). 

Water-Maze Behavioral Assessment

The rd10 mice were >8 weeks old. After MCO-010 injection, the mice were tested with a radial-arm water maze to determine behavioral restoration of vision in rd10 mice with MCO-010–sensitized retinal bipolar cells. Briefly, mice were placed into the center of the maze, and a platform was placed just below the surface of the water at the end of one of the arms. The mice rapidly learned to determine the location of the platform by utilizing visual cues (light-emitting diodies [LEDs] emitting light in the visible spectrum on the platform). The platform (on one of the arms) provided a reward to the mice, as they could rest instead of having to swim continuously. Videorecording was stopped when the mice found the platform or when 60 seconds had elapsed after the mouse was dropped in the water, to prevent the mice from getting tired of swimming. Behavioral restoration of vision was determined by measuring the latency to reach the platform. Because it is known that the rd10 mice undergo progressive retinal degeneration, with advanced degeneration after 12 weeks of age, 6- to 10-week-old mice were chosen for training. The rd10 mice were trained in the radial water maze to find the visually guided cue before intravitreal MCO-010 or vehicle injection. The platform was positioned at the base of the lighted arm. Every time the mouse (with photosensitive retina) was placed close to the center of the maze, it aimed to find the lighted dry platform. The platform provided a reward to the mouse because it could rest instead of having to swim. Each time the mouse arrived at the lighted platform, the mouse was allowed to stay on the platform for ∼30 seconds as its reward. After training 20 to 30 times, the mouse was put back in the cage. This procedure was repeated for 1 week before conducting the visually guided behavioral assessment. The exclusion criterion consisted of events in which the mouse did not swim and simply floated. 

Fast Light-Evoked Electrical Activity Assessment in the Retina

After overnight dark adaptation of the mice, they were anesthetized, the pupil was dilated with a drop of tropicamide, and measurements were made 10 minutes after the tropicamide application. A fast neuroelectrophysiology system (Biopac, Goleta, CA) and gold ring electrode in the back of the eye (without subtenon insertion) were used to evaluate the stimulated retina response from mice before and up to 13 weeks after the MCO-010 injection. In the case of optogenetically treated mice, the conventional electroretinogram (ERG) profile and nomenclature may not apply as they do not have rods and cones. Rather, ON bipolar cells are sensitized with MCO-010, which responds instantaneously to the light stimulus. The mouse was anesthetized with intraperitoneal injection of a mixture of ketamine (90 mg/kg), xylazine (10 mg/kg), and acepromazine (0.5 mg/kg). The electrical measurement was carried out in the dark under dim red light with white-light stimulation. The heating pad was preheated to ∼35°C, and the mouse was placed on the heating pad. The ground needle electrode was inserted in the base of the tail, and the reference needle electrode was placed subdermally between the eyes. After dilation of the pupil, a drop of ALCAINE (proparacaine HCl 0.5%) was applied to the mouse eye. One drop of 1× PBS was applied to the mouse eye, and the contact electrode lens was gently placed on the eye and centered. Any excess moisture was removed using a Kimtech Kimwipe. After each recording, the electrode lens was sanitized by dipping it 10 to 15 times in fresh distilled water. 

Assessment of ERG Response

To evaluate the safety of intravitreal AAV-delivered MCO-010, wild-type mice, after overnight dark adaptation, were anesthetized, the pupil was dilated using tropicamide, and measurements were made 10 minutes after application of the tropicamide. Following the NS-NEEL scotopic ERG protocol and using silver-embedded thread electrodes, we recorded ERG responses from mice before and 4 weeks after MCO-010 injection. Light flashes were elicited by a white LED stimulator providing stimulus intensity fixed at 0.01%, 0.1%, 1%, 10%, or 100% (corresponding to 7 × 10¹⁴ photons/cm²/s) with a stimulus rate of 0.1 Hz and stimulus duration of 5 ms. Signals were amplified through a built-in bias drive amplifier and analog-to-digital converters with a built-in programmable gain amplifier. Signal acquisitions were performed at a 1-kHz sampling rate, and a high-pass filter at 0.1 Hz and a low-pass filter at 250 Hz with a 60-Hz notch filter were used. The ERGs from a sequence of 10 strobe light flashes were averaged to obtain the final waveform. After completion of the experiment, the electrodes from the animal were removed. Mouse eyes were kept moist with a hydrating eye ointment during the recovery period. The mouse was injected intraperitoneally with sterilized saline and placed on a heated pad until it fully recovered. 

Visually Evoked Potential Measurement

The animals were dark adapted overnight. They were then anesthetized with intraperitoneal injections of a mixture of ketamine (90 mg/kg), xylazine (10 mg/kg), and acepromazine (0.5 mg/kg). The visually evoked potential (VEP) measurements were carried out in the dark using a dim red light with the NS-NEEL system. Using surgical tools, the skull was exposed, and a craniotomy was performed by drilling a hole for the recording electrode to access the V1 (anteroposterior [AP] = −3.0 mm, and mediolateral [ML] = +2.5 mm relative to bregma) area of the visual cortex. The mouse was placed on the stereotaxic unit, and the ground electrode was subcutaneously placed in the tail. The reference electrode was placed at AP = +2.5 mm and ML = −1.5 mm. 

Viability of Retinal Cells Subsequent to Chronic Light Exposure

To determine the potential phototoxic effects of chronic high-intensity white-light stimulation, we exposed control (wild-type) and MCO-010–treated rd10 mice to high-intensity white light. Long-term (4 weeks or 4 months) viability of retinal cells in the transduced retina was evaluated after chronic light stimulation (8 hr/day). The mice were placed inside a chamber containing white-light sources providing an intensity of 0.1 mW/mm² on the floor of the chamber. Four weeks after chronic light exposure, the MCO-010–transduced rd10 mice (3.4E9 gc/eye; n = 5), as well as wild-type mice (control; n = 5), were euthanized. Furthermore, 4 months after light exposure, another group of MCO-010–transduced rd10 mice (3.4E8 gc/eye; n = 5), as well as wild-type mice (control; n = 3), were euthanized. The eye tissue was harvested for retina extraction. The explanted retina was immunostained using an apoptotic marker (cleaved caspase 3)/DAPI and imaged using confocal microscopy (FluoView FV1000; Olympus, Tokyo, Japan). The number of total cells versus apoptotic retinal cells was compared between the MCO-010–transduced and control groups. 

Collection of Blood at Different Time Points After Injection of MCO-010 in Mice

After anesthetization, blood (∼0.2 mL) was drawn from the facial vein (using a sterile animal lancet) 1 week before intravitreal injection. After the MCO-010 injection, blood was drawn for analysis. After completion of the study period, the mouse was euthanized. To collect the blood from the facial veins of the mice, the hairless freckle on the side of the jaw was located and pierced with a lancet. 

Immune Assay of Serum Derived From MCO-010–Injected rd10 Mice

Studies on kinetics of immunotoxicity were conducted using mouse Picokine ELISA (Boster Biological Technology, Pleasanton, CA) quantification of different pro- and anti-inflammatory cytokines (interleukin-6 [IL-6] and IL-10) in plasma of MCO-010–treated animals at different time points. The ELISA manufacturer's protocol was followed for analysis of serum (1:10 dilution) derived from mice injected with MCO-010, and a microplate reader (BioTek, Winooski, VT) was used for quantification. 

Statistics

Prism (GraphPad, Boston, MA) was used to analyze the data. The data were plotted as mean ± SD or mean ± SEM. Statistically significant difference analyses were carried out by t-test. P < 0.05 was considered statistically significant. 

Results

Bipolar Cell–Specific Expression Is Highly Efficient After Intravitreal Injection of MCO-010

The transduction of MCO-010 in bipolar cells was evaluated by immunohistochemistry of intravitreally injected retinal sections. Bipolar cell–specific marker protein kinase Cα (PKCα) staining (Fig. 1A, green) was found to overlap with the mCherry (immunofluorescence) expression (Fig. 1B), thus confirming cell-specific expression. The axonal terminals of bipolar cells (which reside within the ganglion cell layer) were positive in PKCα and mCherry images, indicating that MCO-010 expression is on the soma as well as the axonal membrane of the bipolar cells. Though there was a minimal level of diffused fluorescence in the inner plexiform layer, the retinal ganglion cells (RGCs) can be clearly seen to be mCherry-negative (Fig. 1C). The bipolar cell–specific transduction efficiency was calculated as percentage of bipolar (PKCα-positive) cells co-expressing mCherry immunostaining. Approximately 80% of bipolar cells were mCherry positive 8 weeks after intravitreal injection of MCO-010 (Fig. 1D), suggesting robust transduction in a cell-specific manner. 

Figure 1.

 

Intravitreal injection of MCO-010 led to selective expression in rd10 mouse retina. The retina slices were immunostained using primary antibodies (for mCherry and PKCα). (A) PKCα (green) shows the soma and axonal terminals of the bipolar cells. (B) Expression of MCO in bipolar cells visualized by an immunoassay for mCherry (red). (C) Zoomed-in region of the rectangle marked in (A) showing the expression of MCO in bipolar cells. (D) Quantification of percentage of mCherry-positive bipolar cells in MCO-010–injected and control groups. (E) Staining for PKCα in the not injected control mice. (F) Staining for mCherry. Average ± SD; *P < 0.0001.

Intravitreal Injection of MCO-010 Attenuates Retina Degeneration

In the mice model of retinal degeneration, it is important to monitor the change in thickness of the retina of the MCO-010–injected and vehicle-injected control groups to determine any adverse or therapeutic effects of MCO-010 expression. However, a significant challenge exists in monitoring the individual retinal layers due to the severe degeneration of the retinal structure in the rd1 mice. Figure 2A shows a quantitative comparison of SD-OCT–based measurement of retinal thickness at multiple time points, including baseline, day 1, and 1 month and 4 months after intravitreal injection in rd1 mice treated with two different doses of MCO-010 or AAV2 vehicle (no transgene). Although there was an initial increase in retinal thickness (noted on day 1 of injection) in all groups, it decreased close to the baseline values 1 month after injection irrespective of dose or control injection, as shown in the box plots in Figure 2A and scatterplots in Figure 2B. Notably, 4 months after injection, retinal thickness in the MCO-010–injected mice (groups AA and CC) did not decrease as compared to baseline values. However, in the case of the vehicle-injected rd1 mice (group BB), the overall retinal thickness significantly decreased compared to baseline. The mean difference in retinal thickness between baseline and intravitreally injected mice (MCO-010 and vehicle injection) are shown as Gardner–Altman estimation plots in Figure 2C. The OCT-based measurement of retinal thickness showed that MCO-010 sensitization of retinal bipolar cells can effectively attenuate further degeneration of the retina in rd1 mice. 

Figure 2.

 

Intravitreal injection of MCO-010 attenuated retina degeneration, as evaluated by SD-OCT and immunohistology. (A) Box plot of thickness of retina of rd mice before and after different doses of MCO-010 or AAV vehicle injection. Group AA included 3.4E8 gc/eye MCO-010; group BB included 1E9 vg/eye AAV2 (no transgene); and group CC included 3.4E6 gc/eye MCO-010. (B) Scatterplot showing comparisons of retinal thickness before and 4 months after injection in the different groups. (C) The mean difference of retinal thickness between baseline and intravitreally injected mice shown as a Gardner–Altman estimation plot. *P < 0.05 for group BB between baseline and 4 months after vehicle injection. There was no statistically significant difference observed in MCO-010–injected groups AA and CC. (D) MCO-010 treatment led to enhanced connectivity of the synaptic terminals of bipolar cells with RGCs measured by CtBP (red) staining. (E) CtBP immunostaining for control non-treated retina. Right panels of (D) and (E) show composites of CtBP with PKCα (green) and DAPI (blue). (F) Quantification of PKCα and CtBP fluorescence in MCO-treated and non-treated mice (mean ± SEM; *P < 0.05).

Staining for C-terminal binding protein (CtBP), a component of photoreceptor and bipolar cell ribbon synapses,³⁶ demonstrated enhanced punctate labeling in the inner retina of MCO-treated eyes compared to controls (Figs. 2D, 2E). Also, MCO-010–treated rd10 mice retina showed better preservation of bipolar cell axonal terminals (stained using antibodies for the bipolar cell–specific marker PKCα) compared to controls. Quantitative analysis revealed a significantly higher signal for PKCα and CtBP for MCO-injected eyes versus non-treated eyes (Fig. 2F). These findings indicate that optogenetic sensitization of bipolar cells with MCO helps maintain the structural connectivity of bipolar cells and their synaptic terminals. 

Optomotor Response After Intravitreal Injection of MCO-010 in rd1 Mice

Because measurement of the optomotor response is commonly used to determine thresholds of the functional visual system in animals,^34,35 we utilized this tool to evaluate the potential improvement in visual performance of retinal degenerated mice following MCO-010 treatment to photosensitize their retinas. The advantage of this method is that it does not require any previous training of the animal. We validated the assay protocol by taking baseline data for wild-type mice having normal vision and retinal degenerated mice. The number of head movements for retinal degenerated rd1 mice was not significant at any of the spatial frequencies (of stripes in the virtual rotating drum) at low light intensity levels (0.001 mW/mm²), whereas, for wild-type mice, there were variations in head movement with spatial frequency, as expected. The number of head movements for wild-type mice was found to be significantly higher compared to rd1 mice and thus validated the testing system at low light levels. The critical spatial frequency for wild-type mice was determined to be in the range of 0.1 to 0.25 cpd. 

Figure 3 shows the longitudinally measured optomotor response of rd1 mice before and after injection of MCO-010 or AAV2 (vehicle control group). The mean number of head movements for the MCO-010–injected group, in response to rotating stripes at a spatial frequency of 0.25 cpd, increased at 5 weeks from baseline values, and this increase was maintained through the 13-week post-treatment period (Fig. 3A). In contrast, the vehicle-injected control group did not exhibit a detectable change in the mean number of head movements in response to rotating stripes at 0.25-cpd spatial frequency (Fig. 3B). Because photoreceptors of rd1 mice are completely degenerated by the age of 4 weeks, behavioral models, which require training such as radial water maze, could not be used to evaluate possible vision improvement. 

Figure 3.

 

Longitudinal monitoring of optomotor responses of rd1 mice before and after injection of MCO-010 or AAV2 (vehicle control). (A) Number of head movements at 0.25 cpd during baseline and at 13-weeks post-injection for individual mice (n = 3 mice per group; mean ± SD). F, female; M, male. (B) Comparison of baseline-subtracted number of head movements at baseline and different time points after intravitreal injection of MCO-010 and AAV2 vehicle (n = 10 mice per group; mean ± SEM; *P < 0.05). The light intensity at the center of the chamber was 1 µW/mm².

Behavioral Vision Restoration After Intravitreal Injection of MCO-010 in rd10 Mice Lasts for More Than 6 Months

Longitudinal behavioral assessments on MCO-010–injected and vehicle-injected rd10 mice were conducted in order to evaluate the efficacy of MCO-010 in the restoration of vision. Visually guided behavior tests in the radial water-maze system evaluate changes in spatial navigation of animals at baseline and at times after MCO-010 treatment. The water-maze test provides allocentric wayfinding, characterized by the ability of the mice to navigate and swim toward a distal light cue on a platform. The water-maze test system was validated using age-matched wild-type mice with normal vision. As shown in Supplementary Figure S1A, the wild-type mice took less than 20 seconds to find the platform associated with the light cue from the center, side, or arm. In contrast, the rd10 mice with complete retinal photoreceptor degeneration at 12 weeks of age (with AAV2 vehicle injection) required a significantly longer time to reach the lit platform (Supplementary Fig. S1B) than wild-type mice for the same intensity of light. To determine the efficacy of intravitreal injection of MCO-010 in vision restoration, 3-month-old mice with advanced retinal degeneration were subjected to multiple baseline measurements using the visually guided radial water-maze test with LED light intensity of 7 µW /mm². After three baseline measurements (1 week apart), intravitreal injection of MCO-010 was performed in mice at 15 to 16 weeks of age. There was no significant variation in latency (to find the lit platform) among multiple baseline measurements (Figs. 4A, 4B). MCO-010–treated mice were significantly faster at reaching the lit platform in the water maze using the visual cue at 5 weeks after the injections of two different doses of MCO-010 (Figs. 4A, 4B). All MCO-010–injected mice significantly gained visually guided behavior that lasted through the 26-week post-injection period. The multiple intravitreal doses tested were found to be therapeutically effective (as determined by the radial water-maze test). However, for the low-dose group, the vision improvement did not persist over the later time points. For aged mice (6 months old), the latency to reach the platform from the center of the radial-arm water maze reduced significantly after MCO-010 injection (1.2E9 gc/eye) (Fig. 4C) even at 12 months of age, confirming the therapeutic effectiveness of MCO-010 in advanced retinal degeneration stages. Visually guided behavior of aged (6-month-old) rd10 mice also improved after injection of MCO-010 as compared to age-matched mice injected with AAV2 (vehicle control) (Fig. 4D). 

Figure 4.

 

Visually guided dose-dependent behavioral improvement in young and old rd10 mice in radial-water-maze tests after MCO-010 injection. (A, B) Times to reach the platform from the center of the radial-arm water maze at baseline (B1, B2, and B3) and at post-injection evaluations performed at weeks 3 to 26 in 3-month-old mice intravitreally injected with MCO-010: (A) 3E9 gc/eye, (B) 3.4E8 gc/eye (n = 6 mice, 3 male and 3 female, per dose group). (C) Times to reach the platform from the center of the radial-arm water maze at baseline (B1 and B2) and post-injection evaluations at weeks 4 to 24 in 6-month-old mice intravitreally injected with MCO-010 (1.2E9 gc/eye; n = 6 female mice). LED light intensity was 7 µW/mm² (mean ± SEM; *P < 0.05 between baseline and different time points). (D) Visually guided behavior in the water-maze test of 6-month-old rd10 mice improved after injection of MCO-010 as compared to AAV2 (vehicle control)–injected age-matched mice (n = 5 mice per group). The light intensity was 7 µW/mm² (mean ± SEM; *P < 0.05).

Functional Electrophysiology Study Shows Visually Evoked Electrical Response in MCO-010–Sensitized rd Mice

The rd1 mice were used as negative control, and wild-type mice were used as a positive control to detect fast electrical activities near the retina in response to light. The electrical response to 1-ms pulses (3 cd·s/m²) was detected in wild-type mice using the Biopac neuro-electrophysiology system as shown in Figure 5A. In contrast, the rd1 mice retina did not elicit a fast electrical response upon light stimulation (Fig. 5A). Electrical responses to 1-ms pulses (3 cd·s/m²) were detected in MCO-010–injected rd10 mice using the Biopac neuro-electrophysiology system as shown in Figure 5B. As shown in Figure 5B, the light-evoked fast electrical response from the rd10 mice before MCO-010 treatment was flat and did not change in the absence or presence of light (1-ms pulses, red lines). However, 4 weeks after MCO-010 injection, a visually evoked fast electrical response was observed in the rd10 mice (Supplementary Fig. S2). This was similar to the electrical response to the light stimulus in wild-type mice (Fig. 5A). 

Figure 5.

 

Fast electrical responses of retina in MCO-010–injected rd10 mice observed using electrophysiological measurements. (A) Validation of the light-evoked fast electrophysiological responses in rd1 (negative control) and wild-type (positive control) mice. (B) Representative electrical responses of retinas before and 4 weeks after MCO-010 treatment of rd10 mice with and without light stimulation (1 ms, indicated by red bars). (C) Representative longitudinal measurement (baseline and 4 and 13 weeks post-injection) of fast electrical responses in MCO-010–injected OD and OS eyes of rd10 mice in response to 1-ms light pulses (3 cd·s/m²). The timing of the light stimulus is shown by the arrow.

Figure 5C and Supplementary Figure S3 show longitudinal measurements of fast electrical responses of rd10 mice retinas in response to light stimulation. Before the MCO-010 injection, neither OD or OS eyes generated any electrical responses. However, at >4 weeks after MCO-010 injection, light exposure caused both right and left injected eyes to generate evoked electrical responses. Optogenetic modulation using MCO-010 is known to generate fast (ms) electrical activity in cells in response to light.³⁷ The observed fast electrical response was similar to that normally generated during early action potential creation for visual transduction. 

Detection of Significantly Higher Visually Evoked Potential in rd1 Mice Upon Intravitreal Injection of MCO-010

To evaluate if the photosensitization of the retina (after MCO-010 injection) leads to functional activation within the visual cortex, scotopic flash VEP was performed in rd1 mice with and without MCO-010 injection. Figure 6A shows a representative VEP response in a 9-month-old rd1 mouse (without intravitreal injection of MCO-010) at light intensities of 7 × 10¹² photons/cm²/s. The VEP response was flat in the control rd1 mice, indicating no phototransduction in the retina. The rd1 mouse injected with MCO-010 (3.4E8 gc/eye), 8 months after injection, exhibited visually evoked electrical response upon light stimulation at an ambient intensity level (7 × 10¹² photons/cm²/s) as shown in Figure 6B. Figure 6C shows a quantitative comparison of the amplitudes of the VEP negative peaks in rd1 mice receiving intravitreal injections of MCO-010 or AAV control. Significantly higher VEPs in rd1 mice were observed after intravitreal injection of MCO-010. 

Figure 6.

 

VEPs in rd1 mice with and without intravitreal injections of MCO-010. (A) Representative VEPs of rd1 mice at a light intensity of 7 × 10¹² photons/cm²/s, 8 months after AAV2 vehicle injection (1E9 vg/eye). (B) VEPs of rd1 mice 8 months after injection with MCO-010 (3.4E8 gc/eye) at a light intensity of 7 × 10¹² photons/cm²/s. The downward arrow (at 0 ms) indicates the point of light stimulation. (C) Quantitative comparison of the amplitudes of negative peaks of VEPs in rd1 mice receiving intravitreal injections of MCO-010 or AAV control (mean ± SEM; n = 4 mice; *P < 0.05).

Safety of MCO-010 in Wild-Type Mice Evaluated by ERG Monitoring

To evaluate the potential toxicity of intravitreally injected MCO-010 on residual photoreceptors, wild-type mice were evaluated for scotopic ERG before and after injection of the gene therapy. Supplementary Figure S4 shows the raw ERG profiles at different light intensities before (Supplementary Fig. S4A) and after (Supplementary Fig. S4B) MCO-010 injection in OD eyes. Also shown are the ERG profiles for non-injected (OS) eyes at baseline and after OD injection. The a- and b-wave amplitudes were measured at two different visual stimulation intensities: –1.3 and 1.7 log cd·s/m². There were no changes in the a- and b-wave ERG amplitudes after MCO-010 transfection (Supplementary Fig. S4C) in wild-type mice. The natural rod and cone photoreceptors in the MCO-010–sensitized eyes of the wild-type mice were found to function similarly to those observed at baseline. 

Intravitreal Injection of MCO-010 Is Safe and Does Not Increase the IOP of rd1 Mice

Though intravitreal injection is known to be the safest route for the delivery of drugs to the retina, IOP is known to increase with intravitreal injection.³⁸ Furthermore, inflammation due to MCO-010 injection may alter IOP. Tonometric assessments were performed at baseline and in a longitudinal manner to evaluate any adverse effect due to different doses of MCO-010 injected. Longitudinal monitoring of IOP was carried out before and after MCO-010 or AAV2 vehicle injection over time. Both eyes were injected with either AAV2 vehicle control (dose, 1E9 vg/eye) or MCO-010 (high dose, 3.4E8 gc/eye; low dose, 3.4E6 gc/eye). Figure 7 shows no significant (>3 mm Hg) change in IOP from baseline after intravitreal injection of both eyes with a high or low dose of MCO-010 as compared to baseline values (Fig. 7, top and middle rows). The lower panel of Figure 7 shows a maximum change of ∼4 mm Hg in IOP after intravitreal injection with AAV2 vehicle control as compared to baseline. Hence, the injection process itself can transiently alter the IOP, and such a change is not treatment related. IOP fluctuations happen due to many types of ocular perturbations.³⁹ 

Figure 7.

 

Intravitreal injection of MCO-010 did not increase the IOP of rd1 mice. Both eyes were injected with either AAV2 vehicle control or MCO-010. Low dose: 3.4E6 gc/eye (n = 5); high dose: 3.4E8 gc/eye (n = 10); vehicle (AAV2) dose: 1E9 vg/eye (n = 9). Left, OD; right, OS. Average ± SEM.

We also evaluated the IOP in uniocular-injected eyes and in the control eyes. Supplementary Figure S5 shows an IOP change of ∼1 mm Hg from baseline after 2 weeks of intravitreal injection with MCO-010 in OD eyes, which recovered back to baseline values at 4 weeks (Supplementary Fig. S5, left panel). The right panel of Supplementary Figure S5 shows a change of ∼2 mm Hg in IOP for the non-injected contralateral OS eye from baseline at 4 weeks, which recovered back at 9 weeks. Such fluctuation in IOP was not dose related, as the OS eye was not injected, and the injected (OD) eye did not exhibit any change in IOP. To evaluate the toxicity (if any) of the intravitreal injection of MCO-010, we also monitored IOP in wild-type mice. Longitudinal monitoring of IOP was carried out before and after MCO-010 uniocular injection. Supplementary Figure S6 shows no detectable IOP change from baseline after 2 weeks of intravitreal injection with MCO-010 (1.2E9 gc/eye) in the injected (OD) eye or contralateral (OS) eye. Thus, the small changes in the IOP observed are not biologically relevant. 

Lack of Phototoxicity Due to Chronic High-Intensity Light Stimulation in MCO-010–Treated Mice

Determination of the potential phototoxic effects of chronic high-intensity light stimulation of eyes expressing MCO-010–mCherry after intravitreal injection of MCO-010 is necessary before contemplating clinical translation of this gene therapy. Chronic exposure of MCO-010–transduced retina to light may raise concerns about retinal cell viability. Therefore, MCO-010–treated rd10 mice and vehicle-injected wild-type mice were exposed to an order of magnitude higher intensity white light (i.e., 0.1 mW/mm²) than that of ambient light level (i.e., 0.01 mW/mm²). Long-term viability and potential apoptosis of retinal cells in the inner nuclear layer (INL) of MCO-010–injected rd10 mice and vehicle-injected wild-type mice subsequent to chronic intense light exposure was evaluated using a caspase assay. The overlay of fluorescence images of retina stained with PKCα and the caspase-3 marker of MCO-010–treated OD eyes showed no detectable apoptotic cell death in the INL even after 4 months of chronic intense white light illumination (intensity, 0.1 mW/mm²) for 8 hr/day (Fig. 8A). The non-injected contralateral OS eyes also did not show any detectable cell death as measured by the caspase assay. 

Figure 8.

 

Intravitreal injection of MCO-010 in rd10 mice did not cause long-term photo- or immunotoxicity. Figure shows long-term viability of retinal bipolar cells after 4 months of chronic light exposure assessed by caspase assay. (A) Representative fluorescence images of retina stained with caspase-3 (red) overlaid with PKCα (green) for MCO-010–treated mouse retina, 4 months after 8-hr/day illumination of white light (intensity, 0.1 mW/mm²). (Top) OD eye and (bottom) non-treated contralateral OS eye. Scale bar: 50 µm. No caspase-3–positive apoptotic cells were observed in the INL of MCO-010–treated rd10 mice after 4 months of chronic light exposure (n = 4 mice). (B) Long-term immunotoxicity in plasma of MCO-010–injected rd10 mice. (Left) Longitudinal changes in IL-6 (pro-inflammatory marker) levels in the plasma of mice injected with MCO-010 (3.4E8 vg/eye) as compared to baseline values. (Right) Changes in IL-10 (anti-inflammatory marker) levels over the 6-month post-injection period (mean ± SD; n = 6 animals).

Supplementary Figure S7 shows fluorescence images of retina immunostained with PKCα and caspase-3 of PBS-injected and non-injected contralateral eyes of wild-type mice. Similar to MCO-010–injected rd10 mice, no detectable apoptotic cells in the INL of wild-type mice retinas were observed after chronic illumination with intense light. The number of apoptotic retinal cells was quantified, normalized to the total number of cells, and compared between the MCO-010–transduced rd10 and control (wild-type) mice groups. No detectable retinal cell death was observed in the INL in either of the wild-type eyes or MCO-010–injected or non-injected rd10 mice eyes. These results indicate no compromise of retinal cell viability after 4 months of chronic light exposure in MCO-010–treated rd10 mice. No statistically significant difference was found between the chronic light-exposed wild-type (Supplementary Fig. S7) and MCO-010–treated rd10 mice groups (Fig. 8A). 

Quantification of Pro- and Anti-Inflammatory Cytokines

To determine the long-term immunotoxicity in MCO-010–injected rd10 mice, longitudinally collected sera of the mice were analyzed. Figure 8B shows the change in pro- and anti-inflammatory markers (IL-6 and IL-10, respectively) after MCO-010 injection over a 6-month post-injection period with respect to the baseline values. There was no detectable increase in levels of IL-6 and IL-10 within the first 2 weeks after MCO-010 injection as compared to the baseline levels of each cytokine before injection. The nominal increase of the pro- and anti-inflammatory markers during the 3- to 6-month post-injection period was <100 pg/mL, which is close to the baseline value. 

Discussion

rd10 mice are an autosomal recessive model of RP, identified in 2002 by Chang et al.⁴⁰ These mice carry a spontaneous mutation of the rod phosphodiesterase gene, which causes the degeneration of rods beginning around the third week of age. Progressive loss of photoreceptors occurs with aging. Similar to human RP, the rd10 model retina has light sensitivity at an early age that is lost with advancing age. Comprehensive analysis of the morphology by quantitative immunocytochemistry and function by electroretinography of the rd10 mouse retina during the period of maximum photoreceptor degeneration⁴¹ has established that this novel model is relevant to human RP. Photoreceptor death (peaking at around week 4) in rd10 mice is accompanied and followed by dendritic retraction in bipolar cells. An overall slower decay of retinal structure and function observed in rd10 mice allows it to be an excellent model for evaluating the efficacy of therapeutic interventions. Although our earlier publications^27,37 reported behavioral vision restoration for up to 16 weeks following intravitreal MCO-010 injection, this study found such an effect for up to 26 weeks. 

The observed stable retina thickness, measured by SD-OCT, in the MCO-010–injected mice (as compared to the vehicle-injected group) through 4 months after intravitreal injection can be attributed to stabilization of retina thickness by attenuating the loss of remaining photoreceptors (if any) or preventing further disorganization of retinal layers. Thus, an optogenetic treatment has now been shown to demonstrate a disease-modifying aspect (i.e., stabilize the retina from further degeneration), in addition to restoring lost light sensitivity. The results presented in this paper show efficient and stable in vivo expression of MCO-010 reporter protein (visualized by mCherry fluorescence and immunostaining) in mice retina after intravitreal injection of MCO-010. Not only did we see robust MCO-010 expression, but we also observed sustained MCO-010 presence in the soma and axons of retinal bipolar cells. PKCα expression and its distribution in bipolar cells have been previously shown to be activity dependent.⁴² Quantification of fluorescence intensity of PKCα in axonal terminals of bipolar cells shows significantly higher values for MCO-010–treated rd10 mice retina. Because CtBP expression is known to correlate with synapse density and connectivity with ganglion cells, analysis of CtBP was also carried out. CtBP is known to regulate synaptic vesicle recycling by permitting a permissive lipid environment for compensatory endocytosis. The increased CtBP immunoreactivity and preservation of the axonal terminals of bipolar cells suggest that MCO-010 treatments strengthen synaptic signaling between bipolar cell axon terminals and RGC dendrites. To summarize, we see that not only did the retinal structure stabilize but connectivity to the retinal ganglion cells was also enhanced as compared to non-treated mice. 

The results also demonstrate that the expression of MCO-010 in retinal degeneration mice enables behavioral restoration of vision. The directed movement of the mice toward the ambient light-level visual cue in the radial-arm water maze clearly shows repair of the phenotypic deficit in rd10 mice after MCO-010 transduction of targeted retinal bipolar cells. Notably, the improvement in visually guided behavior was observed even at ambient light-intensity levels. Improved visual acuity after transduction with the ambient-light–activatable MCO-010 into ON bipolar cells of rd10 and rd1 mice retina demonstrates the potential of MCO-010 in vision restoration in subjects with retinal degenerative diseases even with the absence of natural photoreceptors. In addition, significant improvement in the optomotor response of mice was observed at spatial frequencies as high as 0.25 cpd at ambient light levels. As demonstrated in the water maze and the optomotor tests, the improvement in visually guided behavior was observed even at light-intensity levels an order of magnitude lower than that required for channelrhodopsin-2 opsin.⁴³ 

MCO-010 optogenetic therapy is based on AAV2 delivery of MCO-010 to the retina, and evaluation of dose-dependent expression is key to establishing a therapeutic dose in conjunction with behavioral studies. Furthermore, determining the kinetics of MCO-010 expression is important to determining the early efficacy time point, peak activity, and stability of expression to support the efficacy outcome. The density of rod bipolar cells in the mouse retina has been estimated to be 15,000 cells/mm²,⁴⁴ which is a factor 2.5 times higher than that in the human retina⁴⁵ due to the large size of bipolar cells in humans. Upon transduction of MCO-010, the large size of retinal bipolar cells in human as compared to murine bipolar cells will allow more efficient optogenetic phototransduction at ambient light levels. With extensive retinal degeneration and remodeling, the activity of mGluR6 promoter becomes reduced in human retinas with advancing age.⁴⁶ To have a long-term benefit, we used a novel hybrid promoter–enhancer (CMV-mGluR6) strategy. Using this approach, we have demonstrated cellular specificity, as the expression was mostly in bipolar cells in rd and wild-type mice. It may be noted that expression of MCO-010 (containing the mCherry reporter/enhancer) in either ON bipolar cells or RGCs is expected to lead to optogenetic vision restoration at ambient light levels, as observed in human subjects in recent clinical trials (NCT04945772). Quantification of mCherry expression in humans by clinically approved scanning laser ophthalmoscopy would provide further insight into the correlation between efficacy and cellular expression. 

MCO-010 sensitization of cells in vitro is known to exhibit ambient light–induced photocurrent.³⁷ Electrophysiological measurement provides objective measurements of retinal function. Longitudinal improvement in electrical response from the degenerated retina was observed in MCO-010–injected rd10 mice upon light stimulation but not in the negative control mice (rd1 mice without MCO-010 transfection) using a fast neuro-electrophysiology platform and placing a gold ring electrode in the back of the eye near the retina. The signal had two components, possibly representing two sequential events in the retina. It is conceivable that the first wave originates from the photosensitive bipolar cells and the second from a volley of action potentials from RGC axons. The negative wave is less consistent than the positive wave. Assuming that the early negative wave represents a photochemical event, the generation of a strong signal depends on the polar orientation of generators, which have to be numerous and similarly oriented. Loss of the negative wave (in longitudinal measurements) may represent a polar disorganization of photochemical generators; however, this does not substantially impact the positive wave. We also hypothesize that the biphasic ERG signal at 4 weeks (after MCO-010 injection) has a biological origin: the transformation phase of ON bipolar cells to photoreceptor cells in which the transformed photosensitive bipolar cells try to overcompensate (exhibited by large downward amplitude) the new signaling pathway in the absence of the rods and cones. Over time, these photosensitive bipolar cells normalize to the new state of photo-excitable retina and thus adjust their early receptor potential. However, as can be seen, the biphasic waveform (with a downward negative) signal still exists at 13 weeks in some traces, albeit with lower amplitude. Evaluating the in vivo efficacy of MCO-010 transduction in the retina with regard to restoration of VEP measurements in the control and MCO-010–injected groups provides additional efficacy measures. The comparison of measured visually evoked electrical responses in rd1 and rd10 mice in the negative control groups and intravitreally injected MCO-010 groups showed a significant increase in the electrical activities in V1 upon visual stimulation with light at ambient light levels (∼10¹²–10¹³ photons/cm²/s). 

No detectable cell death in MCO-010–injected rd10 mice was observed, indicating minimal compromise of cell viability of MCO-010–treated rd10 mice under chronic light exposure. Although avoidance of bright light by the MCO-010–treated mice demonstrated light sensitivity of the MCO-010–sensitized retina, it is possible that the mice were not fully exposed to chronic illumination all the time during this test. Furthermore, because the light sensitivity of MCO-010–expressing cells significantly reduces the required light intensity for generating action potentials, the use of MCO-010 minimizes light toxicity. No reduction of a- and b-wave ERG amplitude was observed after MCO-010 intravitreal injection, which suggests that no damage was inflicted to the natural rod and cone photoreceptors in the MCO-010–expressing eyes. The health of animals was not affected in the long-term studies (>6 months of injection), and retinal cell viability was maintained (assayed by apoptotic marker) even after chronic, high-intensity light exposure for 4 months (8 hr/day). Furthermore, immunotoxicity studies on blood plasma collected from MCO-010–injected rd10 mice at different time points showed no significant increase in pro-/anti-inflammatory cytokines as compared to before the injection. No significant change in IOP was observed in injected or contralateral eyes after intravitreal injection of MCO-010 in rd1 or wild-type mice. 

Gene therapy–based treatments such as optogenetics offer a potentially powerful way to bypass damaged photoreceptors in retinal degenerative diseases and utilize the remaining retinal neuronal cells to achieve photosensitivity. MCO-010 optogenetic therapy can photosensitize retinal cells so that, at ambient light levels, the retina can sense light and provide useful vision. Induction of ambient light responsiveness in remaining healthy bipolar cells in subjects with retinal degeneration will allow the retinal circuitry to regain visual acuity without requiring an active stimulation device. Genes transferred via AAV are known to persist mainly in episomal form with little or no evidence of genomic integration,⁴⁷ and, because MCO-010 is intended to transduce adult (postmitotic) retinal cells, stable transgene expression is expected. Thus, MCO-010 gene transduction in the retinas of affected patients is expected to provide long-lasting improvements in their visual function. The clinical trial data obtained thus far in RP patients using MCO-010 appears to support this contention (NCT04919473, NCT04945772). In addition to partial restoration of vision, the observed attenuation of further retinal degeneration after MCO-010 injection, if translated in humans, will lead to disease-modifying therapy that may have lasting benefit. 

Conclusions

In conclusion, the results of the current studies provide evidence that the expression of MCO-010 in the retinas of mice experiencing photoreceptor degeneration appears to slow down or halt further loss and prevent further disorganization of the retinal cell layers. Behavioral restoration of vision in MCO-010–injected groups of mice was observed during the radial-arm water-maze test. Significant improvements in optomotor responses in MCO-010–treated rd10 mice at ambient light levels were also observed. Similar to wild-type mice, longitudinal improvement in electrical responses from degenerated retinas was observed in MCO-010–injected rd10 mice upon light stimulation but not in the negative control mice. In addition, visually evoked electrical responses in rd mice with intravitreally delivered MCO-010 showed a significant increase in the electrical activities in the V1 area of the visual cortex upon visual stimulation with light at ambient light levels. No significant change in IOP was observed in the injected or contralateral eyes after intravitreal injection of different doses of MCO-010 in rd1 or wild-type mice. Also, no reductions of a- and b-wave ERG amplitudes were recorded after MCO-010 intravitreal injection, indicating a lack of damage to the residual natural rod and cone photoreceptors in wild-type mice. Similarly, the IL-6/IL-10 immune-response study on longitudinally collected sera from mice, with intravitreal injection of MCO-010, showed no limiting immunotoxicity. No detectable retinal cell death in MCO-010–injected rd10 mice was observed under chronic light exposure, indicating preservation of retinal cell viability. The collective safety and efficacy data obtained from the current comprehensive studies in animal models of RP support the promising clinical utility of this novel optogenetic therapeutic approach to help RP patients see and function better in their daily life activities. 

Acknowledgments

The authors thank Vittorio Porciatti, DSc (Bascom Palmer Eye Institute) for help with interpretation of the electrical response from retina. The authors also thank Melissa Galicia and Houssam Al-Saad for assistance with the animal care and behavior experiment. 

Supported by grants from the National Institutes of Health (2R44EY025905-02A1, 1R01EY025717-01A1, and 1R01EY028216-01A1). 

Disclosure: S. Batabyal, Nanoscope Therapeutics (I); S. Kim, Nanoscope Therapeutics (I); M. Carlson, Nanoscope Therapeutics (I); D. Narcisse, None; K. Tchedre, Nanoscope Therapeutics (I); A. Dibas, Nanoscope Therapeutics (I); N.A. Sharif, Nanoscope Therapeutics (I); S. Mohanty, Nanoscope Therapeutics (I), Nanoscope Technologies (I)