Development of an optogenetic gene sensitive to daylight and its implications in vision restoration.
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ABSTRACT:
SUBMITTER: Watanabe Y
PROVIDER: S-EPMC8516861 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.
| S-EPMC7904736 | biostudies-literature
Project description:The advent of optogenetics has ushered in a new era in neuroscience where spatiotemporal control of neurons is possible through light application. These tools used to study neural circuits can also be used therapeutically to restore vision. In order to recapitulate the broad spectral and light sensitivities along with high temporal sensitivity found in human vision, researchers have identified and developed new optogenetic tools. There are two major kinds of optogenetic effectors employed in vision restoration: ion channels and G-protein coupled receptors (GPCRs). Ion channel based optogenetic therapies require high intensity light that can be unsafe at lower wavelengths, so work has been done to expand and red-shift the excitation spectra of these channels. Light activatable GPCRs are much more sensitive to light than their ion channel counterparts but are slower kinetically in terms of both activation and inactivation. This review article examines the latest optogenetic ion channel and GPCR candidates for vision restoration based on light and temporal sensitivity.
| S-EPMC6160748 | biostudies-other
Project description: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.
| S-EPMC7416966 | biostudies-literature
Project description:Inherited progressive degeneration of photoreceptors such as retinitis pigmentosa (RP) is the most common cause of blindness leading to severe vision impairment affecting ~1 in 5,000 people worldwide. Although the function and morphology of the photoreceptors get disrupted, there is evidence that the inner retinal neurons such as bipolar cells and the retinal ganglion cells are left intact until later stages. Among several innovative therapeutic options aiming to restore vision, optogenetic therapy can bestow light sensitivity to remaining retinal neurons by ectopic expression of light-sensitive proteins. Since the advent of this technique, a diverse class of opsins (microbial and mammalian opsins), chimeric proteins, ligand-gated ion channels, and switchable opsins have been used to study their potential in vision restoration. These proteins differ in their excitation spectra, response kinetics, and signal amplification cascade. Although most of the studies have reported high fidelity of responses in the retina, only a handful of them have achieved functional vision in the visual cortex. This review is a summary of the visuocortical and behavioral responses after optogenetic treatment of the degenerated retina. This clarifies to what extent improved and meaningful vision can be obtained for therapeutic efficacy and continued clinical progress.
| S-EPMC10469018 | biostudies-literature
Project description:Retinal disease is one of the most active areas of gene therapy, with clinical trials ongoing in the United States for five diseases. There are currently no treatments for patients with late-stage disease in which photoreceptors have been lost. Optogenetic gene therapies are in development, but, to date, have suffered from the low light sensitivity of microbial opsins, such as channelrhodopsin and halorhodopsin, and azobenzene-based photoswitches. Several groups have shown that photoreceptive G-protein-coupled receptors (GPCRs) can be expressed heterologously, and photoactivate endogenous Gi/o signaling. We hypothesized such a GPCR could increase sensitivity due to endogenous signal amplification. We targeted vertebrate rhodopsin to retinal ON-bipolar cells of blind rd1 mice and observed restoration of: (i) light responses in retinal explants, (ii) visually-evoked potentials in visual cortex in vivo, and (iii) two forms of visually-guided behavior: innate light avoidance and discrimination of temporal light patterns in the context of fear conditioning. Importantly, both the light responses of the retinal explants and the visually-guided behavior occurred reliably at light levels that were two to three orders of magnitude dimmer than required for channelrhodopsin. Thus, gene therapy with native light-gated GPCRs presents a novel approach to impart light sensitivity for visual restoration in a useful range of illumination.
| S-EPMC4817926 | biostudies-literature
Project description:After revolutionizing neuroscience, optogenetic therapy has entered successfully in clinical trials for restoring vision to blind people with degenerative eye diseases, such as retinitis pigmentosa. These clinical trials still have to evaluate the visual acuity achieved by patients and to determine if it reaches its theoretical limit extrapolated from ex vivo experiments. Different strategies are developed in parallel to reduce required light levels and improve information processing by targeting various cell types. For patients with vision loss due to optic atrophy, as in the case of glaucoma, optogenetic cortical stimulation is hampered by light absorption and scattering by the brain tissue. By contrast, ultrasound waves can diffuse widely through the dura mater and the brain tissue as indicated by ultrasound imaging. Based on our recent results in rodents, we propose the sonogenetic therapy relying on activation of the mechanosensitive channel as a very promising vision restoration strategy with a suitable spatiotemporal resolution. Genomic approaches may thus provide efficient brain machine interfaces for sight restoration.
| S-EPMC8762673 | biostudies-literature
Project description:PurposeRetinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter.MethodsFluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration.ResultsA total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration.ConclusionOur findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies.
| S-EPMC8214074 | biostudies-literature
Project description:Optogenetics is the use of genetically encoded light-activated proteins to manipulate cells in a minimally invasive way using light. The most prominent example is channelrhodopsin-2 (ChR2), which allows the activation of electrically excitable cells via light-dependent depolarization. The combination of ChR2 with hyperpolarizing-light-driven ion pumps such as the Cl(-) pump halorhodopsin (NpHR) enables multimodal remote control of neuronal cells in culture, tissue, and living animals. Very soon, it became obvious that this method offers a chance of gene therapy for many diseases affecting vision. Here, we will give a brief introduction to retinal function and retinal diseases; optogenetic vision restoration strategies will be highlighted. We will discuss the functional and structural properties of rhodopsin-based optogenetic tools and analyze the potential for the application of vision restoration.
| S-EPMC5009148 | biostudies-literature
Project description: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.
| S-EPMC5675708 | biostudies-literature
Project description:To develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies.A triple-knockout (TKO) mouse line, Gnat1-/-Cnga3-/-Opn4-/-, and a double-knockout mouse line, Gnat1-/-Cnga3-/-, were employed. The expression of channelrhodopsin-2 (ChR2) and its three more light-sensitive mutants, ChR2-L132C, ChR2-L132C/T159C, and ChR2-132C/T159S, in inner retinal neurons was achieved using rAAV2 vectors via intravitreal delivery. Pupillary constriction was assessed by measuring the pupil diameter. The optomotor response (OMR) was examined using a homemade optomotor system equipped with light-emitting diodes as light stimulation.A robust OMR was restored in the ChR2-mutant-expressing TKO mice; however, significant pupillary constriction was observed only for the ChR2-L132C/T159S mutant. The ability to evoke an OMR was dependent on both the light intensity and grating frequency. The most light-sensitive frequency for the three ChR2 mutants was approximately 0.042 cycles per degree. Among the three ChR2 mutants, ChR2-L132C/T159S was the most light sensitive, followed by ChR2-L132C/T159C and ChR2-L132C. Melanopsin-mediated pupillary constriction resulted in a substantial reduction in the light sensitivity of the ChR2-mediated OMR.The OMR assay using TKO mice enabled the quantitative assessment of the efficacy of different optogenetic tools and the properties of optogenetically restored vision. Thus, the assay can serve as a valuable tool for developing effective optogenetic therapies.
| S-EPMC5839255 | biostudies-literature