Integrated multi-omics single cell atlas of the human retina.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

Secretogranin III Selectively Promotes Vascular Leakage in the Deep Vascular Plexus of Diabetic Retinopathy.

Diabetic retinopathy (DR), a leading cause of vision loss in working-age adults, induces mosaic patterns of vasculopathy that may be associated with spatial heterogeneity of intraretinal endothelial cells. We recently reported that secretogranin III (Scg3), a neuron-derived angiogenic and vascular leakage factor, selectively binds retinal vessels of diabetic but not healthy mice. Here, we investigated endothelial heterogeneity of three retinal vascular plexuses in DR pathogenesis and the therapeutic implications. Our unique in vivo ligand binding assay detected a 22.7-fold increase in Scg3 binding to retinal vessels of diabetic mice relative to healthy mice. Functional immunohistochemistry revealed that Scg3 predominantly binds to the DR-stressed CD31- deep retinal vascular plexus but not to the relatively healthy CD31+ superficial and intermediate plexuses within the same diabetic retina. In contrast, VEGF bound to healthy and diabetic retinal vessels indiscriminately with low activity. FITC-dextran assays indicated that selectively increased retinal vascular leakage coincides with Scg3 binding in diabetic mice that was independent of VEGF, whereas VEGF-induced leakage did not distinguish between diabetic and healthy mice. Dose-response curves showed that the anti-Scg3 humanized antibody (hAb) and anti-VEGF aflibercept alleviated DR leakage with equivalent efficacies, and that the combination acted synergistically. These findings suggest: (i) the deep plexus is highly sensitive to DR; (ii) Scg3 binding to the DR deep plexus coincides with the loss of CD31 and compromised endothelial junctions; (iii) anti-Scg3 hAb alleviates vascular leakage by selectively targeting the DR-stressed deep plexus within the same diabetic retina; (iv) combined anti-Scg3 and anti-VEGF treatments synergistically ameliorate DR through distinct mechanisms.

Retinal organoids derived from rhesus macaque iPSCs undergo accelerated differentiation compared to human stem cells.

PURPOSE: To compare the timing and efficiency of the development of Macaca mulatta, a nonhuman primate (NHP), induced pluripotent stem cell (rhiPSC) derived retinal organoids to those derived from human embryonic stem cells (hESCs). RESULTS: Generation of retinal organoids was achieved from both human and several NHP pluripotent stem cell lines. All rhiPSC lines resulted in retinal differentiation with the formation of optic vesicle-like structures similar to what has been observed in hESC retinal organoids. NHP retinal organoids had laminated structure and were composed of mature retinal cell types including cone and rod photoreceptors. Single-cell RNA sequencing was conducted at two time points; this allowed identification of cell types and developmental trajectory characterization of the developing organoids. Important differences between rhesus and human cells were measured regarding the timing and efficiency of retinal organoid differentiation. While the culture of NHP-derived iPSCs is relatively difficult compared to that of human stem cells, the generation of retinal organoids from NHP iPSCs is feasible and may be less time-consuming due to an intrinsically faster timing of retinal differentiation. CONCLUSIONS: Retinal organoids produced from rhesus monkey iPSCs using established protocols differentiate through the stages of organoid development faster than those derived from human stem cells. The production of NHP retinal organoids may be advantageous to reduce experimental time for basic biology studies in retinogenesis as well as for preclinical trials in NHPs studying retinal allograft transplantation.

Age-related changes in the rhesus macaque eye.

PURPOSE: To assess age-related changes in the rhesus macaque eye and evaluate them to corresponding human age-related eye disease. METHODS: Data from eye exams and imaging tests including intraocular pressure (IOP), lens thickness, axial length, and retinal optical coherence tomography (OCT) images were evaluated from 142 individuals and statistically analyzed for age-related changes. Quantitative autofluorescence (qAF) was measured as was the presence of macular lesions as related to age. RESULTS: Ages of the 142 rhesus macaques ranged from 0.7 to 29 years (mean = 16.4 years, stdev = 7.5 years). Anterior segment measurements such as IOP, lens thickness, and axial length were acquired. Advanced retinal imaging in the form of optical coherence tomography and qAF were obtained. Quantitative assessments were made and variations by age groups were analyzed to compare with established age-related changes in human eyes. Quantitative analysis of data revealed age-related increase in intraocular pressure (0.165 mm Hg per increase in year of age), ocular biometry (lens thickness 7.2 µm per increase in year of age; and axial length 52.8 µm per increase in year of age), and presence of macular lesions. Age-related changes in thicknesses of retinal layers on OCT were observed and quantified, showing decreased thickness of the retinal ganglion cell layer and inner nuclear layer, and increased thickness of photoreceptor outer segment and choroidal layers. Age was correlated with increased qAF by 1.021 autofluorescence units per increase in year of age. CONCLUSIONS: The rhesus macaque has age-related ocular changes similar to humans. IOP increases with age while retinal ganglion cell layer thickness decreases. Macular lesions develop in some aged animals. Our findings support the concept that rhesus macaques may be useful for the study of important age-related diseases such as glaucoma, macular diseases, and cone disorders, and for development of therapies for these diseases.

Advanced Retinal Imaging and Ocular Parameters of the Rhesus Macaque Eye.

Purpose: To determine the range of normal ocular biometry and perform advanced retinal imaging and functional assessment of the rhesus macaque eye. Methods: We performed ocular phenotyping on rhesus macaques at the California National Primate Research Center. This process consisted of anterior and posterior segment eye examination by ophthalmologists, advanced retinal imaging, and functional retinal electrophysiology. Results: Full eye examinations were performed on 142 animals, consisting of pupillary light reflex, tonometry, external examination and photography, anterior slit lamp examination, and posterior segment examination by indirect ophthalmoscopy. Ages of the rhesus macaques ranged from 0.7 to 29 years (mean, 16.4 ± 7.5 years). Anterior segment measurements such as intraocular pressure (n = 142), corneal thickness (n = 84), lens thickness (n = 114), and axial length (n = 114) were acquired. Advanced retinal imaging in the form of fundus photography (n = 78), optical coherence tomography (n = 60), and quantitative autofluorescence (n = 44) was obtained. Electroretinography (n = 75) was used to assay retinal function. Quantitative analyses of the macular structure, retinal layer segmentation, and rod and cone photoreceptor electrical responses are reported. Quantitative assessments were made and variations between sexes were analyzed to compare with established sex changes in human eyes. Conclusions: The rhesus macaque has an ocular structure and function very similar to that of the human eye. In particular macular structure and retinal function is very similar to humans, making this species particularly useful for the study of macular biology and development of therapies for cone photoreceptor disorders. Translational Relevance: Rhesus macaques are an ideal model for future vision science studies of human eye diseases.

A nonhuman primate model of inherited retinal disease.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

Antibiotics and antifungals in silicone oil.

BACKGROUND: Antimicrobials may be injected into silicone oil-filled eyes with endophthalmitis, but the interaction with oil is unclear. The purpose of the experiment is to determine whether vancomycin, amikacin, and amphotericin B mix with silicone oil. METHODS: Using the relative proportions of the human eye, 4 ml of 1000 centistokes silicone oil was centrifuged with 0.1 ml of vancomycin 1 mg/0.1 ml, amikacin 0.4 mg/0.1 ml, or amphotericin B 5 µg/0.1 ml in vitro and eluted. The aqueous was immediately analyzed with a liquid chromatographer/mass spectrometer and after 24 h. RESULTS: Within 24 h, a mean of 26.9 µmol/L of vancomycin, 0 nmol/L of amikacin, and 0 nmol/L of amphotericin B were recovered. When the concentrations of amikacin and amphotericin B were increased 100-fold, 0 nmol/L of amikacin and 75.7 µmol/L of amphotericin B were recovered. CONCLUSIONS: Vancomycin and amphotericin B partially mixed with the silicone oil. Amikacin was not recovered from the antibiotic-silicone oil mixture.

Intracameral moxifloxacin for endophthalmitis prophylaxis after cataract surgery: Cost-effectiveness analysis.

PURPOSE: To determine the cost-effectiveness of intracameral moxifloxacin compared with traditional antibiotic prophylaxis in preventing endophthalmitis after cataract surgery. SETTING: Theoretical surgical center in the United States. DESIGN: Evaluation of technology. METHODS: The incremental cost-effectiveness ratios (ICER) and incremental cost-utility ratios (ICUR) were calculated for patients having cataract surgery with traditional antibiotic prophylaxis (perioperative topical antibiotics) compared with perioperative topical antibiotics with intracameral moxifloxacin. The base case was a healthy binocular 73-year-old man having first-eye cataract surgery. The incidences and costs were derived from PubMed English literature searches, Medicare reimbursement rates, and average wholesale prices. All costs and benefits were adjusted 3% per annum and for inflation to 2017 United States dollars. Deterministic and probabilistic sensitivity analyses were performed to assess uncertainty. RESULTS: Compared with traditional prophylaxis, an adjuvant 500 µg intracameral moxifloxacin (for $20) was cost-saving from a societal perspective in the base case; in probabilistic sensitivity analyses, all the values were within the societal willingness-to-pay threshold of $50 000/quality-adjusted-life-years (QALYs), and 6142 (61%) of 10 000 iterations were cost-saving. From a healthcare sector perspective, intracameral moxifloxacin was cost-effective, with an ICUR of $8275/QALY. In cases with posterior capsule tears, a $20 intracameral moxifloxacin was cost-saving. CONCLUSIONS: From a societal perspective in the U.S., a topical perioperative antibiotic with a 500 µg intracameral moxifloxacin costing $22 dollars or less was cost-effective and cost-saving. From a healthcare sector perspective, a $20 intracameral moxifloxacin was cost-effective but not cost-saving. Adjuvant intracameral moxifloxacin had greater effectiveness in improving QALYs than topical antibiotics.

Results at 5 Years After Gene Therapy for RPE65-Deficient Retinal Dystrophy.

Previously, results at 2 years after subretinal injection of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in eight adults and four children with retinal degeneration caused by RPE65 mutations were reported. Now, results at 5 years after treatment in 11 of these subjects are reported. Subjects received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of two dose levels, and were followed for 5 years after treatment. The primary safety outcomes were ocular and non-ocular adverse events. Efficacy outcomes included changes in best corrected visual acuity, static perimetry hill of vision measurements for the central 30° (V30), and total (VTOT) visual field and kinetic perimetry visual field area. The only adverse events reported during years 3, 4, and 5 were minor intercurrent illnesses. Pediatric subjects had improvement in visual acuity and static perimetry in the treated eye, sometimes with a smaller improvement in the untreated eye, during the first 2 years of the study that persisted during years 3-5, with no consistent changes in kinetic perimetry during the study. Most adult subjects had no consistent changes in visual acuity or static perimetry during the study. Three adult subjects with markedly abnormal baseline kinetic visual field area had improvement in the treated eye during the first 1-2 years after treatment, but the absolute magnitude of the improvement was small and was not sustained at subsequent visits. There were no clinically significant adverse events. Visual acuity and static perimetry testing results suggest that treating patients at a younger age is associated with better visual function outcomes during 5 years after treatment.

Safety and Efficacy of OXB-202, a Genetically Engineered Tissue Therapy for the Prevention of Rejection in High-Risk Corneal Transplant Patients.

Due to both the avascularity of the cornea and the relatively immune-privileged status of the eye, corneal transplantation is one of the most successful clinical transplant procedures. However, in high-risk patients, which account for >20% of the 180,000 transplants carried out worldwide each year, the rejection rate is high due to vascularization of the recipient cornea. The main reason for graft failure is irreversible immunological rejection, and it is therefore unsurprising that neovascularization (NV; both pre and post grafting) is a significant risk factor for subsequent graft failure. NV is thus an attractive target to prevent corneal graft rejection. OXB-202 (previously known as EncorStat®) is a donor cornea modified prior to transplant by ex vivo genetic modification with genes encoding secretable forms of the angiostatic human proteins, endostatin and angiostatin. This is achieved using a lentiviral vector derived from the equine infectious anemia virus called pONYK1EiA, which subsequently prevents rejection by suppressing NV. Previously, it has been shown that rabbit donor corneas treated with pONYK1EiA substantially suppress corneal NV, opacity, and subsequent rejection in an aggressive rabbit model of cornea graft rejection. Here, efficacy data are presented in a second rabbit model, which more closely mirrors the clinical setting for high-risk corneal transplant patients, and safety data from a 3-month good laboratory practice toxicology and biodistribution study of pONYK1EiA-modified rabbit corneas in a rabbit corneal transplant model. It is shown that pONYK1EiA-modified rabbit corneas (OXB-202) significantly reduce corneal NV and the rate of corneal rejection in a dose-dependent fashion, and are tolerated with no adverse toxicological findings or significant biodistribution up to 13 weeks post surgery in these rabbit studies. In conclusion, angiogenesis is a valid target to prevent corneal graft rejection in a high-risk setting, and transplanted genetically modified corneas are safe and well-tolerated in an animal model. These data support the evaluation of OXB-202 in a first-in-human trial.

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy.

PURPOSE: To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. DESIGN: Nonrandomized, multicenter clinical trial. PARTICIPANTS: Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). METHODS: Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. MAIN OUTCOME MEASURES: The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. RESULTS: All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. CONCLUSIONS: Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns.

Update on ocular gene therapy and advances in treatment of inherited retinal diseases and exudative macular degeneration.

PURPOSE OF REVIEW: The purpose of this article is to provide an update on ocular gene therapy and discuss current active clinical trials. RECENT FINDINGS: The main target for ocular gene therapy involves the retinal pigment epithelium or photoreceptors. The most common method to deliver viral vectors to these cells includes intravitreal injection, subretinal injection, or access from the suprachoroidal space. Recombinant adeno-associated virus and lentivirus can be engineered to maximize gene delivery to specific targets. There are several clinical trials currently aimed at treating inherited and retinal diseases with gene therapy via viral vectors. SUMMARY: Recent advances in gene therapy have allowed for a better understanding of inherited and proliferative retinal diseases. New techniques have been developed to improve delivery of viral vectors to their cellular targets. There are currently multiple active clinical trials involving gene therapy underway with promising preliminary results.

Ocular Gene Therapy.

Ocular gene therapy involves the introduction of an exogenous gene product to a host's cellular and genetic machinery for endogenous production of a desired gene product. The eye represents an ideal target organ due to its easy visibility and accessibility, and several trials have demonstrated proof-of-principle safety and efficacy in a subtype of Leber's congenital amaurosis. There are numerous ongoing clinical trials exploring gene therapy in other retinal diseases. In autosomal recessively inherited retinal degenerations, the introduced gene product replaces a known genetically deficient gene product and provides restoration of function. In other disease states, such as neovascular age-related macular degeneration, the delivered gene product modulates existing proteins within a cell, such as vascular endothelial growth factor, for a desired therapeutic effect. This latter approach may have broader applications in other diseases such as diabetes and other retinal vascular diseases that are as yet unrealized. This review summarizes the current state of clinical research in ocular gene therapy focusing on those diseases in which the technology has reached clinical trials.

Bilateral giant retinal tears in a pediatric patient with leukemia inhibitory factor receptor deficiency (Stuve-Wiedemann syndrome).

PURPOSE: To describe a case of retinal detachment in a patient with Stuve-Wiedemann syndrome. METHODS: This report is a retrospective observational case report. The patient's demographics include age, gender, and race, as well as visual acuity, ophthalmic examination, and surgical intervention were extracted from the medical record. For immunohistochemistry studies, a sample of normal human retina from an enucleated specimen was obtained from the Pathology laboratory. A leukemia inhibitory factor receptor/CD118 antibody was obtained from Santa Cruz Biotechnology. RESULTS: A 13-year-old Hispanic boy with known history of Stuve-Wiedemann syndrome (confirmed by genetic testing) presented with bilateral rhegmatogenous retinal detachments secondary to bilateral giant retinal tears. He underwent multiple surgical repairs in both eyes, resulting in successful reattachment in the right eye and an intractable closed funnel detachment in the left eye. CONCLUSION: This is the first case of vitreoretinal pathology reported in Stuve-Wiedemann syndrome. Using immunohistochemistry staining, the authors found ubiquitous expression of leukemia inhibitory factor receptor protein in the normal human retina. They hypothesize that leukemia inhibitory factor receptor mutation may cause intrinsic weakness of the neurosensory retina predisposing it to injury.

BEVACIZUMAB LEVELS IN BREAST MILK AFTER LONG-TERM INTRAVITREAL INJECTIONS.

PURPOSE: The purpose of this study is to determine whether bevacizumab is detectable in the breast milk of nursing mothers. METHODS: Breast milk samples were collected from 2 patients receiving monthly intravitreal bevacizumab injections for choroidal neovascularization over the course of 16 months. Enzyme-linked immunosorbent assay and Western blot analysis was used to determine the levels of bevacizumab in the milk samples. RESULTS: An enzyme-linked immunosorbent assay was developed using antibodies specific to bevacizumab in which the sensitivity threshold was 3 ng/mL. All breast milk samples assayed from the two patients actively undergoing treatment did not have detectable levels of bevacizumab. Samples collected 1.5 hours and 7 hours after an injection and 2 randomly chosen samples were negative by Western blot analysis. CONCLUSION: A sensitive assay to detect bevacizumab in breast milk samples assayed suggests that intravitreal injections do not result in detectable bevacizumab in breast milk.

Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina.

PURPOSE: Simultaneous dual labeling to visualize specific RNA and protein content within the same formalin-fixed paraffin embedded (FFPE) section can be technically challenging and usually impossible, because of variables such as tissue fixation time and pretreatment methods to access the target RNA or protein. Within a specific experiment, ocular tissue sections can be a precious commodity. Thus, the ability to easily and consistently detect and localize cell-specific expression of RNA and protein within a single slide would be advantageous. In this study, we describe a simplified and reliable method for combined in situ hybridization (ISH) and immunohistochemistry (IHC) for detection of mRNA and protein, respectively, within the same FFPE ocular tissue. METHODS: Whole mouse eyes were prepared for 5 micron FFPE sections after fixation for 3, 24, 48 or 72 h. Customized probes from Advanced Cell Diagnostics to detect mRNA for vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (HIF-1α), and hypoxia-inducible factor 2-alpha (HIF-2α) were used for ISH. Various parameters were tested using the novel RNAscope method for ISH and optimized for compatibility with subsequent IHC for glial fibrillary acidic protein (GFAP) or GS-lectin within the same tissue section. Dual fluorescent visualization of Fast Red ISH and Alexa Fluor 488 IHC signal was observed with confocal microscopy. RESULTS: A fixation time of 72 h was found to be optimal for ISH and subsequent IHC. The RNAscope probes for VEGF, HIF-1α, and HIF-2α mRNA all gave a strong Fast Red signal with both 48 h and 72 h fixed tissue, but the optimal IHC signal for either GFAP or GS-lectin within a retinal tissue section after ISH processing was observed with 72 h fixation. A pretreatment boiling time of 15 min and a dilution factor of 1:15 for the pretreatment protease solution were found to be optimal and necessary for successful ISH visualization with 72 h FFPE ocular tissue. CONCLUSIONS: The protocol presented here provides a simple and reliable method to simultaneously detect mRNA and protein within the same paraffin-embedded ocular tissue section. The procedure, after preparation of FFPE sections, can be performed over a 2-day or 4-day period. We provide an optimization strategy that may be adapted for any RNAscope probe set and antibody for determining retinal or ocular cell-specific patterns of expression.

Combination systemic and intravitreal antiviral therapy in the management of acute retinal necrosis syndrome.

BACKGROUND AND OBJECTIVE: Acute retinal necrosis (ARN) may lead to severe visual loss because of its rapid progression and high likelihood of retinal detachment (RD). This study investigates whether combination systemic and intravitreal antiviral therapy is superior to systemic antiviral therapy alone. PATIENTS AND METHODS: Single-center, interventional, comparative case series of patients with ARN treated with combination systemic antiviral and intravitreal foscarnet injection therapy or systemic antiviral therapy alone. Survival analysis and incidence rates of visual acuity (VA) gain of two lines or greater, severe visual loss of 20/200 or worse, and RD were assessed. RESULTS: Twelve patients received combination therapy and 12 received systemic therapy alone. Patients receiving combination therapy were more likely to gain two or more lines of VA and showed decreased incidences of severe visual loss and RD. CONCLUSION: Combination oral and intravitreal antiviral therapy may improve the likelihood for VA gain and decrease the risk of RD in patients with ARN. Clinicians should consider administering combination systemic and intravitreal antiviral therapy for patients with the ARN syndrome.