Optogenetics is a promising approach for restoring vision to the blind after photoreceptor degeneration. The ability to restore vision through AAV-mediated delivery of light-sensitive proteins, especially channelrhodopsins, into retinal ganglion cells has been extensively demonstrated in animal models. For clinical application, knowledge of viral dose-dependent functional efficacy is desired. In this study, using a triple-knockout blind mouse model and a highly light-sensitive channelrhodopsin variant, we evaluated viral dose-dependent vision restoration through retinal ganglion cell expression by using optomotor behavioral assays. Our results show that both the restored light sensitivity and visual acuity reached peak levels at a medial viral dose of 108 vg. With increasing dose, transduction efficiency continued to increase while protein expression peaked at the dose of ~109 vg and declined at higher doses. Also, a significant increase in retinal gliosis and inflammatory responses started at the dose of ~109 vg, and a marked increase was observed at the dose of ~1010. These results provide valuable insights into viral dose design for clinical studies.

Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms.

Purpose: Loss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type–specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors. Methods: Bipolar cell transgene RS1 expression was achieved using two inner retina–specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation. Results: Immunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively. Conclusions: These results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

The loss of photoreceptor cells caused by retinal degenerative diseases leads to blindness. The optogenetic approach for restoring vision involves converting the surviving inner retinal neurons into photosensitive cells, thus imparting light sensitivity to the retina following the loss of photoreceptor cells. Our first demonstration of the feasibility of such an approach involved expressing ChR2 in the retinal ganglion cells of blind mice; since then, optogenetic vision restoration has been demonstrated by using a variety of optogenetic tools, especially microbial channelrhodopsins (ChRs). A ChR-based optogenetic therapy for treating blindness has advanced to clinical trials. In this chapter, we review our early proof-of-concept study of optogenetic vision restoration. We also discuss our studies for developing better ChR tools and for restoring intrinsic visual processing features in retinas with degenerated photoreceptors.

Channelrhodopsin (ChR)-based optogenetics is one promising approach to restore vision in photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Currently, a large number of ChRs from different alga species as well as engineered variants are available. They vary with their light response properties like peak sensitive wavelength (λmax), current amplitude, and kinetics. Therefore, it is important to choose an appropriate ChR for practical applications, such as vision restoration. Here we describe a standard laboratory protocol for characterizing properties of ChRs in in vitro in human embryonic kidney (HEK) cells. Based on such characterization, we also discuss the criteria for selecting optimal ChRs for optogenetic vision restoration.

Background: Adeno-associated Virus (AAV) vectors are the most promising vehicles for therapeutic gene delivery to the retina. To develop a practical gene delivery tool, achieving high AAV transduction efficiency in specific cell types is often required. AAV-mediated targeted expression in retinal bipolar cells is needed in certain applications such as optogenetic therapy, however, the transduction efficiency driven by endogenous cell-specific promoters is usually low. Methods that can improve AAV transduction efficiency in bipolar cells need to be developed. Objective: The study aimed to examine the effect of proteasome inhibitors on AAV-mediated transduction efficiency in retinal bipolar cells. Methods: Quantitative analysis of fluorescent reporter protein expression was performed to assess the effect of two proteasome inhibitors, doxorubicin and MG132, on AAV-mediated transduction efficiency in retinal bipolar cells in mice. Results: Our results showed that doxorubicin can increase the AAV transduction efficiency in retinal bipolar cells in a dose-dependent manner. We also observed doxorubicin-mediated cytotoxicity in retinal neurons, but the cytotoxicity could be mitigated by the coapplication of dexrazoxane. Three months after the coapplication of doxorubicin (300 μM) and dexrazoxane, the AAV transduction efficiency in retinal bipolar cells increased by 33.8% and no cytotoxicity was observed in all the layers of the retina. Conclusion: Doxorubicin could enhance the AAV transduction efficiency in retinal bipolar cells in vivo. The potential long-term cytotoxicity caused by doxorubicin to retinal neurons could be partially mitigated by dexrazoxane. The coapplication of doxorubicin and dexrazoxane may serve as a potential adjuvant regimen for improving AAV transduction efficiency in retinal bipolar cells.

The loss of photoreceptors in individuals with retinal degenerative diseases leads to partial or complete blindness. Optogenetic therapy is a promising approach for restoring vision to the blind. Multiple strategies have been employed by targeting genetically encoded light sensors, particularly channelrhodopsins, to surviving retinal neurons in animal models. In particular, the strategy of targeting retinal bipolar cells has commonly been expected to result in better vision than ubiquitous expression in retinal ganglion cells. However, a direct comparison of the channelrhodopsin-restored vision between these two strategies has not been performed. Here, we compared the restored visual functions achieved by adenoassociated virus (AAV)-mediated expression of a channelrhodopsin in ON-type bipolar cells and retinal ganglion cells driven by an improved mGluR6 promoter and a CAG promoter, respectively, in a blind mouse model by performing electrophysiological recordings and behavioral assessments. Unexpectedly, the efficacy of the restored vision based on light sensitivity and visual acuity was much higher following ubiquitous retinal ganglion cell expression than that of the strategy targeting ON-type bipolar cells. Our study suggests that, at least based on currently available gene delivery techniques, the expression of genetically encoded light sensors in retinal ganglion cells is likely a practical and advantageous strategy for optogenetic vision restoration.

Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6β proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

Purpose: To develop an animal behavioral assay for the quantitative assessment of the functional efficacy of optogenetic therapies. Methods: 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. Results: 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. Conclusions: 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.

The retina is a thin neural tissue sitting on the backside of the eye, composed of light-sensing cells, interneurons, and output ganglion neurons. The latter send electrical signals to higher visual centers in the brain. Transgenic mouse lines are becoming one of the most valuable mammalian animal models for the study of visual signal processing within the retina. Especially, the generation of Cre recombinase transgenic mouse lines provides a powerful tool for genetic manipulation. A key step for the utilization of transgenic lines is the characterization of their transgene expression patterns in the retina. Here we describe a standard protocol for characterizing the expression pattern of the Cre recombinase or fluorescent proteins in the retina with an immunohistochemical approach.

To characterize recombinant AAV2 (rAAV2)-mediated expression of L132C/T159C ChR2 mutant in retinal ganglion cells (RGCs) of young adult cynomolgus monkeys. rAAV2 vectors carrying a fusion construct of the ChR2 mutant and GFP (ChR2-GFP) were delivered to the vitreous chamber by intravitreal injection. Expression patterns of the ChR2 mutant in RGCs were examined by immunohistochemical methods three months after injection. The RNA-binding protein with multiple splicing (RBPMS) was used as an RGC specific marker to differentiate RGCs from other retinal neurons and non-neuronal cells. The numbers of RBPMS+ and GFP+ double-labeled RGCs in the central foveal varied with the eccentricity. The expression peaked within 100 μm from the edge of the foveola and drastically decreased to a single superficial RGC layer approximately 300 μm from the edge. On average, the ratio of the double-labeled RGCs versus RBPMS+ RGCs approached 0.32±0.15 (n=14 fields) at the central foveal region (0.1 to 0.53 mm). We observed that the ratio reached 0.78±0.16 (n=21 fields) at peripheral retinal locations (eccentricity >7 mm). This investigation demonstrates that RBPMS could serve as a valuable RGC specific marker for future investigations in this field.

Purpose: Recombinant adeno-associated virus (rAAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell-type specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in targeted gene expression in retinal bipolar cells, especially for optogenetic gene therapy for vision restoration. However, rAAV-mediated transduction efficiency in retinal bipolar cells is relatively low. The transduction efficiency could be affected by a number of factors, one of which is the proteasome-dependent virus degradation. In this study, we are examining the effect of proteasome inhibitors on the transduction efficiency of rAAV vectors in retinal bipolar cells. Methods: The expression of the transgene, mCherry, was used to evaluate the AAV transduction efficiency. Targeted expression of mCherry in retinal bipolar cells was achieved by rAAV2 vectors carrying an mGluR6 promoter. rAAV vectors at the concentration of 5 × 1012 vg/ml with or without containing proteasome inhibitors were intravitreally injected into the eyes of C57BL/6J mice at about one month of age. Animals were euthanized about one month after virus injection for assessing the expression of mCherry. Results: We tested the effects of three proteasome inhibitors, MG132, doxorubicin, and aclarubicin, on rAAV-mediated transduction efficiency in retinal bipolar cells. Retinas treated with doxorubicin from 200 µM to 800 µM exhibited a concentration-dependent increase in the transduction efficiency. Doxorubicin at the concentration of 2000 µM produced cytotoxicity as evidenced by the thinning of the retinas and decreased the number of mCherry-expressing bipolar cells. The optimal concentration of doxorubicin to enhance the AAV transduction efficiency was 500 µM. MG132 (100 µM, 200 µM, 500 µM) and aclarubicin (50 µM, 100 µM) were not found to enhance the transduction efficiency. Conclusions: Doxorubicin, a proteasome inhibitor, is effective in enhancing rAAV transduction in retinal bipolar cells in mice. Investigation of the mechanism of doxorubicin action may help to further improve the rAAV transduction efficiency in retinal bipolar cells in particular and rAAV-mediated gene delivery in general.

Adeno-associated virus (AAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell type-specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in AAV-mediated gene targeting to specific retinal bipolar cell types. A 200-bp enhancer in combination with a basal SV40 promoter has been commonly used to target transgenes into ON-type bipolar cells. In the current study, we searched for additional cis-regulatory elements in the mGluR6 gene for improving AAV-mediated transduction efficiency into retinal bipolar cells. Our results showed that the combination of the endogenous mGluR6 promoter with additional enhancers in the introns of the mGluR6 gene markedly enhanced AAV transduction efficiency as well as made the targeting more selective for rod bipolar cells in mice. Furthermore, the AAV vectors with the improved promoter could target to ON bipolar cells with robust transduction efficiency in the parafovea and the far peripheral retina of marmoset monkeys. The improved mGluR6 promoter constructs could provide a valuable tool for genetic manipulation in rod bipolar cells in mice and facilitate clinical applications for ON bipolar cell-based gene therapies.