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Purpose: To determine whether dilation status has a clinically meaningful effect on sensitivity in normal subjects undergoing two-color dark-adapted perimetry, which can be useful to assess rod function., Methods: A perimeter measured naturally and pharmacologically dilated scotopic sensitivities using a test grid consisting of 16 points across the horizontal meridian ranging from 60° temporal to 45° nasal using cyan (500 nm wavelength) or red (650 nm wavelength) stimuli. The primary outcome was average overall sensitivity based on dilation status, which was compared using a linear mixed effect model for each color stimuli. A difference of 2 dB or more was considered clinically significant., Results: Twenty-nine eyes from 15 subjects (nine female) ages 23 to 63 with no known retinal pathology were included. Pharmacologically dilated eyes were 0.54 dB (95% confidence interval [CI], 0.05 dB to 1.03 dB; P = 0.032) more sensitive to a red stimulus than naturally dilated eyes, but this was not statistically significant after correction for multiple comparisons. Pharmacologically dilated eyes were 0.03 dB (95% CI, -0.20 dB to 0.14 dB; P = 0.734) less sensitive to a cyan stimulus compared to naturally dilated eyes., Conclusions: These findings show no clinically significant differences in sensitivity of scotopic perimetry in eyes without retinal pathology based on dilation status for both cyan and red stimuli., Translational Relevance: In this study, pharmacological dilation did not have a clinically meaningful effect on sensitivity, suggesting that this is not necessary when using two-color dark-adapted perimetry to assess for rod function.

Atypical Usher syndrome (USH) is poorly defined with a broad clinical spectrum. Here, we characterize the clinical phenotype of disease caused by variants in CEP78, CEP250, ARSG , and ABHD12 .Chart review evaluating demographic, clinical, imaging, and genetic findings of 19 patients from 18 families with a clinical diagnosis of retinal disease and confirmed disease-causing variants in CEP78, CEP250, ARSG , or ABHD12 . CEP78 -related disease included sensorineural hearing loss (SNHL) in 6/7 patients and demonstrated a broad phenotypic spectrum including: vascular attenuation, pallor of the optic disc, intraretinal pigment, retinal pigment epithelium mottling, areas of mid-peripheral hypo-autofluorescence, outer retinal atrophy, mild pigmentary changes in the macula, foveal hypo-autofluorescence, and granularity of the ellipsoid zone. Nonsense and frameshift variants in CEP250 showed mild retinal disease with progressive, non-congenital SNHL. ARSG variants resulted in a characteristic pericentral pattern of hypo-autofluorescence with one patient reporting non-congenital SNHL. ABHD12 -related disease showed rod-cone dystrophy with macular involvement, early and severe decreased best corrected visual acuity, and non-congenital SNHL ranging from unreported to severe.This study serves to expand the clinical phenotypes of atypical USH. Given the variable findings, atypical USH should be considered in patients with peripheral and macular retinal disease even without the typical RP phenotype especially when SNHL is noted. Additionally, genetic screening may be useful in patients who have clinical symptoms and retinal findings even in the absence of known SNHL given the variability of atypical USH.

Objective: Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is associated with pathogenic variants in BEST1 , which typically causes visual impairment in the late stage of disease. We present a pedigree with variable expressivity and the youngest case in the literature with visual impairment in early childhood., Methods and Analysis: This is a retrospective, observational, case series describing multigenerational members of one family affected with ADVIRC. Patients underwent examination, ultra-widefield fundus photography and angiography, optical coherence tomography, full-field electroretinography (ffERG) and full-field perimetry., Results: Three affected members of the pedigree, one from each successive generation, were found to harbour a mutation, c.715G>A:p.Val239Met, in BEST1 . The proband characterised in this report is, to our knowledge, the youngest documented case of ADVIRC in early childhood. Yet, this patient has the most severe retinal dysfunction compared with the father and paternal grandmother, whom exhibit classic characteristics of ADVIRC. Longitudinal data from the paternal grandmother showed that there was a rapid decline in ffERG responses (photopic decline worse than scotopic) from the fourth to fifth decade of life, which correlated with severe concentric constriction of visual fields., Conclusion: This multigenerational case series provides new insights into the ADVIRC disease spectrum and rate of progression. While ADVIRC typically causes a slowly progressive disease, we show that variable phenotypic expressivity is possible among affected members of the same family with the same mutation in BEST1 . Thus, ADVIRC must also be considered in the differential diagnosis of paediatric patients with severe retinal dystrophy in early childhood., Competing Interests: Competing interests: RGW, Oregon Health & Science University, in the name of RGW, holds US patent no. 8657446, Method and apparatus for visual field monitoring, also known as Visual Field Modelling and Analysis or VFMA (P), which is licensed but no royalties have yet accrued to RGW. RGW is on advisory boards for AGTC, Janssen Research and the Foundation Fighting Blindness (F)., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To describe the detailed retinal phenotype of KCNV2-associated retinopathy., Study Design: Multicenter international retrospective case series., Methods: Review of retinal imaging including fundus autofluorescence (FAF) and optical coherence tomography (OCT), including qualitative and quantitative analyses., Results: Three distinct macular FAF features were identified: (1) centrally increased signal (n = 35, 41.7%), (2) decreased autofluorescence (n = 27, 31.1%), and (3) ring of increased signal (n = 37, 44.0%). Five distinct FAF groups were identified based on combinations of those features, with 23.5% of patients changing the FAF group over a mean (range) follow-up of 5.9 years (1.9-13.1 years). Qualitative assessment was performed by grading OCT into 5 grades: (1) continuous ellipsoid zone (EZ) (20.5%); (2) EZ disruption (26.1%); (3) EZ absence, without optical gap and with preserved retinal pigment epithelium complex (21.6%); (4) loss of EZ and a hyporeflective zone at the foveola (6.8%); and (5) outer retina and retinal pigment epithelium complex loss (25.0%). Eighty-six patients had scans available from both eyes, with 83 (96.5%) having the same grade in both eyes, and 36.1% changed OCT grade over a mean follow-up of 5.5 years. The annual rate of outer nuclear layer thickness change was similar for right and left eyes., Conclusions: KCNV2-associated retinopathy is a slowly progressive disease with early retinal changes, which are predominantly symmetric between eyes. The identification of a single OCT or FAF measurement as an endpoint to determine progression that applies to all patients may be challenging, although outer nuclear layer thickness is a potential biomarker. Findings suggest a potential window for intervention until 40 years of age., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors declare that they have no conflicts of interest to disclose.

Purpose: To compare foveal hypoplasia and the appearance of the ellipsoid zone (EZ) at the fovea in patients with genetically confirmed achromatopsia (ACHM) and blue cone monochromacy (BCM)., Design: Retrospective, multi-center observational study., Subjects: Molecularly confirmed patients with ACHM (n = 89) and BCM (n = 33)., Methods: We analyzed high-resolution spectral domain optical coherence tomography (SD-OCT) images of the macula from aforementioned patients with BCM. Three observers independently graded SD-OCT images for foveal hypoplasia (i.e. retention of one or more inner retinal layers at the fovea) and four observers judged the integrity of the EZ at the fovea, based on an established grading scheme. These measures were compared with previously published data from the ACHM patients., Main Outcome Measures: Presence of foveal hypoplasia and EZ grade., Results: Foveal hypoplasia was significantly more prevalent in ACHM than in BCM (p<0.001). In addition, we observed a significant difference in the distribution of EZ grades between ACHM and BCM, with grade II EZ being by far the most common phenotype in BCM (61% of patients). In contrast, ACHM patients had a relatively equal prevalence of EZ grades I, II, and IV. Interestingly, grade IV EZ was 2.6 times more prevalent in ACHM compared to BCM, while grade V EZ (macular atrophy) was present in 3% of both the ACHM and BCM cohorts., Conclusions: The higher incidence of foveal hypoplasia in ACHM than BCM supports a role for cone activity in foveal development. Although there are differences in EZ grades between these conditions, the degree of overlap suggests EZ grade is not sufficient for definitive diagnosis, in contrast to previous reports. Analysis of additional OCT features in similar cohorts may reveal differences with greater diagnostic value. Finally, the extent to which foveal hypoplasia or EZ grade is prognostic for therapeutic potential in either group remains to be seen, but motivates further study.

Competing Interests: Equity and salary from Clearside to T.C., MD, MBA. Consultant for Optos, Adverum, Regenxbio, Gyroscope, Regeneron, Genentech/Roche; research support from Optos; equity from Fortress Bio; and intellectual property related to gene and cellular therapy to S.K., MD. Data Safety Monitoring Board for Akous and Gensight; clinical trial support for FFB; consultant for 4D Molecular Therapetuics, Adverum, Astellas Pharmaceuticals, Blue Rock, IVERIC, Novartis, Ora, PYC Therapeutics, RegenexBio, Roche, Viewpoint Therapeutics; consultant and scientific advisory board for Eyevensys, Horama, Nayan, Sparing Vision, Vedere; consultant and clinical trial support for Biogen, AGTC, Editas, ProQR, Sanofi; consultant and equity with Endogena; consultant, equity, and clinical advisory board for Atsena; consultant, equity, and scientific advisory board for DTx, Nacuity Pharmaceuticals, Ocugen to M.E.P, MD, PhD. E.T.C. declares that there is no conflicts of interest to disclose.

Competing Interests: The authors declare that they have no conflicts of interest to disclose.

Purpose: The purpose of this study was to characterize the phenotypic spectrum of ophthalmic findings in patients with Alagille syndrome., Methods: We conducted a retrospective, observational, multicenter, study on 46 eyes of 23 subjects with Alagille syndrome. We reviewed systemic and ophthalmologic data extracted from medical records, color fundus photography, fundus autofluorescence, optical coherence tomography, visual fields, electrophysiological assessments, and molecular genetic findings., Results: Cardiovascular abnormalities were found in 83% of all cases (of those, 74% had cardiac murmur), whereas 61% had a positive history of hepatobiliary issues, and musculoskeletal anomalies were present in 61% of all patients. Dysmorphic facies were present in 16 patients, with a broad forehead being the most frequent feature. Ocular symptoms were found in 91%, with peripheral vision loss being the most frequent complaint. Median (range) Snellen visual acuity of all eyes was 20/25 (20/20 to hand motion [HM]). Anterior segment abnormalities were present in 74% of the patients; of those, posterior embryotoxon was the most frequent finding. Abnormalities of the optic disc were found in 52%, and peripheral retinal abnormalities were the most frequent ocular finding in this series, found in 96% of all patients. Fifteen JAG1 mutations were identified in 16 individuals; of those, 6 were novel., Conclusions: This study reports a cohort of patients with Alagille syndrome in which peripheral chorioretinal changes were more frequent than posterior embryotoxon, the most frequent ocular finding according to a number of previous studies. We propose that these peripheral chorioretinal changes are a new hallmark to help diagnose this syndrome.

Purpose: Adaptive optics scanning light ophthalmoscopy (AOSLO) imaging in patients with achromatopsia (ACHM) and albinism is not always successful. Here, we tested whether optical coherence tomography (OCT) measures of foveal structure differed between patients for whom AOSLO images were either quantifiable or unquantifiable., Methods: The study included 166 subjects (84 with ACHM; 82 with albinism) with previously acquired OCT scans, AOSLO images, and best-corrected visual acuity (BCVA, if available). Foveal OCT scans were assessed for outer retinal structure, outer nuclear layer thickness, and hypoplasia. AOSLO images were graded as quantifiable if a peak cone density could be measured and/or usable if the location of peak density could be identified and the parafoveal mosaic was quantifiable., Results: Forty-nine percent of subjects with ACHM and 57% of subjects with albinism had quantifiable AOSLO images. Older age and better BCVA were found in subjects with quantifiable AOSLO images for both ACHM (P = 0.0214 and P = 0.0276, respectively) and albinism (P = 0.0073 and P < 0.0004, respectively). There was a significant trend between ellipsoid zone appearance and ability to quantify AOSLO (P = 0.0028). In albinism, OCT metrics of cone structure did not differ between groups., Conclusions: Previously reported AOSLO-based cone density measures in ACHM may not necessarily reflect the degree of remnant cone structure in these patients., Translational Relevance: Until AOSLO is successful in all patients with ACHM and albinism, the possibility of the reported data from a particular cohort not being representative of the entire population remains an important issue to consider when interpreting results from AOSLO studies.

Purpose: To investigate genetics, electrophysiology, and clinical course of KCNV2-associated retinopathy in a cohort of children and adults., Study Design: This was a multicenter international clinical cohort study., Methods: Review of clinical notes and molecular genetic testing. Full-field electroretinography (ERG) recordings, incorporating the international standards, were reviewed and quantified and compared with age and recordings from control subjects., Results: In total, 230 disease-associated alleles were identified from 117 patients, corresponding to 75 different KCNV2 variants, with 28 being novel. The mean age of onset was 3.9 years old. All patients were symptomatic before 12 years of age (range, 0-11 years). Decreased visual acuity was present in all patients, and 4 other symptoms were common: reduced color vision (78.6%), photophobia (53.5%), nyctalopia (43.6%), and nystagmus (38.6%). After a mean follow-up of 8.4 years, the mean best-corrected visual acuity (BCVA ± SD) decreased from 0.81 ± 0.27 to 0.90 ± 0.31 logarithm of minimal angle of resolution. Full-field ERGs showed pathognomonic waveform features. Quantitative assessment revealed a wide range of ERG amplitudes and peak times, with a mean rate of age-associated reduction indistinguishable from the control group. Mean amplitude reductions for the dark-adapted 0.01 ERG, dark-adapted 10 ERG a-wave, and LA 3.0 30 Hz and LA3 ERG b-waves were 55%, 21%, 48%, and 74%, respectively compared with control values. Peak times showed stability across 6 decades., Conclusion: In KCNV2-associated retinopathy, full-field ERGs are diagnostic and consistent with largely stable peripheral retinal dysfunction. Report 1 highlights the severity of the clinical phenotype and established a large cohort of patients, emphasizing the unmet need for trials of novel therapeutics., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Foundation Fighting Blindness, a 501(c)(3) nonprofit organization, established an international consortium of inherited retinal disease specialists in 2016, with a mission to accelerate the development of treatments for rare, inherited retinal degenerations, such as retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis, Usher syndrome, choroideremia, and achromatopsia. The Consortium accomplishes its mission by evaluating novel outcome measures, sharing standardized study protocols and datasets, and disseminating findings. Having established research infrastructure in the first 3 years, including 39 global research sites, the network is now poised to expand its infrastructure for trials of new therapies in partnership with industry. This model represents an innovative approach to overcome challenges of therapeutic development for rare diseases.

Purpose: Despite the extensive use of next-generation sequencing (NGS) technology to identify disease-causing genomic variations, a major gap in our understanding of Mendelian diseases is the unidentified molecular lesion in a significant portion of patients. For inherited retinal degenerations (IRDs), although currently close to 300 disease-associated genes have been identified, the mutations in approximately one-third of patients remain unknown. With mounting evidence that noncoding mutations might contribute significantly to disease burden, we aimed to systematically investigate the contributions of noncoding regions in the genome to IRDs., Methods: In this study, we focused on RPGRIP1 , which has been linked to various IRD phenotypes, including Leber congenital amaurosis (LCA), retinitis pigmentosa (RP), and macular dystrophy (MD). As several noncoding mutant alleles have been reported in RPGRIP1, and we observed that the mutation carrier frequency of RPGRIP1 is higher in patient cohorts with unsolved IRDs, we hypothesized that mutations in the noncoding regions of RPGRIP1 might be a significant contributor to pathogenicity. To test this hypothesis, we performed whole-genome sequencing (WGS) for 25 patients with unassigned IRD who carry a single mutation in RPGRIP1 ., Results: Three noncoding variants in RPGRIP1 , including a 2,890 bp deletion and two deep-intronic variants (c.2710+233G>A and c.1468-263G>C), were identified as putative second hits of RPGRIP1 in three patients with LCA. The mutant alleles were validated with direct sequencing or in vitro assays., Conclusions: The results highlight the significance of the contribution of noncoding pathogenic variants to unsolved IRD cases., (Copyright © 2021 Molecular Vision.)

High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort of 571 IRD patients who lack a confident molecular diagnosis, and potential deep intronic variants that affect proper splicing were identified using SpliceAI. A total of six deleterious deep intronic variants were identified in eight patients. An in vitro minigene system was applied to further validate the effect of these variants on the splicing pattern of the associated genes. The prediction scores assigned to splice-site disruption positively correlated with the impact of mutations on splicing, as those with lower prediction scores demonstrated partial splicing. Through this study, we estimated the contribution of deep-intronic splice mutations to unassigned IRD patients and leveraged in silico and in vitro methods to establish a framework for prioritizing deep intronic variant candidates for mechanistic and functional analyses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qian, Wang, Wang, Igelman, Jones, Li, Wang, Goetz, Birch, Yang, Pennesi and Chen.)

Purpose: To determine whether artifacts in optical coherence tomography (OCT) images are associated with the success or failure of adaptive optics scanning light ophthalmoscopy (AOSLO) imaging in subjects with achromatopsia (ACHM)., Methods: Previously acquired OCT and non-confocal, split-detector AOSLO images from one eye of 66 subjects with genetically confirmed achromatopsia (15 CNGA3 and 51 CNGB3 ) were reviewed along with best-corrected visual acuity (BCVA) and axial length. OCT artifacts in interpolated vertical volumes from CIRRUS macular cubes were divided into four categories: (1) none or minimal, (2) clear and low frequency, (3) low amplitude and high frequency, and (4) high amplitude and high frequency. Each vertical volume was assessed once by two observers. AOSLO success was defined as sufficient image quality in split-detector images at the fovea to assess cone quantity., Results: There was excellent agreement between the two observers for assessing OCT artifact severity category (weighted kappa = 0.88). Overall, AOSLO success was 47%. For subjects with OCT artifact severity category 1, AOSLO success was 65%; for category 2, 47%; for category 3, 11%; and for category 4, 0%. There was a significant association between OCT artifact severity category and AOSLO success ( P = 0.0002). Neither BCVA nor axial length was associated with AOSLO success ( P = 0.07 and P = 0.75, respectively)., Conclusions: Artifacts in OCT volumes are associated with AOSLO success in ACHM. Subjects with less severe OCT artifacts are more likely to be good candidates for AOSLO imaging, whereas AOSLO was successful in only 7% of subjects with category 3 or 4 OCT artifacts. These results may be useful in guiding patient selection for AOSLO imaging., Translational Relevance: Using OCT to prescreen patients could be a valuable tool for clinical trials that utilize AOSLO to reduce costs and decrease patient testing burden., Competing Interests: Disclosure: K.M. Litts, None; E.N. Woertz, None; M. Georgiou, MeiraGTx (C); E.J. Patterson, None; B.L. Lam, AGTC (F), G.A. Fishman, AGTC (F); M.E. Pennesi, AGTC (F); C.N. Kay, AGTC (F); W.W. Hauswirth, AGTC (I, R); M. Michaelides, MeiraGTx (C); J. Carroll, MeiraGTx (C, F), OptoVue (F), AGTC (F), Translational Imaging Innovations (I), (Copyright 2021 The Authors.)

PNPLA6-related disorders include several phenotypes, such as Boucher-Neuhäuser syndrome, Gordon Holmes syndrome, spastic paraplegia, photoreceptor degeneration, Oliver-McFarlane syndrome and Laurence-Moon syndrome. In this study, detailed clinical evaluations and genetic testing were performed in five (4 Chinese and 1 Caucasian/Chinese) syndromic retinal dystrophy patients. Genotype-phenotype correlations were analyzed based on review of the literatures of previously published PNPLA6-related cases. The mean age of patients and at first visit were 20.8 years (11, 12, 25, 28, 28) and 14.2 years (4, 7, 11, 24, 25), respectively. They all presented with severe chorioretinal dystrophy and profoundly decreased vision. The best corrected visual acuity (BCVA) ranged from 20/200 to 20/2000. Systemic manifestations included cerebellar ataxia, hypogonadotropic hypogonadism and hair anomalies. Six novel and three reported pathogenic variants in PNPLA6 (NM&#95;001166111) were identified. The genotypes of the five cases are: c.3134C > T (p.Ser1045Leu) and c.3846+1G > A, c.3547C > T (p.Arg1183Trp) and c.1841+3A > G, c.3436G > A (p.Ala1146Thr) and c.2212-10A > G, c.3436G > A (p.Ala1146Thr) and c.2266C > T (p.Gln756*), c.1238&#95;1239insC (p.Leu414Serfs*28) and c.3130A > G (p.Thr1044Ala). RT-PCR confirmed that the splicing variants indeed led to abnormal splicing. Missense variants p.Thr1044Ala, p.Ser1045Leu, p.Ala1146Thr, p.Arg1183Trp and c.3846+1G > A are located in Patatin-like phospholipase (Pat) domain. In conclusion, we report the phenotypes in five patients with PNPLA6 associated syndromic retinal dystrophy with variable systemic involvement and typical choroideremia-like fundus changes. Ocular manifestations may be the first and the only findings for years. All of our patients carried one severe deleterious variant (stop-gain or splicing variant) and one milder variant (missense variant). Retinal involvement was significantly correlated with severe deleterious variants and variants in Pat domain., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Optical coherence tomographic angiography (OCTA) is a novel technology capable of imaging retinal vasculature three-dimensionally at capillary scale without the need to inject any extrinsic dye contrast. However, projection artifacts cause superficial retinal vascular patterns to be duplicated in deeper layers, thus interfering with the clean visualization of some retinal plexuses and vascular pathologies. Projection-resolved OCTA (PR-OCTA) uses post-processing algorithms to reduce projection artifacts. With PR-OCTA, it is now possible to resolve up to 4 distinct retinal vascular plexuses in the living human eye. The technology also allows us to detect and distinguish between various retinal and optic nerve diseases. For example, optic nerve diseases such as glaucoma primarily reduces the capillary density in the superficial vascular complex, which comprises the nerve fiber layer plexus and the ganglion cell layer plexus. Outer retinal diseases such as retinitis pigmentosa primarily reduce the capillary density in the deep vascular complex, which comprises the intermediate capillary plexus and the deep capillary plexus. Retinal vascular diseases such as diabetic retinopathy and vein occlusion affect all plexuses, but with different patterns of capillary loss and vascular malformations. PR-OCTA is also useful in distinguishing various types of choroidal neovascularization and monitoring their response to anti-angiogenic medications. In retinal angiomatous proliferation and macular telangiectasia type 2, PR-OCTA can trace the pathologic vascular extension into deeper layers as the disease progress through stages. Plexus-specific visualization and measurement of retinal vascular changes are improving our ability to diagnose, stage, monitor, and assess treatment response in a wide variety of optic nerve and retinal diseases. These applications will be further enhanced with the continuing improvement of the speed and resolution of the OCT platforms, as well as the development of software algorithms to reduce artifacts, improve image quality, and make quantitative measurements., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to analyze the natural history and phenotypic overlap of patients with microcephaly and a chorioretinopathy or familial exudative vitreoretinopathy (FEVR) ocular phenotype caused by mutations in KIF11, TUBGCP4, or TUBGCP6., Methods: Patients diagnosed with congenital microcephaly and chorioretinopathy or FEVR were included. Molecular investigations consisted of targeted genetic sequencing. Data from medical records, ophthalmologic examination and imaging, electroretinography, and visual fields were analyzed for systemic and ophthalmic features and evidence of posterior segment disease progression., Results: Twelve patients from 9 families were included and had a median of 8 years of follow-up. Nine patients had KIF11 variants, two had heterozygous TUBGCP6 variants, and one had heterozygous variants in TUBGCP4. All patients had reduced visual function and multiple individuals and families showed features of both chorioretinopathy and FEVR. Progression of posterior segment disease was highly variable, with some degree of increased atrophy of the macula or peripheral retina or increased vitreoretinal traction observed in 9 of 12 patients., Conclusions: Microcephaly due to mutations in KIF11, TUBGCP4, or TUBGCP6 can be associated with retinal disease on a spectrum from chorioretinal atrophy to FEVR-like posterior segment changes. Visually significant disease progression can occur and patients should be monitored closely by a team experienced in ophthalmic genetics.

Purpose: To report baseline visual fields in the Rate of Progression in USH2A-related Retinal Degeneration (RUSH2A) study., Design: Cross-sectional study within a natural history study., Methods: Setting: multicenter, international., Study Population: Usher syndrome type 2 (USH2) (n = 80) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP) (n = 47) associated with biallelic disease-causing sequence variants in USH2A., Observation Procedures: Repeatability of full-field static perimetry (SP) and between-eye symmetry of kinetic perimetry (KP) were evaluated with intraclass correlation coefficients (ICCs). The association of demographic and clinical characteristics with total hill of vision (V TOT ) was assessed with general linear models. Associations between V TOT and other functional and morphologic measures were assessed using Spearman correlation coefficients and t tests., Main Outcome Measures: V TOT (SP) and III4e isopter area (KP)., Results: USH2 participants had more severe visual field loss than ARRP participants (P < .001, adjusting for disease duration, age of enrollment). Mean V TOT measures among 3 repeat tests were 32.7 ± 24.1, 31.2 ± 23.4, and 31.7 ± 23.9 decibel-steradians (intraclass correlation coefficient [ICC] = 0.96). Better VA, greater photopic ERG 30-Hz flicker amplitudes, higher mean microperimetry sensitivity, higher central subfield thickness, absence of macular cysts, and higher III4e seeing area were associated with higher V TOT (all r > .48; P < .05). Mean III4e isopter areas for left (4561 ± 4426 squared degrees) and right eyes (4215 ± 4300 squared degrees) were concordant (ICC = 0.94)., Conclusions: USH2 participants had more visual field loss than participants with USH2A-related ARRP, adjusting for duration of disease and age of enrollment. V TOT was repeatable and correlated with other functional and structural metrics, suggesting it may be a good summary measure of disease severity in patients with USH2A-related retinal degeneration., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene therapy is now an effective approach to treat many forms of retinal degeneration. Delivery agents that are cell-specific, allow for multiple dosing regimens, and have low immunogenicity are needed to expand the utility of gene therapy for the retina. We generated eight novel lipid nanoparticles (LNPs) ranging in size from 50 nm to 150 nm by changing the PEG content from 5% to 0.5%, respectively. Subretinal injections of LNP-mRNA encoding luciferase revealed that 0.5% PEG content within nanoparticles elicits the highest expression. Similar injections of LNP delivered cre mRNA into Ai9 mice revealed cell-specific protein expression in the retinal pigment epithelium (RPE), confirmed by fundus photography and immunohistochemistry of whole globe cross-sections. To investigate mechanisms of LNP delivery to the eye, we injected mCherry mRNA using the subretinal approach in apoE-/- and Mertk-/- mice. RPE transfection was observed in both mouse models suggesting that LNP intracellular delivery is not solely dependent on apolipoprotein adsorption or phagocytosis. To investigate LNP penetration, particles were delivered to the vitreous chamber via an intravitreal injection. The 0.5% PEG particles mediated the highest luciferase activity and expression was observed in the Müller glia, the optic nerve head and the trabecular meshwork, but failed to reach the RPE. Overall, particles containing less PEG (~150 nm in size) mediated the highest expression in the eye. Thus far, these particles successfully transfect RPE, Müller cells, the optic nerve head and the trabecular meshwork based on route of administration which can expand the utility of LNP-mediated gene therapies for the eye., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Treatments that delay retinal cell death regardless of genetic causation are needed for inherited retinal degeneration (IRD) patients. The ketogenic diet is a high-fat, low-carbohydrate diet, used to treat epilepsy, and has beneficial effects for neurodegenerative diseases. This study aimed to determine whether the ketogenic diet could slow retinal degeneration., Methods: Early weaned, rd10 and wild-type (WT) mice were placed on either standard chow, a ketogenic diet, or a ketogenic & low-protein diet. From postnatal day (PD) 23 to PD50, weight and blood β-hydroxybutyrate levels were recorded. Retinal thickness, retinal function, and visual performance were measured via optical coherence tomography, electroretinography (ERG), and optokinetic tracking (OKT). At PD40, serum albumin, rhodopsin protein, and phototransduction gene expression were measured., Results: Both ketogenic diets elicited a systemic induction of ketosis. However, rd10 mice on the ketogenic & low-protein diet had significant increases in photoreceptor thickness, ERG amplitudes, and OKT thresholds, whereas rd10 mice on the ketogenic diet showed no photoreceptor preservation. In both rd10 and WT mice, the ketogenic & low-protein diet was associated with abnormal weight gain and decreases in serum albumin levels, 27% and 56%, respectively. In WT mice, the ketogenic & low-protein diet was also associated with an ∼20% to 30% reduction in ERG amplitudes., Conclusions: The ketogenic & low-protein diet slowed retinal degeneration in a clinically relevant IRD model. In WT mice, the ketogenic & low-protein diet was associated with a decrease in phototransduction and serum albumin, which could serve as a protective mechanism in the rd10 model. Although ketosis was associated with protection, its role remains unclear., Translational Relevance: Neuroprotective mechanisms associated with the ketogenic & low-protein diet have potential to slow retinal degeneration., Competing Interests: Disclosure: R.C. Ryals, None; S.J. Huang, None; D. Wafai, None; C. Bernert, None; W. Steele, None; M. Six, None; S. Bonthala, None; H. Titus, None; P. Yang, None; M. Gillingham, None; M.E. Pennesi, None, (Copyright 2020 The Authors.)

Purpose: The purpose of this study was to evaluate baseline best corrected visual acuity (BCVA), full-field electroretinography (ERG), full-field stimulus thresholds (FST), and their relationship with baseline demographic and clinical characteristics in the Rate of Progression in Usher syndrome type 2 ( USH2A )-related Retinal Degeneration (RUSH2A) multicenter study., Methods: Participants had Usher syndrome type 2 (USH2, N = 80) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP, N  = 47) associated with biallelic variants in the USH2A gene. Associations of demographic and clinical characteristics with BCVA, ERG, and FST were assessed with regression models., Results: In comparison to ARRP, USH2 had worse BCVA (median 79 vs. 82 letters; P < 0.001 adjusted for age), lower rod-mediated ERG b-wave amplitudes (median 0.0 vs. 6.6 µV; P < 0.001) and 30 Hz flicker cone-mediated ERG amplitudes (median 1.5 vs. 3.1 µV; P  = 0.001), and higher (white, blue, and red) FST thresholds (means [-26, -31, -23 dB] vs. [-39, -45, -28 dB]; P < 0.001 for all stimuli). After adjusting for age, gender, and duration of vision loss, the difference in BCVA between diagnosis groups was attenuated ( P  = 0.09). Only diagnosis was associated with rod- and cone-mediated ERG parameters, whereas both genders ( P  = 0.04) and duration of visual loss ( P < 0.001) also were associated with FST white stimulus., Conclusions: USH2 participants had worse BCVA, ERG, and FST than ARRP participants. FST was strongly associated with duration of disease; it remains to be determined whether it will be a sensitive measure of progression., Translational Relevance: Using standardized research protocols in RUSH2A, measures have been identified to monitor disease progression and treatment response and differentiate features of prognostic relevance between USH2 and ARRP participants with USH2A mutations., Competing Interests: Disclosure: D.G. Birch, Biogen/Nightstarx (F), Acucela (C), ProQR (F, C), Nacuity (C), AGTC (F, C), Editas (C), 4D Therapeutics (F, C); P. Cheng None; J.L. Duncan, 4D Therapeutics (C), Acucela (F), AGTC (C), DTx Pharma, Inc. (C), Editas (C), Eloxx (C), Gyroscope (C), Biogen/Nightstarx (F, C), ProQR (C), Spark (C), SparingVision (S), Vedere Bio (S), Horama (C); A.R. Ayala None; M.G. Maguire Genentech/Roche (C), Regenera (C); I. Audo Novartis (C), SparingVision (C); J.K. Cheetham, AbbVie (I); T.A. Durham None; A.T. Fahim None; F.L. Ferris III, Bausch and Lomb (P, R), Janssen Research & Development, LLC (C) Viewpoint Therapeutics, Inc (C), Genetech (C), Norvo Nordisk (C), Apellis (C), Roche (C), SemaThera PTC Therapeutics (C), Regenera (C), Novartis (C), Eyevensys (C), Kodiak (C), 4D Molecular (C), Clear Side Biomedical (C); E. Heon None; R.M. Huckfeldt, AGTC (F), Spark (F), Biogen (F), ProQR (F), MeiraGTx (F); A. Iannaccone, Alia Therapeutics (S), ClearView Healthcare Partners (C), Teladoc Health (C), GLG Group (C), Guidepoint (C), Astellas Institute for Regenerative Medicine (C), Roivant Pharma (C), Editas (C), Rhythm Pharmaceuticals (C), IQVIA (C), Gyroscope (C), Ocugen (C), AGTC (F), AbbVie (F), Acucela (F), ProQR (F), Retinagenix (F), 4D Molecular Therapeutics (F), BridgeBio Pharma/Retinagenix (F); N.W. Khan None; E.M. Lad, Biogen/Nightstarx (C) and Novartis (C); M. Michaelides, MeiraGTx (F), Stargazer Pharma (F), Acucela (F), Astellas (F), ProQR (F), 2CTech (F); M.E. Pennesi, AbbVie/Editas (C), Spark Therapeutics (C), Wave Biosciences (C), Astellas Pharmaceuticals (C), RegenexBio (C), Iveric (C), Biogen (C), Novartis (C), Adverum (C), Gensight (F), ProQR (F), Horama (F), Eyevensys (F), Nayan (F) (I), Nacuity (F, I), Ocugen (F, I), Vedere Bio (F, I), SparingVision (F, I), AGTC (C), Sanofi (C); K. Stingl, ProQR (F); A. Vincent, ADVERUM Biotechnologies INC (C); C.Y. Weng, Allergan/AbbVie, Inc. (C), Alcon (C), Alimera Sciences (C), Regeneron (C), Novartis (C), Dutch Ophthalmic Research Center (C), (Copyright 2020 The Authors.)

Purpose : To determine the interocular symmetry of foveal cone topography in achromatopsia (ACHM) using non-confocal split-detection adaptive optics scanning light ophthalmoscopy (AOSLO). Methods : Split-detector AOSLO images of the foveal cone mosaic were acquired from both eyes of 26 subjects (mean age 24.3 years; range 8-44 years, 14 females) with genetically confirmed CNGA3 - or CNGB3 -associated ACHM. Cones were identified within a manually delineated rod-free zone. Peak cone density (PCD) was determined using an 80 × 80 μm sampling window within the rod-free zone. The mean and standard deviation (SD) of inter-cell distance (ICD) were calculated to derive the coefficient of variation (CV). Cone density difference maps were generated to compare cone topography between eyes. Results : PCD (mean ± SD) was 17,530 ± 9,614 cones/mm 2 and 17,638 ± 9,753 cones/mm 2 for right and left eyes, respectively ( p = .677, Wilcoxon test). The mean (± SD) for ICD was 9.05 ± 2.55 µm and 9.24 ± 2.55 µm for right and left eyes, respectively ( p = .410, paired t -test). The mean (± SD) for CV of ICD was 0.16 ± 0.03 µm and 0.16 ± 0.04 µm for right and left eyes, respectively ( p = .562, paired t -test). Cone density maps demonstrated that cone topography of the ACHM fovea is non-uniform with local variations in cone density between eyes. Conclusions : These results demonstrate the interocular symmetry of the foveal cone mosaic (both density and packing) in ACHM. As cone topography can differ between eyes of a subject, PCD does not completely describe the foveal cone mosaic in ACHM. Nonetheless, these findings are of value in longitudinal monitoring of patients during treatment trials and further suggest that both eyes of a given subject may have similar therapeutic potential and non-study eye can be used as a control.

Novel therapeutics for inherited retinal dystrophies (IRDs) have rapidly evolved since groundbreaking clinical trials for LCA due to RPE65 mutations led to the first FDA-approved in vivo gene therapy. Since then, advancements in viral vectors have led to more efficient AAV transduction and developed other viral vectors for gene augmentation therapy of large gene targets. Furthermore, significant developments in gene editing and RNA modulation technologies have introduced novel capabilities for treatment of autosomal dominant diseases, intronic mutations, and/or large genes otherwise unable to be treated with current viral vectors. We highlight strategies currently being evaluated in gene therapy clinical trials and promising preclinical developments for IRDs., (© 2020 Wiley Periodicals LLC.)

Purpose: To determine the effect of mycophenolate mofetil (MMF) on retinal degeneration on two mouse models of retinitis pigmentosa., Methods: Intraperitoneal injections of MMF were administered daily in rd10 and c57 mice starting at postoperative day 12 (P12) and rd1 mice starting at P8. The effect of MMF was assessed with optical coherence tomography, immunohistochemistry, electroretinography, and OptoMotry. Whole retinal cyclic guanosine monophosphate (cGMP) and mycophenolic acid levels were quantified with mass spectrometry. Photoreceptor cGMP cytotoxicity was evaluated with cell counts of cGMP immunostaining., Results: MMF treatment significantly delays the onset of retinal degeneration and cGMP-dependent photoreceptor cytotoxicity in rd10 and rd1 mice, albeit a more modest effect in the latter. In rd10 mice, treatment with MMF showed robust preservation of the photoreceptors up to P22 with associated suppression of cGMP immunostaining and microglial activation; The neuroprotective effect diminished after P22, but outer retinal thickness was still significantly thicker by P35 and OptoMotry response was significantly better up to P60. Whereas cGMP immunostaining of the photoreceptors were present in rd10 and rd1 mice, hyperphysiological whole retinal cGMP levels were observed only in rd1 mice., Conclusions: Early treatment with MMF confers potent neuroprotection in two animal models of RP by suppressing the cGMP-dependent common pathway for photoreceptor cell death. The neuroprotective effect of MMF on cGMP-dependent cytotoxicity occurs independently of the presence of hyperphysiological whole retinal cGMP levels. Thus our data suggest that MMF may be an important new class of neuroprotective agent that could be useful in the treatment of patients with RP.

Importance: Recent studies have linked a vision-threatening maculopathy with long-term use of pentosan polysulfate sodium (PPS)., Objective: To evaluate the disease course in PPS-associated maculopathy after drug cessation., Design, Setting, and Participants: In this retrospective case series, patients diagnosed with PPS-associated maculopathy with at least 6 months of follow-up after drug cessation who were treated at the Emory Eye Center, Atlanta, Georgia, or the Casey Eye Institute, Portland, Oregon, were included. Data were collected from April 2014 through November 2019., Main Outcomes and Measures: Change in visual acuity and retinal imaging characteristics over time., Results: Of the 11 included patients, all were female, and the median (interquartile range [IQR]) age was 53 (44-63) years. Participants had a baseline visit at a median (IQR) of 2 (0-4) months after drug cessation and were subsequently observed for a median (IQR) of 12 (8-26) months. The median (IQR) cumulative PPS exposure was 1.97 (1.55-2.18) kg. No eyes exhibited a demonstrable improvement in disease after discontinuing PPS. A total of 9 of 11 patients (82%) reported worsening visual symptoms at the final visit. The mean (SD) logMAR visual acuity was 0.14 (0.23) and 0.14 (0.34) at the baseline and final visit, respectively. Visual acuity improved by 2 or more Snellen lines in 1 eye (5%) and declined by 2 or more Snellen lines in 2 eyes of 1 patient (9%). There was evolution in the pattern of fundus autofluorescence changes and/or optical coherence tomography findings in all eyes. A total of 17 eyes (77%) exhibited expansion of the area of involved tissue. A total of 7 eyes (32%) had macular retinal pigment epithelium atrophy at the baseline visit, and atrophy enlarged after discontinuation of PPS in all 7 eyes, with a median (IQR) growth rate of 0.32 (0.13-0.38) mm per year., Conclusions and Relevance: These retrospective data among 11 patients suggest PPS-associated maculopathy continues to evolve after drug cessation for at least 10 years. In some cases, progressive retinal pigment epithelium atrophy encroaches on the foveal center and thus may pose a long-term threat to central vision.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety., Competing Interests: Disclosure: D.A. Thompson, MeiraGTx (F), filed patent on gene therapy (P); A. Iannaccone, Editas Medicine (C), Astellas (C), Roivant Pharma (C), IQVIA (C), Gyroscope/Orbit Biomedical (C), Rhythm Pharmaceuticals (C), Applied Genetic Technologies Corp (F), Allergan (F), Acucela (F), ProQR Therapeutics (F), Foundation Fighting Blindness Clinical Research Institute (F), Alia Therapeutics (S); R.R. Ali, MeiraGTx (S), filed patents on gene and cell therapies (P); V.Y. Arshavsky, None; I. Audo, None; J.W.B. Bainbridge, MeiraGTx (S), filed patents on gene and cell therapies (P); C.G. Besirli, MeiraGTx (F), Spark Therapeutics (F); D.G. Birch, Nightstar Therapeutics/Biogen (C), Applied Genetic Technologies Corp (S), Nacuity Pharmaceuticals (C), Editas Medicine (C), Acucela (S), Foundation Fighting Blindness (C), ProQR Therapeutics (C); K.E. Branham, ProQR Therapeutics (C); A.V. Cideciyan, Patents on gene therapies for RPGR, RHO, and NPHP5-associated IRDs (P); ProQR Therapeutics (F), IVERIC bio (F); S.P. Daiger, Applied Genetic Technologies Corp (S); D. Dalkara, Patent on AAV virions with variant capsid and methods of use (P); J.L. Duncan, 4D Therapeutics (C), Applied Genetic Technologies Corp (C), Editas Medicine (C), Gyroscope (C), Horama (C), Nightstar Therapeutics/Biogen (C), ProQR Therapeutics (C), SparingVision (S), Spark Therapeutics (C), Vedere Bio (C), Acucela (F), Allergan (F), Neurotech (F), Second Sight Medical Products (F); A.T. Fahim, None; J.G. Flannery, None; R. Gattegna, None; J.R. Heckenlively, None; E. Heon, None; K.T. Jayasundera, Editas Medicine (C); N.W. Khan, None; H. Klassen, jCyte (S, I); B.P. Leroy, Bayer (C), Nightstar Therapeutics/Biogen (C), IVERIC bio (C), Novartis Pharma (C), RegenX Bio (C), Vedere Bio (C); Novartis (R), Spark Therapeutics (R); GenSight Biologics (F), ProQR Therapeutics (F); R.S. Molday, Applied Genetic Technologies Corp (S); D.C. Musch, Belite Bio (S), Mactel Group NTMT-03 trial (S), NEI intramural branch clinical trials (S); M.E. Pennesi, Adverum (C), Allergan (C), Astellas (C), Pharmaceuticals (C), Biogen (C), IVERIC bio (C), Novartis (C), Regenix Bio (C), Spark Therapeutics (C), Wave Biosciences (C), Eyevensys (S), Horama (S), Nayan (S), Nacuity Pharmaceuticals (S, I), Ocugen (S, I), Verede (S), ProQR Therapeutics (S), Gensight (S), Applied Genetic Technologies Corp (F), Sanofi (F), Foundation Fighting Blindness (F), ProQR Therapeutics (F), Allergan (F); S.M. Petersen-Jones, None; E.A. Pierce, Astellas (C), Merck (C), Sanofi-Genzyme (C), Wave Therapeutics (C); GenSight Biologics (S), Odylia Therapeutics (S), Opsis Therapeutics (S), Sana Biotechnology (S); CRISPR Therapeutics (F), Spark Therapeutics (F); Editas Medicine (F), MeiraGTx (F), ProQR Therapeutics (F), filed patents on gene and genetic therapies (P); R.C. Rao, None; T.A. Reh, Nayan Pharmaceuticals (C, I); J.A. Sahel, Pixium Vision (S, I), GenSight Biologics (S, I), SparingVision (S, I); Prophesee (I), Chronolife (I); D. Sharon, None; P.A. Sieving, None; E. Strettoi, None; P. Yang, Astellas (C), Applied Genetic Technologies Corp (C); D.N. Zacks, ONL Therapeutics (C, I, P), (Copyright 2020 The Authors.)

Purpose: To evaluate the phenotypic spectrum of autosomal recessive RP1- associated retinal dystrophies and assess genotypic associations., Methods: A retrospective multicenter study was performed of patients with biallelic RP1 -associated retinal dystrophies. Data including presenting symptoms and age, visual acuity, kinetic perimetry, full field electroretinogram, fundus examination, multimodal retinal imaging, and RP1 genotype were evaluated., Results: Nineteen eligible patients from 17 families were identified and ranged in age from 10 to 56 years at the most recent evaluation. Ten of the 21 unique RP1 variants identified were novel, and mutations within exon 2 accounted for nearly half of alleles across the cohort. Patients had clinical diagnoses of retinitis pigmentosa (13), cone-rod dystrophy (3), Leber congenital amaurosis (1), early-onset severe retinal dystrophy (1), and macular dystrophy (1). Macular atrophy was a common feature across the cohort. Symptom onset occurred between 4 and 30 years of age (mean 14.9 years, median 13 years), but there were clusters of onset age that correlated with the effects of RP1 mutations at a protein level. Patients with later-onset disease, including retinitis pigmentosa, had at least one missense variant in an exon 2 DCX domain., Conclusions: Biallelic RP1 mutations cause a broad spectrum of retinal disease. Exon 2 missense mutations are a significant contributor to disease and can be associated with a considerably later onset of retinitis pigmentosa than that typically associated with biallelic RP1 mutations., (Copyright © 2020 Molecular Vision.)

Background: To analyze intraoperative OCT (iOCT) findings during subretinal gene therapy., Methods: A single-center, retrospective, observational, case series study of twenty one eyes submitted to subretinal gene therapy. Intrasurgical high definition videos were included for analyzes. Cases with absence of iOCT video or unsuccessful bleb creation were excluded. Sharp needle tip (SNT) or blunted needle tip (BNT) and their interaction with neurosensory retina were evaluated. Presence of subretinal air bubbles, visible opened retinotomy, and medication reflux were also correlated and analyzed., Results: Nineteen of twenty-one eyes were included. Of the two excluded eyes, subretinal bleb creation was unsuccessful in one and technical issues prevented OCT image acquisition in the other. Immediately before subretinal injection, needle indention/penetration of the neurosensory retina with temporary indentation of the RPE/choroid was evident in 16 (84%) of the 19 eyes. Complete RPE/choroid indentation was needed with BNT use compared to SNT (p = 0.0114). An open retinotomy was identified in 14 (74%) of 19 eyes at the conclusion of bleb injection and was more commonly associated with SNT (p = 0.0108)., Conclusions: iOCT provides valuable real-time feedback of cross-sectional retinal anatomy during subretinal gene therapy surgeries. The type of needle tip and its use during the gene therapy procedure seems to influence in the bleb creation and presence of visible open retinotomy. Further studies of iOCT findings during gene therapy delivery procedures are likely to help refine the surgical technique., Competing Interests: Competing interestsHMV—No competing interests. BJL—Translational Imaging Innovations, Inc: advisor/stock; University of California, Berkeley: patent/IP. MEP—Editas/Allergan: consultant; ProQR Therapeutics: consultant; Nacuity Pharmaceuticals: consultant; Astellas Pharmaceuticals: consultant; Gensight: consultant; Horama: consultant; Sparks Therapeutics: consultant; Wavemetrics: consultant; Eyevensys: consultant; Nayan: consultant; AGTC: consultant/clinical trial support; Sanofi: consultant/clinical trial support; Foundation Fighting Blindness: consultant; Nightstar/Biogen: consultant/clinical trial support. PY—Sparks Therapeutics: clinical trial support; AGTC: clinical trial support; Sanofi: clinical trial support; Foundation Fighting Blindness: clinical trial support; Nightstar/Biogen: clinical trial support; Acucela: clinical trial support. AKL – Editas/Allergan: clinical trial support; Genentech: clinical trial support; IVERIC bio: clinical trial support; Nightstar/Biogen: clinical trial support; Sanofi: clinical trial support., (© The Author(s) 2020.)

Background: Alström syndrome (AS) is a rare monogenic disorder characterized by progressive multi-organ pathology including retinal degeneration, hearing impairment and type 2 diabetes. Here we present clinical features in two siblings diagnosed with Alström syndrome associated with two novel changes in ALMS1., Case Presentation: Two siblings originally diagnosed as having achromatopsia presented with mild light sensitivity, nonspecific otitis media, and mild developmental delay during the first decade of life with a relatively stable ocular appearance during second decade, late onset of nystagmus and dyschromatopsia (after 20 years) and preserved vision during the third decade of life. One sibling had late onset hearing loss and both siblings had symmetric high myopia, normal stature, and ptosis. Clinical findings revealed structural and functional tests consistent with a cone-rod dystrophy. Novel variants c.9894dupC (p.S3298 fs) and c.10769delC (p.T3590 fs) in ALMS1 gene were found., Conclusions: Two North American siblings who presented with a mild clinical phenotype of Alström syndrome were found to have novel mutations in ALMS1. These two frame-shift mutations segregated with the disease phenotype lending evidence to their pathogenicity.

Background: Defects in retinol dehydrogenase 12 ( RDH12 ) account for 3.4%-10.5 % of Leber congenital amaurosis and early-onset severe retinal dystrophy (EOSRD) and are a potential target for gene therapy. Clinical trials in inherited retinal diseases have unique challenges, and natural history studies are critical to successful trial design. The purpose of this study was to characterise the natural history of RDH12 -associated retinal degeneration., Methods: A retrospective chart review was performed in individuals with retinal degeneration and two likely disease-causing variants in RDH12 ., Results: 57 subjects were enrolled from nine countries. 33 subjects had clinical records available from childhood. The data revealed an EOSRD, with average age of onset of 4.1 years. Macular atrophy was a universal clinical finding in all subjects, as young as 2 years of age. Scotopic and photopic electroretinography (ERG) responses were markedly reduced in all subjects, and a non-recordable ERG was documented as young as 1 year of age. Assessment of visual acuity, visual field and optical coherence tomography revealed severe loss of function and structure in the majority of subjects after the age of 10 years. Widefield imaging in 23 subjects revealed a unique, variegated watercolour-like pattern of atrophy in 13 subjects and sparing of the peripapillary area in 18 subjects., Conclusions: This study includes the largest collection of phenotypic data from children with RDH12 -associated EOSRD and provides a comprehensive description of the timeline of vision loss in this severe, early-onset condition. These findings will help identify patients with RDH12 -associated retinal degeneration and will inform future design of therapeutic trials., Competing Interests: Competing interests: ATF reports a grant from the Vitreoretinal Surgery Foundation and a K12 grant from the National Institute of Health, during the conduct of the study; other from Ionis Pharamceuticals, outside the submitted work. RRA reports a patent on RDH12 gene therapy. DAT reports a grant from the Foundation Fighting Blindness, during the conduct of the study; In addition, DAT has a patent for viral vectors comprising RDH12 coding regions and methods of treating retinal dystrophies pending., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background : Ring 14 syndrome is a rare chromosomal disorder characterized by a ring-shaped appearance of chromosome 14. Classically findings include distinct facial characteristics, refractory epilepsy, global development delay, muscular hypotonia and ocular abnormalities. Here we report a retinal multimodal imaging analyses of a ring chromosome 14 syndrome patient with associated macular pigmentary changes. Materials and Methods : Case report of an 11-year-old female with a history of refractory epilepsy since 3 months of age was diagnosed with ring 14 syndrome after karyotype at 8 months old. She presented with muscle weakness, mild intellectual delay, associated hyperopia and punctiform yellowish lesions. Multimodal imaging including fundus photography, red-free fundus photography, fundus auto-fluorescence and spectral-domain optical coherence tomography were used to assess this patient. Results : An 11-year-old female with ring 14 syndrome caused by the fusion of terminal breakpoints in both the short arm and long arm of chromosome 14 at p11.1 and q32.3, respectively. At eye exam, the best corrected visual acuity was 20/20 at both eyes with associated hyperopia. Macula showing scattered punctiform yellowish lesions, bright on red-free fundus photography and hyperautofluorescence dots in the same area. The SD-OCT showed normal characteristics at both eyes with the exception of localized irregularity of the RPE in an area associated with a macular yellow dots. Conclusions : Ring 14 syndrome can cause hyperopia and associated macular yellow dots visible at multimodal imaging analyses. Our data support regular eye examination for all patients with ring chromosome 14 syndrome.

Purpose: Choroideremia is an incurable, X-linked, recessive retinal dystrophy caused by loss of function mutations in the CHM gene. It is estimated to affect approximately 1 in 50,000 male patients. It is characterized by progressive degeneration of the retinal pigment epithelium, choroid, and photoreceptors, resulting in visual impairment and blindness. There is an unmet need in choroideremia, because currently, there are no approved treatments available for patients with the disease., Methods: We review the patient journey, societal impact, and emerging treatments for patients with choroideremia., Results: Its relative rarity and similarities with other retinal diseases in early years mean that diagnosis of choroideremia can often be delayed. Furthermore, its impact on affected individuals, and wider society, is also likely underestimated. AAV2-mediated gene therapy is an investigational treatment that aims to replace the faulty CHM gene. Early-phase studies reported potentially important visual acuity gains and maintenance of vision in some patients, and a large Phase 3 program is now underway., Conclusion: Choroideremia is a disease with a significant unmet need. Interventions that can treat progression of the disease and improve visual and functional outcomes have the potential to reduce health care costs and enhance patient quality of life.

Importance: A unique pigmentary maculopathy was recently described in 6 patients with long-term exposure to pentosan polysulfate sodium (PPS), a long-standing oral therapy for interstitial cystitis., Objective: To characterize the exposure characteristics and clinical manifestations of PPS-associated maculopathy., Design, Setting, and Participants: In this multi-institutional case series, medical records of patients who exhibited the characteristic maculopathy in the setting of prior PPS exposure were retrospectively reviewed. Data were collected from August 1, 2012, to October 1, 2018, and data were analyzed from October 2018 to January 2019., Main Outcomes and Measures: Drug exposure, visual acuity, and retinal imaging characteristics., Results: Of the 35 included patients (70 eyes), 34 (97%) were female, and the median (range) age was 60 (37-79) years. The median (range) duration of PPS intake was 15 (3-22) years, and the median (range) cumulative exposure was 1.61 (0.44-4.31) kg. The leading visual symptoms were metamorphopsia, blurred vision, and prolonged dark adaptation. Median (range) logMAR visual acuity of all eyes was 0.10 (-0.12 to 1.18). Fundus examination often revealed hyperpigmented macular spots (34 of 64 eyes [53%]) with interspersed pale-yellow deposits, although less commonly in eyes that exhibited retinal pigment epithelial atrophy (6 of 26 eyes [23%]; P < .001). Optical coherence tomography showed foci of retinal pigment epithelium elevation or thickening associated with hyperreflectance on near-infrared reflectance imaging. Fundus autofluorescence imaging typically revealed a symmetric, confluent pattern of hyperautofluorescent and hypoautofluorescent spots that involved the fovea in all eyes and extended to the retinal periphery in 24 eyes (36%). Longitudinal evaluation demonstrated dynamic changes in pigmentary abnormalities., Conclusions and Relevance: These findings suggest that PPS-associated maculopathy is a vision-threatening condition that can manifest in the setting of long-term exposure to the drug. Multimodal imaging posits a distinctive clinical phenotype, characterized in this cohort by dynamic alterations within the retinal pigment epithelium and at the retinal pigment epithelium-photoreceptor interface. Ongoing work might explore causality and direct screening guidelines.

Purpose: To use projection-resolved optical coherence tomographic angiography (PR-OCTA) to characterize the microvascular changes in 3 distinct retinal plexuses in retinitis pigmentosa (RP) patients., Design: Prospective cross-sectional study., Methods: A commercial 70-kHz spectral-domain optical coherence tomography (OCT) system was used to acquire 6-mm macular scans from RP patients and age-matched healthy participants at a tertiary academic center. Blood flow was detected using a commercial version of split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. The PR-OCTA algorithm was used to suppress projection artifacts and resolve microvasculature in 3 plexuses around the macula. Vessel density was calculated from en face OCTA of the parafoveal and perifoveal regions in each of the 3 plexuses, as well as the all-plexus inner retinal slab. Inner and outer retinal thicknesses were measured form structural OCT scans. Generalized estimating equations and Spearman's rank correlation statistical methods were used., Results: Forty-four eyes from 26 RP patients and 34 eyes from 26 healthy subjects were included. Significant reduction in vessel density was detected in the perifovea but not the parafovea of inner retinal slab of RP patients (P = .001 and P = .56, respectively) compared to controls. We also found deeper retinal plexuses (intermediate and deep capillary plexuses, ICP and DCP) were primarily damaged by RP, compared to superficial vascular complex (SVC). Significant thickening of the inner retina and thinning of the outer retina were also observed. Strong correlation was found between the vessel density in the perifoveal ICP and DCP and outer retinal thickness in RP patients with no history of cystoid macular edema., Conclusions: PR-OCTA enables the detection of microvascular changes in the perifoveal regions of the ICP and DCP in RP, with relative sparing of the SVC. OCT and OCTA parameters might be able to provide better understanding of the pathophysiology of the disease, as well as monitoring disease progression and the response to experimental treatments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background/aims: To better understand the pattern of degeneration progression in cases with choroideremia., Methods: A cohort of genotypically confirmed choroideremia cases who underwent optical coherence tomography (OCT) and fundus autofluorescence (FAF) imaging was studied. Using HEYEX review software, the foveal centre was marked on FAF images under guidance of corresponding OCT images, followed by application of an ETDRS grid. The boundaries of preserved autofluorescence (AF) were manually segmented in each individual ETDRS subfield. The regional distribution of preserved AF was assessed by comparing its area among the various subfields., Results: A total of 168 eyes from 84 choroideremia cases were enrolled. There was a statistically significant difference in the amount of preserved AF area between inner subfields as determined by one-way analysis of variance (F (3,668)=9.997, p<0.001) and also between outer subfields (F (3,668)=8.348, p<0.001). A Tukey posthoc test revealed that the preserved AF area in the nasal subfields in both the inner and outer subfields was significantly smaller compared with analogue subfields., Conclusion: The asymmetric spatial distribution of preserved AF in choroideremia (corresponding to the stellate shaped nature of these regions) suggests that the progression of degeneration has directional preference., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Retinal gene therapy has had unprecedented success in generating treatments that can halt vision loss. However, immunogenic response and long-term toxicity with the use of viral vectors remain a concern. Non-viral vectors are relatively non-immunogenic, scalable platforms that have had limited success with DNA delivery to the eye. Messenger RNA (mRNA) therapeutics has expanded the ability to achieve high gene expression while eliminating unintended genomic integration or the need to cross the restrictive nuclear barrier. Lipid-based nanoparticles (LNPs) remain at the forefront of potent delivery vectors for nucleic acids. Herein, we tested eleven different LNP variants for their ability to deliver mRNA to the back of the eye. LNPs that contained ionizable lipids with low pKa and unsaturated hydrocarbon chains showed the highest amount of reporter gene transfection in the retina. The kinetics of gene expression showed a rapid onset (within 4 h) that persisted for 96 h. The gene delivery was cell-type specific with majority of the expression in the retinal pigmented epithelium (RPE) and limited expression in the Müller glia. LNP-delivered mRNA can be used to treat monogenic retinal degenerative disorders of the RPE. The transient nature of mRNA-based therapeutics makes it desirable for applications that are directed towards retinal reprogramming or genome editing. Overall, non-viral delivery of RNA therapeutics to diverse cell types within the retina can provide transformative new approaches to prevent blindness., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: This study measured and correlated degeneration of the junction between the inner and outer segments (IS/OS), the retinal pigment epithelium (RPE), and the choriocapillaris (CC) in Stargardt disease (STGD)., Design: Prospective cross-sectional study., Methods: This study was conducted at the Casey Eye Institute. A total of 23 patients with STGD were enrolled and underwent optical coherence tomography angiography (OCTA). Scans were centered on the fovea. OCT slab projections and en face boundary maps were used to create masks to measure total IS/OS loss or RPE atrophy as well as regions of isolated IS/OS loss, isolated RPE atrophy, and matched IS/OS and RPE degeneration or intact IS/OS junction and RPE. CC vascular density (CCVD) was quantified from the CC angiogram. Outcomes included the area of loss, and the CCVD of degeneration in different areas was quantified and correlated., Results: The total area of IS/OS loss was strongly correlated with the total area of RPE atrophy (r = 0.96; P < 0.0001) by a 1.6:1 ratio (r 2  = 0.90). CCVD within regions of matched degeneration (85.6% ± 2.7%; P < 0.0001), isolated IS/OS junction loss (93.6% ± 1.0%; P = 0.0011), and isolated RPE atrophy (94.1% ± 1.1%; P = 0.0065) were all significantly lower than normal (99.0% ± 0.17%). There was a trend for CCVD within intact areas (97.6% ± 0.38%) to decline as the area diminished (r = 0.68)., Conclusions: Photoreceptor and RPE degeneration exhibited a strong relationship wherein the IS/OS loss was 1.6-fold greater than that of RPE atrophy, supporting the theory that photoreceptor degeneration precedes RPE in STGD. Both the photoreceptors and the RPE degeneration contributed synergistically to CCVD attenuation, but extralesional CCVD also tended to be abnormal. The findings and techniques in this study may be of utility in developing endpoints for clinical trials., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: We determine the intersession repeatability of cone measurements via flood-illuminated adaptive optics (AO) imaging in patients with retinitis pigmentosa (RP), to better differentiate variation due to imaging inaccuracies versus pathology-driven change., Methods: A total of 25 4° × 4° AO images were acquired three times on the same day in 10 subjects with RP, registered in i2K Retina, and cones were identified using a custom-built MATLAB algorithm. Nine equally spaced regions of interest were selected for each imaging set. A subset of subjectively "poor" and "good" quality images was selected by three independent graders, analyzed using cone density, cone location similarity (CLS) and cone spacing, and compared to age-matched normals., Results: The coefficient of variation (CoV), repeatability, and percent repeatability of automated cone density were slightly higher in patients with RP compared to age-matched normals, but showed no statistically significant difference. The standard deviation of CLS and cone spacing of nearest-neighbor distance demonstrated a statistically significant difference between good- and poor-quality images., Conclusions: Repeatability of automated cone density measurements in patients with RP is comparable to normals. Misidentification of cones due to image quality variability is a major limitation of automated cone counting algorithms in patients with RP. Our study suggests that CLS and cone spacing metrics could be used to help define image quality and, thus, increase confidence in automated cone counts in patients with RP., Translational Relevance: The novel AO image quality assessment metrics described in our study could help to improve patient image interpretation, prognosis, and longitudinal care.

Purpose: To delineate the natural history of visual parameters over time in individuals with biallelic RPE65 mutation-associated inherited retinal dystrophy (IRD); describe the range of causative mutations; determine potential genotype/phenotype relationships; and describe the variety of clinical diagnoses., Design: Global, multicenter, retrospective chart review., Methods: Study Population: Seventy individuals with biallelic RPE65 mutation-associated IRD., Procedures: Data were extracted from patient charts., Measurements: Visual acuity (VA), Goldmann visual field (GVF), optical coherence tomography, color vision testing, light sensitivity testing, and electroretinograms (retinal imaging and fundus photography were collected and analyzed when available)., Results: VA decreased with age in a nonlinear, positive-acceleration relationship (P < .001). GVF decreased with age (P < .0001 for both V4e and III4e), with faster GVF decrease for III4e stimulus vs V4e (P = .0114, left eye; P = .0076, right eye). On average, a 1-year increase in age decreased III4e GVF by ∼25 sum total degrees in each eye while V4e GVF decreased by ∼37 sum total degrees in each eye, although individual variability was observed. A total of 78 clinical diagnoses and 56 unique RPE65 mutations were recorded, without discernible RPE65 mutation genotype/phenotype relationships., Conclusions: The number of clinical diagnoses and lack of a consistent RPE65 mutation-to-phenotype correlation underscore the need for genetic testing. Significant relationships between age and worsening VA and GVF highlight the progressive loss of functional retina over time. These data may have implications for optimal timing of treatment for IRD attributable to biallelic RPE65 mutations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: We determine if monomethyl fumarate (MMF) can protect the retina in mice subjected to light-induced retinopathy (LIR)., Methods: Albino BALB/c mice were intraperitoneally injected with 50 to 100 mg/kg MMF before or after exposure to bright white light (10,000 lux) for 1 hour. Seven days after light exposure, retinal structure and function were evaluated by optical coherence tomography (OCT) and electroretinography (ERG), respectively. Retinal histology also was performed to evaluate photoreceptor loss. Expression levels of Hcar2 and markers of microglia activation were measured by quantitative PCR (qPCR) in the neural retina with and without microglia depletion. At 24 hours after light exposure, retinal sections and whole mount retinas were stained with Iba1 to evaluate microglia status. The effect of MMF on the nuclear factor kB subunit 1 (NF-kB) and Nrf2 pathways was measured by qPCR and Western blot., Results: MMF administered before light exposure mediated dose-dependent neuroprotection in a mouse model of LIR. A single dose of 100 mg/kg MMF fully protected retinal structure and function without side effects. Expression of the Hcar2 receptor and the microglia marker Cd14 were upregulated by LIR, but suppressed by MMF. Depleting microglia reduced Hcar2 expression and its upregulation by LIR. Microglial activation, upregulation of proinflammatory genes (Nlrp3, Caspase1, Il-1β, Tnf-α), and upregulation of antioxidative stress genes (Hmox1) associated with LIR were mitigated by MMF treatment., Conclusions: MMF can completely protect the retina from LIR in BALB/c mice. Expression of Hcar2, the receptor of MMF, is microglia-dependent in the neural retina. MMF-mediated neuroprotection was associated with attenuation of microglia activation, inflammation and oxidative stress in the retina.