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1.
Commun Biol ; 4(1): 125, 2021 01 27.
Article in English | MEDLINE | ID: mdl-33504896

ABSTRACT

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.


Subject(s)
Optogenetics , Photic Stimulation , Retinal Degeneration/therapy , Vision, Ocular/physiology , Animals , Equipment and Supplies , Female , Humans , Macaca fascicularis , Male , Optogenetics/instrumentation , Optogenetics/methods , Pattern Recognition, Visual/physiology , Photic Stimulation/instrumentation , Photic Stimulation/methods , Primates , Retinal Degeneration/physiopathology , Retinal Degeneration/rehabilitation , Therapies, Investigational/instrumentation , Therapies, Investigational/methods
2.
PLoS Comput Biol ; 16(7): e1007857, 2020 07.
Article in English | MEDLINE | ID: mdl-32667921

ABSTRACT

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.


Subject(s)
Blindness , Optogenetics/methods , Retinal Ganglion Cells/physiology , Animals , Blindness/physiopathology , Blindness/therapy , Genetic Therapy , Macaca , Mice , Models, Biological , Retina/physiology , Visual Acuity/physiology
3.
Nat Commun ; 8(1): 1964, 2017 12 06.
Article in English | MEDLINE | ID: mdl-29213097

ABSTRACT

In the early visual system, cells of the same type perform the same computation in different places of the visual field. How these cells code together a complex visual scene is unclear. A common assumption is that cells of a single-type extract a single-stimulus feature to form a feature map, but this has rarely been observed directly. Using large-scale recordings in the rat retina, we show that a homogeneous population of fast OFF ganglion cells simultaneously encodes two radically different features of a visual scene. Cells close to a moving object code quasilinearly for its position, while distant cells remain largely invariant to the object's position and, instead, respond nonlinearly to changes in the object's speed. We develop a quantitative model that accounts for this effect and identify a disinhibitory circuit that mediates it. Ganglion cells of a single type thus do not code for one, but two features simultaneously. This richer, flexible neural map might also be present in other sensory systems.


Subject(s)
Computer Simulation , Retina/cytology , Retina/physiology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/physiology , Amacrine Cells/physiology , Animals , Female , Male , Models, Theoretical , Motion Perception/physiology , Photic Stimulation/methods , Rats , Visual Fields
4.
Mol Ther ; 25(11): 2546-2560, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28807567

ABSTRACT

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.


Subject(s)
Channelrhodopsins/genetics , Genetic Vectors/administration & dosage , Promoter Regions, Genetic , Recovery of Function , Retinal Degeneration/therapy , Animals , Channelrhodopsins/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Disease Models, Animal , Gene Expression , Genetic Therapy/methods , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Intravitreal Injections , Light , Macaca fascicularis , Mice , Mice, Inbred C57BL , Mice, Transgenic , Optogenetics , Photoreceptor Cells, Vertebrate/metabolism , Photoreceptor Cells, Vertebrate/pathology , Retinal Degeneration/genetics , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Transduction, Genetic , Transgenes , Vision, Ocular/physiology
5.
Invest Ophthalmol Vis Sci ; 58(3): 1768-1778, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28334377

ABSTRACT

Purpose: Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). Using a cCSNB mouse model lacking Lrit3 (nob6), we recently have shown that LRIT3 has a role in the correct localization of TRPM1 (transient receptor potential melastatin 1) to the dendritic tips of ON-bipolar cells (BCs), contacting both rod and cone photoreceptors. Furthermore, postsynaptic clustering of other mGluR6 cascade components is selectively eliminated at the dendritic tips of cone ON-BCs. The purpose of this study was to further define the role of LRIT3 in structural and functional organization of cone synapses. Methods: Exhaustive electroretinogram analysis was performed in a patient with LRIT3 mutations. Multielectrode array recordings were performed at the level of retinal ganglion cells in nob6 mice. Targeting of GluR1 and GluR5 at the dendritic tips of OFF-BCs in nob6 retinas was assessed by immunostaining and confocal microscopy. The ultrastructure of photoreceptor synapses was evaluated by electron microscopy in nob6 mice. Results: The patient with LRIT3 mutations had a selective ON-BC dysfunction with relatively preserved OFF-BC responses. In nob6 mice, complete lack of ON-pathway function with robust, yet altered signaling processing in OFF-pathways was detected. Consistent with these observations, molecules essential for the OFF-BC signaling were normally targeted to the synapse. Finally, synaptic contacts made by ON-BC but not OFF-BC neurons with the cone pedicles were disorganized without ultrastructural alterations in cone terminals, horizontal cell processes, or synaptic ribbons. Conclusions: These results suggest that LRIT3 is likely involved in coordination of the transsynaptic communication between cones and ON-BCs during synapse formation and function.


Subject(s)
DNA/genetics , Eye Diseases, Hereditary/genetics , Genetic Diseases, X-Linked/genetics , Membrane Proteins/genetics , Mutation , Myopia/genetics , Night Blindness/genetics , Retinal Bipolar Cells/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Synapses/ultrastructure , Animals , DNA Mutational Analysis , Dendrites/metabolism , Dendrites/ultrastructure , Electroretinography , Eye Diseases, Hereditary/metabolism , Eye Diseases, Hereditary/pathology , Female , Genetic Diseases, X-Linked/metabolism , Genetic Diseases, X-Linked/pathology , Humans , Immunohistochemistry , Male , Membrane Proteins/metabolism , Mice , Mice, Knockout , Microscopy, Electron , Myopia/metabolism , Myopia/pathology , Night Blindness/metabolism , Night Blindness/pathology , Retinal Bipolar Cells/ultrastructure , Retinal Cone Photoreceptor Cells/ultrastructure , Retrospective Studies , Synapses/metabolism , Synaptic Transmission/genetics , Young Adult
6.
EMBO Mol Med ; 8(11): 1248-1264, 2016 11.
Article in English | MEDLINE | ID: mdl-27679671

ABSTRACT

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.


Subject(s)
Genetic Therapy/methods , Phototherapy/methods , Retina/physiology , Retinal Degeneration/therapy , Rhodopsin/genetics , Animals , Dependovirus/genetics , Genetic Vectors , Humans , Lentivirus/genetics , Light , Macaca , Mice , Rhodopsin/metabolism , Transduction, Genetic , Treatment Outcome
7.
Cell Rep ; 13(5): 990-1002, 2015 Nov 03.
Article in English | MEDLINE | ID: mdl-26565912

ABSTRACT

OTX2 (orthodenticle homeobox 2) haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2(+/GFP) heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2(+/GFP) mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2(+/GFP) mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.


Subject(s)
Mitochondria/drug effects , Otx Transcription Factors/pharmacology , Retinal Bipolar Cells/drug effects , Retinal Dystrophies/drug therapy , Adenosine Triphosphate/metabolism , Animals , Intravitreal Injections , Mice , Mitochondria/metabolism , Otx Transcription Factors/administration & dosage , Otx Transcription Factors/therapeutic use , Retinal Bipolar Cells/metabolism
8.
Invest Ophthalmol Vis Sci ; 56(4): 2639-48, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25814000

ABSTRACT

PURPOSE: Sildenafil (Viagra), a cGMP-specific phosphodiesterase type 5 inhibitor, is widely used for the treatment of erectile dysfunction and pulmonary hypertension. Clinical studies have reported transient visual impairments in patients after single-dose sildenafil use, suggesting neural involvement in several retinal layers, and also, possibly, retinal ganglion cells (RGCs), which provide the unique output of visual information to the brain. However, the effect of sildenafil on the RGC light responses is poorly understood. We therefore evaluated its effect on RGC spiking activity. METHODS: We measured spontaneous and light-induced RGC spiking activity in Long-Evans rat ex vivo retinas by using the multielectrode array technique. Sildenafil citrate (0.3-30 µM) was applied to retinal preparations under continuous perfusion, during 10 to 60 minutes, followed by sildenafil washout. RESULTS: A high concentration (30 µM) of sildenafil decreased the magnitudes of both ON- and OFF-type RGC light responses, to 26.3% ± 17% and 18.3% ± 7%, respectively, of the initial value, in a reversible and concentration-dependent fashion, while in 50% of RGCs all light responses were completely suppressed. Sildenafil also greatly increased the latency of both types of light responses. In this study, we provided evidence that extended exposure to both sildenafil and repeated light stimulation potentiates drug effects and delays recovery. CONCLUSIONS: We found transient and concentration-dependent alterations of light responses at the RGC level after sildenafil exposure that are relevant for a better understanding of the acute visual effects of administration of this compound in humans.


Subject(s)
Piperazines/pharmacology , Retinal Ganglion Cells/drug effects , Sulfonamides/pharmacology , Vision Disorders/drug therapy , Animals , Disease Models, Animal , Phosphodiesterase 5 Inhibitors/pharmacology , Purines/pharmacology , Rats , Rats, Long-Evans , Retinal Ganglion Cells/pathology , Sildenafil Citrate , Vision Disorders/pathology , Vision Disorders/physiopathology
9.
Mol Ther ; 23(1): 7-16, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25095892

ABSTRACT

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.


Subject(s)
Blindness/therapy , Dependovirus/genetics , Genetic Therapy/methods , Retinal Bipolar Cells/metabolism , Retinal Degeneration/therapy , Animals , Behavior, Animal , Blindness/genetics , Blindness/pathology , Channelrhodopsins , Female , Gene Expression , Gene Transfer Techniques , Genetic Engineering , Genetic Vectors , Intravitreal Injections , Light , Mice , Mice, Inbred C57BL , Mice, Transgenic , Promoter Regions, Genetic , Retinal Bipolar Cells/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/pathology , Visual Perception/genetics , Vitreous Body
10.
Mol Cell Neurosci ; 43(4): 414-21, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20132888

ABSTRACT

The anti-epileptic drug vigabatrin induces an irreversible constriction of the visual field, but is still widely used to treat infantile spasms and some forms of epilepsy. We recently reported that vigabatrin-induced cone damage is due to a taurine deficiency. However, optic atrophy and thus retinal ganglion cell degeneration was also reported in children treated for infantile spasms. We here show in neonatal rats treated from postnatal days 4 to 29 that the vigabatrin treatment triggers not only cone photoreceptor damage, disorganisation of the photoreceptor layer and gliosis but also retinal ganglion cell loss. Furthermore, we demonstrate in these neonatal rats that taurine supplementation partially prevents these retinal lesions and in particular the retinal ganglion cell loss. These results provide the first evidence of retinal ganglion cell neuroprotection by taurine. They further confirm that taurine supplementation should be administered with the vigabatrin treatment for infantile spasms or epilepsy.


Subject(s)
Cell Death/drug effects , Optic Atrophy/chemically induced , Photoreceptor Cells/pathology , Retinal Ganglion Cells/pathology , Taurine/deficiency , Vigabatrin/pharmacology , Analysis of Variance , Animals , Animals, Newborn , Anticonvulsants/pharmacology , Cell Count , Electroretinography , Fluorescent Antibody Technique , Neuroprotective Agents/administration & dosage , Optic Atrophy/pathology , Photoreceptor Cells/drug effects , Rats , Rats, Wistar , Retinal Ganglion Cells/drug effects , Taurine/administration & dosage
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