In vivo optogenetic stimulation of the primate retina activates the visual cortex after long-term transduction.

Over the last 15 years, optogenetics has changed fundamental research in neuroscience and is now reaching toward therapeutic applications. Vision restoration strategies using optogenetics are now at the forefront of these new clinical opportunities. But applications to human patients suffering from retinal diseases leading to blindness raise important concerns on the long-term functional expression of optogenes and the efficient signal transmission to higher visual centers. Here, we demonstrate in non-human primates continued expression and functionality at the retina level ∼20 months after delivery of our construct. We also performed in vivo recordings of visually evoked potentials in the primary visual cortex of anesthetized animals. Using synaptic blockers, we isolated the in vivo cortical activation resulting from the direct optogenetic stimulation of primate retina. In conclusion, our work indicates long-term transgene expression and transmission of the signal generated in the macaque retina to the visual cortex, two important features for future clinical applications.

Partial recovery of visual function in a blind patient after optogenetic therapy.

Optogenetics may enable mutation-independent, circuit-specific restoration of neuronal function in neurological diseases. Retinitis pigmentosa is a neurodegenerative eye disease where loss of photoreceptors can lead to complete blindness. In a blind patient, we combined intraocular injection of an adeno-associated viral vector encoding ChrimsonR with light stimulation via engineered goggles. The goggles detect local changes in light intensity and project corresponding light pulses onto the retina in real time to activate optogenetically transduced retinal ganglion cells. The patient perceived, located, counted and touched different objects using the vector-treated eye alone while wearing the goggles. During visual perception, multichannel electroencephalographic recordings revealed object-related activity above the visual cortex. The patient could not visually detect any objects before injection with or without the goggles or after injection without the goggles. This is the first reported case of partial functional recovery in a neurodegenerative disease after optogenetic therapy.

Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy.

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.

Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 1015 photons.cm-2.s-1. Vector doses of 5 × 1010 and 5 × 1011 vg/eye transfected up to 7000 RGCs/mm2 in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.

Generation of a Transplantable Population of Human iPSC-Derived Retinal Ganglion Cells.

Optic neuropathies are a major cause of visual impairment due to retinal ganglion cell (RGC) degeneration. Human induced-pluripotent stem cells (iPSCs) represent a powerful tool for studying both human RGC development and RGC-related pathological mechanisms. Because RGC loss can be massive before the diagnosis of visual impairment, cell replacement is one of the most encouraging strategies. The present work describes the generation of functional RGCs from iPSCs based on innovative 3D/2D stepwise differentiation protocol. We demonstrate that targeting the cell surface marker THY1 is an effective strategy to select transplantable RGCs. By generating a fluorescent GFP reporter iPSC line to follow transplanted cells, we provide evidence that THY1-positive RGCs injected into the vitreous of mice with optic neuropathy can survive up to 1 month, intermingled with the host RGC layer. These data support the usefulness of iPSC-derived RGC exploration as a potential future therapeutic strategy for optic nerve regeneration.

cAMP-Dependent Co-stabilization of Axonal Arbors from Adjacent Developing Neurons.

Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.

Towards optogenetic vision restoration with high resolution.

In many cases of inherited retinal degenerations, ganglion cells are spared despite photoreceptor cell death, making it possible to stimulate them to restore visual function. Several studies have shown that it is possible to express an optogenetic protein in ganglion cells and make them light sensitive, a promising strategy to restore vision. However the spatial resolution of optogenetically-reactivated retinas has rarely been measured, especially in the primate. Since the optogenetic protein is also expressed in axons, it is unclear if these neurons will only be sensitive to the stimulation of a small region covering their somas and dendrites, or if they will also respond to any stimulation overlapping with their axon, dramatically impairing spatial resolution. Here we recorded responses of mouse and macaque retinas to random checkerboard patterns following an in vivo optogenetic therapy. We show that optogenetically activated ganglion cells are each sensitive to a small region of visual space. A simple model based on this small receptive field predicted accurately their responses to complex stimuli. From this model, we simulated how the entire population of light sensitive ganglion cells would respond to letters of different sizes. We then estimated the maximal acuity expected by a patient, assuming it could make an optimal use of the information delivered by this reactivated retina. The obtained acuity is above the limit of legal blindness. Our model also makes interesting predictions on how acuity might vary upon changing the therapeutic strategy, assuming an optimal use of the information present in the retinal activity. Optogenetic therapy could thus potentially lead to high resolution vision, under conditions that our model helps to determinine.

AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.

Opsins for vision restoration.

Optogenetics is a biological technique that combines the advantageous spatial-temporal resolution of optics and genetic cell targeting to control cellular activity with unprecedented precision. It has found vast applications both in neurosciences and therapy, particularly in view of its application to restore vision in blind patients. Optogenetics requires the ectopic expression of a so-called opsin to render neurons sensitive to light. There are two types of opsins for modulating membrane potential of neurons: (i) microbial opsins from unicellular organisms that respond to a light stimulus by mediating a flow of ions across the membrane (ii) animal opsins that are naturally present in mammalian retinas that initiate G protein coupled signaling in response to light. The former category has been extensively employed for vision restoration in the past decade with two ongoing clinical trials employing microbial opsins to restore light sensation in retinitis pigmentosa patients. The latter subtype of animal opsins is emerging more recently as strong candidates to restore vision with the promise of greater light sensitivity and tolerability. In this review we will discuss each approach in view of its utility for vision restoration in retinal blindness.

Restoration of visual function by transplantation of optogenetically engineered photoreceptors.

A major challenge in the treatment of retinal degenerative diseases, with the transplantation of replacement photoreceptors, is the difficulty in inducing the grafted cells to grow and maintain light sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). Here, for an RPE-independent treatment approach, we introduce a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplant them into blind mice lacking the photoreceptor layer. These optogenetically-transformed photoreceptors are light responsive and their transplantation leads to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generate cone photoreceptors from human induced pluripotent stem cells, expressing the chloride pump Jaws. After transplantation into blind mice, we observe light-driven responses at the photoreceptor and ganglion cell levels. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics.

Optogenetic Light Sensors in Human Retinal Organoids.

Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.

Characterization and Transplantation of CD73-Positive Photoreceptors Isolated from Human iPSC-Derived Retinal Organoids.

Photoreceptor degenerative diseases are a major cause of blindness for which cell replacement is one of the most encouraging strategies. For stem cell-based therapy using human induced pluripotent stem cells (hiPSCs), it is crucial to obtain a homogenous photoreceptor cell population. We confirmed that the cell surface antigen CD73 is exclusively expressed in hiPSC-derived photoreceptors by generating a fluorescent cone rod homeobox (Crx) reporter hiPSC line using CRISPR/Cas9 genome editing. We demonstrated that CD73 targeting by magnetic-activated cell sorting (MACS) is an effective strategy to separate a safe population of transplantable photoreceptors. CD73+ photoreceptor precursors can be isolated in large numbers and transplanted into rat eyes, showing capacity to survive and mature in close proximity to host inner retina of a model of photoreceptor degeneration. These data demonstrate that CD73+ photoreceptor precursors hold great promise for a future safe clinical translation.

Mini-Review: Cell Type-Specific Optogenetic Vision Restoration Approaches.

The expression of light-sensitive microbial opsins is a promising mutation-independent approach to restore vision in retinal degenerative diseases. Using viral vectors, optogenetic tools can be genetically expressed in various subpopulations of retinal neurons. The choice of cell type depends on the availability of surviving retinal cells. If cones are still alive but they lack outer segments, they can be targeted with optogenetic inhibitors, such as halorhodopsin. Alternatively, it is possible to bypass the photoreceptors and to target bipolar cells. In late-stage degeneration, when bipolar cells degenerate, "artificial photoreceptors" can be made from retinal ganglion cells, but with this approach, upstream retinal processing cannot be utilized. However, when ganglion cells are stimulated directly, higher brain regions might be able to compensate for some loss of retinal processing, which is indicated by clinical studies with epiretinal implants, where patients can perform simple visual tasks. Finally, optogenetics in combination with neuroprotective approaches could serve as a valuable strategy to restore the function of remaining cells, as well as to rescue retinal neurons from progressive degeneration.

A spike sorting toolbox for up to thousands of electrodes validated with ground truth recordings in vitro and in vivo.

In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here, we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain 'ground truth' data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal-to-noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes.

Noninvasive gene delivery to foveal cones for vision restoration.

Intraocular injection of adeno-associated viral (AAV) vectors has been an evident route for delivering gene drugs into the retina. However, gaps in our understanding of AAV transduction patterns within the anatomically unique environments of the subretinal and intravitreal space of the primate eye impeded the establishment of noninvasive and efficient gene delivery to foveal cones in the clinic. Here, we establish new vector-promoter combinations to overcome the limitations associated with AAV-mediated cone transduction in the fovea with supporting studies in mouse models, human induced pluripotent stem cell-derived organoids, postmortem human retinal explants, and living macaques. We show that an AAV9 variant provides efficient foveal cone transduction when injected into the subretinal space several millimeters away from the fovea, without detaching this delicate region. An engineered AAV2 variant provides gene delivery to foveal cones with a well-tolerated dose administered intravitreally. Both delivery modalities rely on a cone-specific promoter and result in high-level transgene expression compatible with optogenetic vision restoration. The model systems described here provide insight into the behavior of AAV vectors across species to obtain safety and efficacy needed for gene therapy in neurodegenerative disorders.

A New Promoter Allows Optogenetic Vision Restoration with Enhanced Sensitivity in Macaque Retina.

The majority of inherited retinal degenerations converge on the phenotype of photoreceptor cell death. Second- and third-order neurons are spared in these diseases, making it possible to restore retinal light responses using optogenetics. Viral expression of channelrhodopsin in the third-order neurons under ubiquitous promoters was previously shown to restore visual function, albeit at light intensities above illumination safety thresholds. Here, we report (to our knowledge, for the first time) activation of macaque retinas, up to 6 months post-injection, using channelrhodopsin-Ca2+-permeable channelrhodopsin (CatCh) at safe light intensities. High-level CatCh expression was achieved due to a new promoter based on the regulatory region of the gamma-synuclein gene (SNCG) allowing strong expression in ganglion cells across species. Our promoter, in combination with clinically proven adeno-associated virus 2 (AAV2), provides CatCh expression in peri-foveolar ganglion cells responding robustly to light under the illumination safety thresholds for the human eye. On the contrary, the threshold of activation and the proportion of unresponsive cells were much higher when a ubiquitous promoter (cytomegalovirus [CMV]) was used to express CatCh. The results of our study suggest that the inclusion of optimized promoters is key in the path to clinical translation of optogenetics.

Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73+ photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73+ photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188.

Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina.

Targeting the photosensitive ion channel channelrhodopsin-2 (ChR2) to the retinal circuitry downstream of photoreceptors holds promise in treating vision loss caused by retinal degeneration. However, the high intensity of blue light necessary to activate channelrhodopsin-2 exceeds the safety threshold of retinal illumination because of its strong potential to induce photochemical damage. In contrast, the damage potential of red-shifted light is vastly lower than that of blue light. Here, we show that a red-shifted channelrhodopsin (ReaChR), delivered by AAV injections in blind rd1 mice, enables restoration of light responses at the retinal, cortical, and behavioral levels, using orange light at intensities below the safety threshold for the human retina. We further show that postmortem macaque retinae infected with AAV-ReaChR can respond with spike trains to orange light at safe intensities. Finally, to directly address the question of translatability to human subjects, we demonstrate for the first time, AAV- and lentivirus-mediated optogenetic spike responses in ganglion cells of the postmortem human retina.

The Thioredoxin Encoded by the Rod-Derived Cone Viability Factor Gene Protects Cone Photoreceptors Against Oxidative Stress.

AIMS: Rod-derived cone viability factor long (RdCVFL) is an enzymatically active thioredoxin encoded by the nucleoredoxin-like-1 (Nxnl1) gene. The second product of the gene, RdCVF, made by alternative splicing is a novel trophic factor secreted by rods that protects cones in rodent models of retinitis pigmentosa, the most prevalent inherited retinal disease. It acts on cones by stimulating aerobic glycolysis through its interaction with a complex containing basigin-1 and the glucose transporter GLUT1. We studied the role of Nxnl1 in cones after its homologous recombination using a transgenic line expressing Cre recombinase under the control of a cone opsin promoter. RESULTS: We show that the cones of these mice are dysfunctional and degenerate by 8 months of age. The age-related deficit in cones is exacerbated in young animals by exposure to high level of oxygen. In agreement with this phenotype, we found that the cones express only one of the two Nxnl1 gene products, the thioredoxin RdCVFL. Administration of RdCVFL to the mouse carrying a deletion of the Nxnl1 gene in cones reduces the damage produced by oxidative stress. Silencing the expression of RdCVFL in cone-enriched culture reduces cell viability, showing that RdCVFL is a cell-autonomous mechanism of protection. INNOVATION: This novel mode of action is certainly relevant for the therapy of retinitis pigmentosa since the delivery into cones of the rd10 mouse, a recessive model of the disease, rescues cones. CONCLUSION: Our work highlights the duality of the Nxnl1 gene, which protects the cones by two distinct mechanisms. Antioxid. Redox Signal. 24, 909-923.

Optogenetics.

PURPOSE OF REVIEW: In this review, we will discuss the recent developments in optogenetics and their potential applications in ophthalmology to restore vision in retinal degenerative diseases. RECENT FINDINGS: In recent years, we have seen major advances in the field of optogenetics, providing us with novel opsins for potential applications in the retina. Microbial opsins with improved light sensitivity and red-shifted action spectra allow optogenetic stimulation at light levels well below the safety threshold in the human eye. In parallel, remarkable success in the development of highly efficient viral vectors for ocular gene therapy led to new strategies of using these novel optogenetic tools for vision restoration. SUMMARY: These recent findings show that novel optogenetic tools and viral vectors for ocular gene delivery are now available providing many opportunities to develop potential optogenetic strategies for vision restoration.