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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.

PurposeTo 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).ResultsGeneration 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.ConclusionsRetinal 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.

PurposeTo assess age-related changes in the rhesus macaque eye and evaluate them to corresponding human age-related eye disease.MethodsData 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.ResultsAges 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 (0.165mm Hg per increase in year of age), ocular biometry (lens thickness 7.2μm per increase in year of age; and axial length 52.8μm 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.ConclusionsThe 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.

Purpose To investigate the natural history and genetic associations of drusenoid pigment epithelial detachment (DPED) associated with age-related macular degeneration (AMD). Design Retrospective analysis of a prospective cohort study. Participants Of the 4203 Age-Related Eye Disease Study 2 (AREDS2) participants, 391 eyes (325 participants) had DPED without late AMD at the time of DPED detection. Genetic analyses included 120 white AREDS2 participants and 145 Age-Related Eye Disease Study (AREDS) participants with DPED. Methods Baseline and annual stereoscopic fundus photographs were graded centrally to detect DPED, a well-defined yellow elevated mound of confluent drusen ≥433 μm in diameter, and to evaluate progression rates to late AMD: geographic atrophy (GA) and neovascular (NV)-AMD. Five single nucleotide polymorphisms (CFH [rs10611670], C3 [rs2230199], CFI [rs10033900], C2/CFB [rs114254831], ARMS2 [rs10490924]) and genetic risk score (GRS) group were investigated for association with DPED development. Kaplan–Meier analyses and multivariable proportional hazard regressions were performed. Main Outcome Measures Progression rates to late AMD and decrease of ≥3 lines in visual acuity (VA) from the time of DPED detection; association of rate of DPED development with genotype. Results Mean (standard deviation [SD]) follow-up time from DPED detection was 4.7 (0.9) years. DPED was associated with increased risk of progression to late AMD (hazard ratio [HR], 2.36; 95% confidence interval [CI], 1.98–2.82; P Conclusions This study replicates the results of previous natural history studies of eyes with DPED including the high rates of progression to late AMD and vision loss (regardless of progression to late AMD). The genetic associations are consistent with genes associated with AMD progression.

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.

Purpose To analyze the prevalence, incidence, and clinical characteristics of eyes with geographic atrophy (GA) in age-related macular degeneration (AMD), including clinical and genetic factors affecting enlargement. Design Prospective cohort study within a controlled clinical trial. Participants Age-Related Eye Disease Study 2 (AREDS2) participants, aged 50–85 years. Methods Baseline and annual stereoscopic color fundus photographs were evaluated for GA presence and area. Analyses included GA prevalence and incidence rates, Kaplan-Meier rates, mixed-model regression, and multivariable analysis of the square root of GA, area adjusted for covariates, including clinical/imaging characteristics and genotype. Main Outcome Measures (1) Presence or development of GA; (2) change in the square root of GA area over time. Results At baseline, 517 eyes (6.2%) of 411 participants (9.8%) had pre-existing GA (without neovascular AMD), with the following characteristics: 33% central, 67% noncentral; and the following configurations: 36% small, 26% solid/unifocal, 24% multifocal, 9% horseshoe/ring, and 6% indeterminate. Of the remaining 6530 eyes at risk, 1099 eyes (17.3%) of 883 participants developed incident GA without prior neovascular disease during mean follow-up of 4.4 years. The Kaplan-Meier rate of incident GA was 19% of eyes at 5 years. In eyes with incident GA, 4-year risk of subsequent neovascular AMD was 29%. In eyes with incident noncentral GA, 4-year risk of central involvement was 57%. GA enlargement rate (following square root transformation) was similar in eyes with pre-existing GA (0.29 mm/year; 95% confidence interval 0.27–0.30) and incident GA (0.28 mm/year; 0.27–0.30). In the combined group, GA enlargement was significantly faster with noncentrality, multifocality, intermediate baseline size, and bilateral GA (P Conclusions Analyses of AREDS2 data on natural history of GA provide representative data on GA evolution and enlargement. GA enlargement, which was influenced by lesion features, was relentless, resulting in rapid central vision loss. The genetic variants associated with faster enlargement were partially distinct from those associated with risk of incident GA. These findings are relevant to further investigations of GA pathogenesis and clinical trial planning.

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.

Purpose:To describe a series of retinal arterial occlusions in healthy children.Methods:Chart review from multiple US and international institutions.Results:Five episodes affecting otherwise health...

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.

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.

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.

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.

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. [ Ophthalmic Surg Lasers Imaging Retina . 2014;45:399–407.]

Yevgeniy V Sychev,1 Elizabeth A Verner-Cole,2 Eric B Suhler,2 J Timothy Stout,2 G Atmaram Vemulakonda1 1Department of Ophthalmology, University of Washington, Seattle, WA, USA; 2Casey Eye Institute, Oregon Health and Science University, Portland, OR, USA Abstract: Intraocular foreign bodies (IOFBs) can complicate globe trauma and are associated with a high incidence of severe vision loss. Occult IOFBs present a particular challenge as they are not diagnosed promptly and tend to present with advanced complications, including endophthalmitis and retinal detachment. In this report, we present three cases of occult nonmetallic IOFBs presenting as fulminant uveitis, and we also review the literature. Keywords: intraocular foreign body, endophthalmitis, vitrectomy, trauma

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1 , a gene on chromosome 2q31 that encodes α2-chimaerin, a Rac guanosine triphosphatase–activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase α2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance α2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant α2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that α2-chimaerin has a critical developmental function in ocular motor axon pathfinding.

Cultured endothelial cells of the human retina and choroid demonstrate distinct patterns of gene expression. We hypothesized that differential gene expression reflected differences in the interactions of transcription factors and respective cis-regulatory motifs(s) in these two endothelial cell subpopulations, recognizing that motifs often exist as modules. We tested this hypothesis in silico by using TRANSFAC Professional and CisModule to identify cis-regulatory motifs and modules in genes that were differentially expressed by human retinal versus choroidal endothelial cells, as identified by analysis of a microarray data set. Motifs corresponding to eight transcription factors were significantly (p

Age-related macular degeneration (AMD), a complex multigenic disorder and the most common cause of vision loss in the elderly, is associated with polymorphisms in the LOC387715/ARMS2 and HTRA1 genes on 10q26. Like humans, macaque monkeys possess a macula and develop age-related macular pathologies including drusen, the phenotypic hallmark of AMD. We genotyped a cohort of 137 unrelated rhesus macaques with and without macular drusen. As in humans, one variant within LOC387715/ARMS2 and one in HTRA1 were significantly associated with affected status. HTRA1 and the predicted LOC387715/ARMS2 gene were both transcribed in rhesus and human retina and retinal pigment epithelium. Among several primate species, orthologous exons for the human LOC387715/ARMS2 gene were present only in Old World monkeys and apes. In functional analyses, the disease-associated HTRA1 polymorphism resulted in a 2-fold increase in gene expression, supporting a role in pathogenesis. These results demonstrate that two genes associated with AMD in humans are also associated with macular disease in rhesus macaques and that one of these genes is specific to higher primates. This is the first evidence that humans and macaques share the same genetic susceptibility factors for a common complex disease.

To observe temporal changes in retinal physiology and histology in a rat model of laser-induced retinal vein occlusion (RVO).Ophthalmoscopy, fundus photography, and fluorescein angiography (FA) were performed following laser-induced central retinal vein occlusion (CRVO) and branch retinal vein occlusion (BRVO) immediately after laser treatment and at 3 and 6 hr and 2, 4, 7, 14, and 21 days. Retinal histology was examined at 4, 7, 14, and 21 days. Full-field electroretinogram was recorded from both eyes simultaneously at day 4.For CRVO and BRVO, reperfusion of occluded branch veins was observed 1 to 2 days after treatment. Despite complete reperfusion of branch veins, retinal edema and hemorrhages peaked on day 4, and by day 14, treated retinas appeared pale and edematous upon ophthalmoscopy. In BRVO animals, retinal hemorrhages were limited to the vein-occluded region, although edema was more widespread and, to a limited extent, involved the untreated hemi-retina. Significant GCL cell loss was observed in both CRVO and BRVO groups after day 14. Regional analysis showed that relative GCL loss was greatest in the peripheral retina in BRVO group. Electroretinography disclosed moderate to severe functional deficits in photoreceptors, bipolar, and amacrine and ganglion cells.Laser-induced RVO in rats results in targeted vascular occlusion that persisted for 1 to 2 days. Functional deficits were evident and significant GCL cell loss was seen, notably within peripheral retina of the BRVO model. This reproducible model provides a valuable tool for the study of the molecular events associated with retinal ischemia and cell death.

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 (V 30 ) and total visual field hill of vision (V TOT ), 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, V 30 increased in 6 patients, V TOT 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.

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.

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.

The soluble neuropilin-1 (sNRP-1) gene employs an extremely short dual function polyadenylation (pA) signal/stop codon that is efficient for termination of gene transcription and translation in vivo. However, the functionality and usefulness of this signal in regard to other genes is unknown. This quantitative study compares the levels of humanized Renilla green fluorescent protein (hrGFP) mRNA polyadenylated with either the sNRP-1 pA signal or the much larger, more widely used SV40 pA signal. We show that the overall starting copy number of hrGFP for equally loaded RNAs is equivalent between the two groups. Our data show little to no difference between levels of mRNA generated by the sNRP-1 polyadenylation signal and the SV40 polyadenylation signal despite a remarkable size difference in signal length. An extremely short polyadenylation signal could potentially alleviate gene insert size restrictions associated with cloning or with therapeutic vectors such as adeno-associated virus.

Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) were given a single intravitreous injection of an E1-, partial E3-, E4-deleted adenoviral vector expressing human pigment epithelium- derived factor (AdPEDF.11). Doses ranging from 10(6) to 10(9.5) particle units (PU) were investigated. There were no serious adverse events related to AdPEDF.11 and no dose-limiting toxicities. Signs of mild, transient intraocular inflammation occurred in 25% of patients, but there was no severe inflammation. Six patients experienced increased intraocular pressure that was easily controlled by topical medication. All adenoviral cultures were negative. At 3 and 6 months after injection, 55 and 50%, respectively, of patients treated with 10(6)-10(7.5) PU and 94 and 71% of patients treated with 10(8)-10(9.5) PU had no change or improvement in lesion size from baseline. The median increase in lesion size at 6 and 12 months was 0.5 and 1.0 disk areas in the low-dose group compared with 0 and 0 disk areas in the high-dose group. These data suggest the possibility of antiangiogenic activity that may last for several months after a single intravitreous injection of doses greater than 10(8) PU of AdPEDF.11. This study provides evidence that adenoviral vector-mediated ocular gene transfer is a viable approach for the treatment of ocular disorders and that further studies investigating the efficacy of AdPEDF.11 in patients with neovascular AMD should be performed.

Proliferative ocular diseases encompass a wide variety of pathological processes with adverse cellular differentiation, proliferation and migration as common features. Pathologies may involve neovascular responses associated with diabetic retinopathy, retinopathy of prematurity or age-related macular degeneration. These diseases are quite prevalent and account for substantial visual impairment and blindness worldwide. Although treatment strategies are largely surgical, advances in our understanding of the proteins crucial to cell transdifferentiation, proliferation and migration, along with better gene transfer techniques, have greatly increased the potential for biological treatment options. In this report, the most common proliferative ocular vascular diseases and existing therapeutic modalities will be reviewed and an overview of possible gene therapy options will be discussed, along with potential candidate genes.

Myotonic dystrophy (DM) is an autosomal dominant disorder characterized by skeletal muscle wasting, myotonia, cardiac arrhythmia, hyperinsulinaemia, mental retardation and ocular cataracts1. The genetic defect in DM is a CTG repeat expansion located in the 3′ untranslated region of DMPK and 5′ of a homeodomain-encoding gene, SIX5 (formerly DMAHP; refs 2–5). There are three mechanisms by which CTG expansion can result in DM. First, repeat expansion may alter the processing or transport of the mutant DMPK mRNA and consequently reduce DMPK levels6. Second, CTG expansion may establish a region of heterochromatin 3′ of the repeat sequence and decrease SIX5 transcription7,8,9. Third, toxic effects of the repeat expansion may be intrinsic to the repeated elements at the level of DNA or RNA (refs 10,11). Previous studies have demonstrated that a dose-dependent loss of Dm15 (the mouse DMPK homologue) in mice produces a partial DM phenotype characterized by decreased development of skeletal muscle force and cardiac conduction disorders12,13,14,15. To test the role of Six5 loss in DM, we have analysed a strain of mice in which Six5 was deleted. Our results demonstrate that the rate and severity of cataract formation is inversely related to Six5 dosage and is temporally progressive. Six5+/− and Six5−/− mice show increased steady-state levels of the Na+/K+-ATPase α-1 subunit and decreased Dm15 mRNA levels. Thus, altered ion homeostasis within the lens may contribute to cataract formation. As ocular cataracts are a characteristic feature of DM, these results demonstrate that decreased SIX5 transcription is important in the aetiology of DM. Our data support the hypothesis that DM is a contiguous gene syndrome associated with the partial loss of both DMPK and SIX5.

Purpose: The purpose of the study was to characterize the clinical development of proliferative vitreoretinopathy (PVR) after trauma in the human eye. Methods: A chart review was performed on the records of 1564 patients with ocular trauma seen at a large metropolitan hospital. The frequency, type of ocular trauma, time to onset, potential risk factors, and visual outcome for PVR were evaluated. Results: Proliferative vitreoretinopathy occurred in 71 (4%) of 1654 injured eyes. Of these 71 injured eyes, 30 (42%) resulted from rupture, 15 (21 %) from penetration, 13 (18%) from perforation, and 7 (10%) from contusion. Six (9%) were associated with an intraocular foreign body (IOFB). The frequency of PVR following perforation, rupture, penetration, IOFB, and contusion was 43%, 21%, 15%, 11 %, and 1 %, respectively. Overall, those eyes that developed PVR had a poorer visual outcome, with PVR being the primary reason for visual loss. The time from injury to onset of PVR was shortest after perforation (median, 1.3 months), followed by rupture (2.1 months), IOFB (3.1 months), penetration (3.2 months), and contusion (5.7 months). Vitreous hemorrhage was the strongest independent predictive factor for the development of PVR. A long, posteriorly located wound and persistent intraocular inflammation were also important risk factors for PVR. Conclusions: These results suggest that PVR is a common complication following a variety of ocular injuries, and that it is associated with a poor visual outcome. Its frequency, onset, and outcome are strongly dependent on the nature of the trauma. Specific high-risk groups are identified as candidates for more aggressive therapy.

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.

Cytomegalovirus (CMV) retinitis typically presents as a hemorrhagic, full-thickness retinitis in immunosuppressed individuals, often in the setting of HIV infection. The management of CMV retinitis includes systemic and locally administered intravitreal antivirals (i.e. foscarnet or ganciclovir), as well as the surgical intravitreal ganciclovir implant. In chronically immunosuppressed patients (i.e. transplant recipients, cancer chemotherapy) and in HIV/AIDS patients who fail to immune reconstitute, chronic CMV prophylaxis with valganciclovir may lead to ganciclovir- and foscarnet-resistant CMV strains. Moreover, the identification of drug-resistant CMV may influence the choice or dosing of antiviral medication.1–2 Ganciclovir-resistance is classified into genotypic resistance defined as CMV DNA harboring a mutation known to confer antiviral resistance or phenotypic resistance meaning that ganciclovir at a therapeutic dose fails to exceed the concentration required to inhibit 50% of CMV growth on viral culture media. We characterize a series of patients with CMV retinitis who were evaluated for genotypic ganciclovir-resistance using PCR-based analysis of ocular fluids and describe its influence on management.

Gene and cell therapy holds tremendous promise for treating a variety of acquired and inherited disorders of the retina that impact on vision. Much has been written about the impact of vectors in delivering genes to cells of the retina in vivo. A critically important component of these kinds of therapies is the procedure used to introduce the cells or vectors. Drs. Stout and Francis provide a broad overview of gene and cell therapies for diseases of the retina, focusing on the procedures that could be used for delivery.

In the past decade, advances in vector design and delivery have helped guide gene therapy technologies from the laboratory to clinical trials. These technologies are poised to tackle a key problem in opthalmology: pathologic angiogenesis. Gene transfer techonologies offer the potential for highly regulated and sustained delivery of a therapeutic agent, and may have the potential to arrest a pathological process prior to the evolution of a soluble angiogenic factor. Multiple biologic effectors of agiogenesis have been investigated as potential therapeutic targets, including Vascular endothelial cell growth factor (VEGF) and the VEGF receptor, angiopoietin 1 and 2, tissue inhibitor of matrix metalloproteinases-3 (Timp-3), angiostatin, Pigment Epithelial-Derived Factor (PEDF), and endostatin. Data from experimental models in these systems has been encouraging, and the experience gained from ongoing ocular gene therapy trials in non-angiogenic disease will be instructive. In the first human angiogenic gene therapy trial for a neovascular retinopathy, PEDF expressed in an adenoviral vector demonstrated a reasonable safety profile and proof of concept. Cell-based and siRNA technologies are also under clinical investigation, and, together with gene therapy, may provide breakthrough treatments for diseases that cause much of the world’s blindness.

PURPOSE: To evaluate the etiology of a unilateral hemangioblastoma noted in a male with a family history remarkable only for spine surgery in the proband’s father. METHODS: Genomic DNA was isolated from peripheral blood of family members, and the three exons of the von Hippel–Lindau gene were examined for mutations by direct sequencing. RESULTS: A three base pair (bp) deletion in exon 1 of the VHL gene was found in the father and both sons. This in-frame deletion results in the loss of a phenylalanine residue from the von Hippel–Lindau protein product, at amino acid position 76. CONCLUSION: Genetic screening has confirmed that von Hippel–Lindau syndrome is responsible for the hemangioblastoma in the proband. Magnetic resonance imaging scans performed as a consequence of these results indicated spinal tumors present in the father and tumors present in the cerebellum of the proband’s sibling. As close, lifelong follow-up is warranted with this disease, this case demonstrates the value of DNA testing in patients with ocular findings consistent with von Hippel–Lindau disease in the absence of a recognized family history.

Development of novel molecular biology techniques in the 1970s and 1980s furnished scientists with new tools to advance the study and treatment of human disease. Progress in the understanding of bacterial and viral biology led to innovations in molecular cloning and chimeric plasmid construction. Advances in nucleic acid sequencing allowed researchers to gain a better understanding of genes and the ability to study gene mutations. Unraveling the minutiae of molecular events involved in gene transcription and translation furthered the analysis of cellular pathways and their complex interrelationships. Production of proteins ex vivo allowed physicians to treat diseases such as diabetes with synthetic human insulin, ending the dependency on animal sources.

Retinal hemangioblastoma is a clinically and histopathologically distinct tumor that may occur sporadically or as a manifestation of von Hippel-Lindau (VHL) disease.1 von Hippel-Lindau disease is transmitted in an autosomal dominant fashion due to germline mutations in the VHL gene, a tumor suppressor gene on the short arm of chromosome 3 (3p25.5).2 It consists of 3 exons, which encode a messenger RNA (mRNA) transcript expressed in many tissues and translated into a 213-amino acid protein. Originally identified as a tumor suppressor, the VHL protein is now known to repress expression of mRNAs that are normally induced under hypoxic conditions.3,4 The hypoxia-inducible factor is a key transcription factor responsible for up-regulation of expression of hypoxia-inducible genes. When mutated, the VHL gene produces a protein that is unable to regulate hypoxia-inducible factor, permitting accumulation of hypoxia-inducible factor and subsequent activation of vascular endothelial growth factor and other hypoxiainducible genes.5,6 This can result in growth of retinal hemangioblastomas and other tumors associated with VHL disease. Clinically, retinal hemangioblastomas are characterized by their vascular appearance, often with dilated feeder vessels. They may increase in size with endophytic, exophytic, or sessile growth patterns. Vision loss may occur due to leakage of fluid and proteins from the tumor and traction retinal detachment secondary to associated vitreous and retinal changes. Various treatment options have been used, including observation, thermal laser, cryotherapy, brachytherapy, photodynamic therapy, transpupillary thermotherapy, external beam radiation therapy, and systemic and intravitreal vascular endothelial growth factor inhibitors.7-18 Treatment depends on the size and location of the tumor. Smaller tumors are generally treated using laser if posterior in location or using cryotherapy if in the retinal periphery. These methods are less effective on larger tumors (>4 mm in diameter), which may be more effectively treated using radiation therapy. However, tumors greater than 5 mm in diameter or with exudative or tractional retinal detachment have a poor prognosis even using radiation therapy. We describe the treatment of 3 patients with large solitary retinal hemangioblastomas using internal en bloc surgical resection. All patients lack a germline mutation in the VHL gene, as determined by sequence analysis of leukocyte cell DNA. To analyze the molecular basis for sporadically occurring retinal hemangioblastoma, 2 of the resected tumors were evaluated for loss of heterozygosity (LOH) and for VHL gene mutations.

BACKGROUND To evaluate macular thickness measured by optical coherence tomography (OCT) during pegaptanib therapy for neovascular age-related macular degeneration (AMD). METHODS For this prospective, nonrandomized, observational case series, 41 eyes from 41 patients with neovascular AMD received intravitreous pegaptanib (1 mg) injections repeated every 6 weeks. The primary outcome measure was central foveal thickness measured by OCT. Secondary outcomes were fluorescein angiographic leakage and visual acuity. RESULTS Mean thickness of the central area on OCT decreased from 340 +/- 24 microm to 299 +/- 14 microm after 12 weeks of pegaptanib injections. This represents a reduction in thickening of 32%. Fluorescein angiograms with definite leakage decreased from 100% to 81%, and mean visual acuity decreased from 20/116 to 20/120. CONCLUSIONS Intravitreal injections of pegaptanib at 6-week intervals result in a moderate reduction of central foveal thickness in eyes with subfoveal neovascular AMD. This presents a modest effect relative to that reported with other anti-angiogenic agents.

Purpose To report the change in visual acuity and central retinal thickness by optical coherence tomography (OCT) after intravitreal injections of bevacizumab for the treatment of neovascular age-related macular degeneration (AMD). Methods A retrospective case series in a university-based practice evaluated patients with subfoveal choroidal neovascularization (CNV) due to AMD. Patients received intravitreal injections (1.25 mg) of bevacizumab and were monitored monthly with determination of best-corrected ETDRS visual acuity and OCT for persistence of retinal thickening. Eyes were retreated on an "as needed" basis, defined by presence of intraretinal or subretinal fluid. Patients were monitored every 2 months to 3 months for persistence of angiographic leakage. Results Seventy-nine eyes of 74 consecutive patients received the initial injection of bevacizumab between August 1, 2005, and January 30, 2006. Sixty-eight eyes (86%) of 64 patients had at least 3 months of follow-up. Mean central retinal thickness +/- SD decreased from 304 +/- 83 microm at baseline to 237 +/- 105 microm at 3 months (P = 0.00002). Mean ETDRS visual acuity gained 4 letters from 20/100 at baseline to 20/80-1 at 3 months (P = 0.040). Twenty eyes (25%) appeared to have a sustained response to a single injection and did not require further injections through 3 months. Two patients had a potentially drug-related adverse event (ischemic stroke and myocardial infarction). No serious injection-related adverse events occurred. Conclusions Intravitreal bevacizumab injection affects a rapid decrease in retinal thickness to normal or near-normal levels and improvement in visual acuity in eyes with CNV due to AMD. The sustainability of changes in retinal thickness and visual acuity in response to bevacizumab treatment warrant further investigation and long-term follow-up.

Purpose: To report the ocular presentation and histopathology of a patient with diffuse neonatal hemangiomatosis. Methods: Case report. A 3.7-kg female was born at term. Multiple hemangiomas were present, and the patient died 39 days after birth. Results: Widespread cutaneous and visceral hemangiomas were present. Ophthalmic examination disclosed bilateral eyelid and conjunctival hemangiomas. The right eye had multiple iris hemangiomas, hyphema, vitreous hemorrhage, and discrete subretinal lesions. Histopathology of the right eye demonstrated hemangiomas involving the iris, ciliary body, and ciliary processes. Conclusion: Diffuse neonatal hemangiomatosis was associated with ophthalmic findings of multiple hemangiomas involving eyelid, conjunctiva, iris, ciliary body, and ciliary processes.

PURPOSE. Mutations in the frizzled-4 gene (FZD4) have recently been associated with autosomal dominant familial exudative vitreoretinopathy (FEVR) in families linking to the EVR1 locus on the long arm of chromosome 11. The purpose of this study was to screen FZD4 in a panel of 40 patients with FEVR to identify the types and location of mutations and to calculate what proportion of this heterogeneous condition is attributable to FZD4 mutations. METHODS. PCR products were generated from genomic DNA with primers designed to amplify the coding sequence of FZD4. The PCR products were screened for mutations by single-strand conformational polymorphism-heteroduplex analysis (SSCP-HA) and by direct sequencing. RESULTS. In total, eight mutations were identified, seven of which were novel. Three were deletions (c957delG, c1498delA, and c1501-1502delCT), one was a nonsense mutation (Q505X), and four were missense mutations (G36D, M105T, M157V, and S497F). CONCLUSIONS. Eight mutations have been identified in the FZD4 gene in a cohort of 40 unrelated patients with FEVR. This result indicates that FZD4 mutations are responsible for only 20% of FEVR index cases and suggests that the other FEVR loci may account for more cases than previously anticipated.

To describe the clinical features, histologic changes, and genetic analysis of Avellino corneal dystrophy in an African-American woman.Interventional case report.A 79-year-old African-American woman with corneal deposits consistent with Avellino corneal dystrophy was studied with histologic and genetic analysis.The patient had multiple crumb-like opacities in the anterior stroma of both eyes. Deep to these lesions were numerous faint, stellate lattice lesions. Corneal scraping confirmed the presence of Masson trichrome and Congo red positive subepithelial deposits. Genetic analysis revealed a heterozygous CGC/CAC change in exon 4 of the beta iG-H3 gene, resulting in an arginine to histidine substitution at codon 124.This case reveals several novel findings, including surface changes resembling vortex dystrophy and large granular deposits protruding through the anterior corneal surface. This is the first case described in an African-American patient.