Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

Variability of Vascular Reactivity in the Retina and Choriocapillaris to Oxygen and Carbon Dioxide Using Optical Coherence Tomography Angiography.

Purpose: To investigate the regional and layer-specific vascular reactivity of the healthy human retina and choriocapillaris to changes in systemic carbon dioxide or oxygen. Methods: High-resolution 3 × 3-mm2 optical coherence tomography angiography (OCTA) images were acquired from the central macula, temporal macula, and peripapillary retina while participants were exposed to three gas breathing conditions-room air, 5%CO2, and 100% O2. OCTA from all three regions were extracted and the apparent skeletonized vessel density (VSD) was assessed. The mean flow deficit sizes (MFDSs) of the choriocapillaris were also assessed. Repeated-measures analysis of variance was used to compare the ratio of intrasubject VSD change induced by the gas conditions from baseline in the superficial retinal layer (SRL) and deep retinal layer (DRL) for each retinal region independently, as well as the MFDS of the choriocapillaris. We also compared the vessel reactivity between the retinal capillaries and the choriocapillaris. Results: The cumulative intrasubject response to the gas conditions differed significantly among regions of the SRL (F(2, 7) = 28.22, P < 0.001), with the temporal macula showing the largest response (15%) compared to the macula (8%) and radial peripapillary capillaries (7%). A similar trend was found in the DRL. The choriocapillaris reactivity was similar between the macula (5.8%) and temporal macula (5.6%). There was also a significant heterogeneity in the layer-specific gas responses, with the DRL showing the largest response (28.2%) and the choriocapillaris showing the smallest response (2.8%). Conclusions: Capillary reactivity to changes in inhaled O2 and CO2 is spatially heterogeneous across the retina but not choriocapillaris.

Optical Coherence Tomography Angiography-Derived Flux As a Measure of Physiological Changes in Retinal Capillary Blood Flow.

Purpose: To compare optical coherence tomography angiography (OCTA)-derived flux with conventional OCTA measures of retinal vascular density in assessment of physiological changes in retinal blood flow. Methods: Healthy subjects were recruited, and 3 × 3-mm2 fovea-centered scans were acquired using commercially available swept-source OCTA (SS-OCTA) while participants were breathing room air, 100% O2, or 5% CO2. Retinal perfusion was quantified using vessel area density (VAD) and vessel skeleton density (VSD), as well as novel measures of retinal perfusion, vessel area flux (VAF) and vessel skeleton flux (VSF). Flux is proportional to the number of red blood cells moving through a vessel segment per unit time. The percentage change in each measure was compared between the O2 and CO2 gas conditions for images of all vessels (arterioles, venules, and capillaries) and capillary-only images. Statistical significance was determined using paired t-tests and a linear mixed-effects model. Results: Eighty-four OCTA scans from 29 subjects were included (age, 45.9 ± 19.5 years; 14 male, 48.3%). In capillary-only images, the change under the CO2 condition was 168% greater in VAF than in VAD (P = 0.002) and 124% greater in VSF than in VSD (P = 0.004). Similarly, under the O2 condition, the change was 94% greater in VAF than in VAD (P = 0.004) and 57% greater in VSF than in VSD (P = 0.01). Flux measures showed significantly greater change in capillary-only images compared with all-vessels images. Conclusions: OCTA-derived flux measures quantify physiological changes in retinal blood flow at the capillary level with a greater effect size than conventional vessel density measures. Translational Relevance: OCTA-derived flux is a useful measure of subclinical changes in retinal capillary perfusion.

Functional characteristics of glaucoma related arcuate defects seen on OCT en face visualisation of the retinal nerve fibre layer.

PURPOSE: To assess continuity of perimetric defects corresponding to arcuate defects seen on optical coherence tomography (OCT) en face reflectance images of the retinal nerve fibre layer (RNFL) in patients with glaucoma. METHODS: Seven patients with glaucoma who had arcuate structural defects on OCT RNFL en face images were recruited. Static suprathreshold stimuli were presented along different meridians to localise perimetric defects in the corresponding hemifield. Then two contrasts, one 6 dB greater than the other, were used with kinetic perimetry to assess the slope of the defect. Findings with kinetic and 24-2 perimetry were compared. RESULTS: Static perimetry found that regions of perimetric abnormality spatially corresponded with the regions of en face RNFL hyporeflectivity. Kinetic perimetry found that the slopes of the edges of the defects ranged from 3-12 dB degree-1 , and that the functional abnormalities were continuous with the physiologic blind spot even when the 24-2 protocol only showed paracentral defects. CONCLUSIONS: Perimetric abnormalities and arcuate RNFL en face defects were spatially correspondent. Perimetric testing guided by OCT en face reflectance images can reveal greater functional detail of glaucomatous abnormality than 24-2 testing.

Impaired layer specific retinal vascular reactivity among diabetic subjects.

PURPOSE: To investigate layer specific retinal vascular reactivity (RVR) in capillaries of diabetic subjects without DR or with only mild non-proliferative diabetic retinopathy (NPDR). METHODS: A previously described nonrebreathing apparatus was used to deliver room air, 5% CO2, or 100% O2 to 41 controls and 22 diabetic subjects (with mild or no NPDR) while simultaneously acquiring fovea-centered 3x3mm2 Swept-Source Optical Coherence Tomography Angiography (SS-OCTA) images. Vessel skeleton density (VSD) and vessel diameter index (VDI) were calculated for each gas condition for the superficial retinal layer (SRL) and deep retinal layer (DRL). The superficial layer analysis excluded arterioles and venules. Data analysis was performed using mixed factorial analysis of covariance stratified by diabetic status. All models were adjusted for age, gender, and hypertension, and statistical significance for multiple comparisons from posthoc comparisons were defined at p<0.017. RESULTS: Among controls, there was a significant difference in capillary VSD between all gas conditions (p<0.001). This difference was present in both the SRL and DRL. Among diabetics, there was no significant difference in response to CO2 conditions in the SRL (p = 0.072), and a blunted response to both CO2 (p = 0.9) and O2 in the DRL (p = 0.019). A significant gas effect was detected in the capillary VDI in the SRL of controls (p = 0.001), which was driven by higher VDI in the oxygen condition compared to that of carbon dioxide. CONCLUSIONS: Impairment in RVR in diabetic subjects is characterized by a paradoxical response to CO2 in both the SRL and DRL as well as an attenuated response to O2 in the DRL. These layer and gas specific impairments in diabetics seem to occur early in the disease and to be driven primarily at the capillary level.

Lower retinal capillary density in minimal cognitive impairment among older Latinx adults.

INTRODUCTION: We investigated the hypothesis that retinal capillary perfusion is a biomarker of early cognitive decline and cerebrovascular perfusion associated with small vessel disease in a pilot data set of Latinx adults at high risk for vascular cognitive impairment and dementia. METHODS: High-resolution optical coherence tomography angiography (OCTA) images were acquired from dilated eyes of Latinx subjects using a 3 × 3 mm2 scan pattern from a commercially available device. A previously validated method was used to quantify the density of perfused retinal capillaries as the retinal vessel skeleton density (VSD). The association of VSD with Clinical Dementia Rating Sum of Boxes, total Montreal Cognitive Assessment (MoCA) score, and individual MoCA test elements were analyzed using multivariate statistics that adjusted for confounders. VSD was also compared with magnetic resonance imaging (MRI) measures of cerebrovascular reactivity (CVR) and perfusion in the middle cerebral artery perforator (MCA-Perf) territory. RESULTS: The mean (± SD) age of the subjects was 68 (± 6) years. For every 0.01-unit lower VSD, the risk of having a CDR-SOB >0 was 20% higher (95%CI = 5%-90%; P = .031). Similarly, a lower VSD was associated with lower total MoCA score (r = 0.3; P = .038). The Visuospatial/Executive domain of the MoCA assessment showed the strongest association with VSD ( ß  = 0.02; P = .022). Lower retinal VSD was associated with worse MRI measure of CVR (r = 0.7, P = .04) and less perfusion in the MCA-Perf territory (r = 0.45, P = .02). DISCUSSION: Impaired retinal capillary perfusion is associated with cognitive impairment and abnormalities in cerebrovascular perfusion and function. OCTA-based retinal capillary assessment holds promise for identifying and quantifying retinal correlates of neurovascular abnormalities associated with vascular cognitive impairment.

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

Quantifying Subclinical and Longitudinal Microvascular Changes Following Episcleral Plaque Brachytherapy Using Spectral Domain-Optical Coherence Tomography Angiography.

PURPOSE: To assess longitudinal microvascular changes in eyes treated with I-125 episcleral plaque brachytherapy (EPB). METHODS: High resolution OCT angiograms of the central 3×3mm macula were obtained from I-125 episcleral plaque brachytherapy treated and untreated fellow eyes of 61 patients. Capillary density (vessel skeleton density, VSD) and caliber (vessel diameter index, VDI) were quantified using previously validated semi-automated algorithms. Nonperfusion was also quantified as flow impairment regions (FIR). Exams from treated and fellow eyes obtained pre-treatment and at 6-month, 1-year, and 2-year intervals were compared using generalized estimating equation linear models. Dosimetry maps were used to evaluate spatial correlation between radiation dose and microvascular metrics. RESULTS: At 6 months, treated eyes had significantly lower VSD (0.145 ± 0.003 vs 0.155 ± 0.002; p = 0.009) and higher FIR (2.01 ± 0.199 vs 1.46 ± 0.104; p = 0.010) compared to fellow eyes. There was a significant decrease in VSD and a corresponding increase in FIR even for treated eyes without clinically identifiable retinopathy at 6 months. VDI was significantly higher in treated eyes than in fellow eyes at 2 years (2.92 ± 0.025 vs 2.84 ± 0.018; p < 0.001). When our cohort was categorized into low dose radiation (<15Gy) and high dose radiation (>45Gy) to the fovea, there were significant differences in VSD and FIR between groups. CONCLUSIONS: OCTA can be used to quantify and monitor EPB induced retinopathy, and can detect vascular abnormalities even in the absence of clinically observable retinopathy. OCTA may therefore be useful in investigating treatment interventions that aim to delay EPB-induced radiation retinopathy.

Impaired Retinal Vascular Reactivity in Diabetic Retinopathy as Assessed by Optical Coherence Tomography Angiography.

Purpose: To assess retinal vascular reactivity in healthy controls and subjects with diabetic retinopathy (DR). Methods: A total of 22 healthy control eyes and 16 eyes with DR were enrolled. Images were acquired using a commercially available swept-source optical coherence tomography angiography (SS-OCTA) system. Three conditions were tested for each patient (hyperoxia, hypercapnia, and room-air) by employing a non-rebreathing apparatus that delivered appropriate gas mixtures (100% O2, 5% CO2, room air). Vessel skeleton density (VSD) and vessel diameter index (VDI) were compared between the conditions using mixed-model ANOVA adjusting for age and hypertension. Significant gas or interaction effects were followed by a Bonferroni adjusted pairwise post hoc analysis. Statistical significance was defined at P < 0.05. Results: The mixed-model ANOVA of the VSD found a significant intraindividual gas effect (F[2, 70] = 20.3, P < 0.001) and intergroup effect (F[1, 35] = 6.9, P = 0.001), and interaction effects (F[2, 70] = 4.6, P = 0.03). The post hoc pairwise comparison found significant differences among all three gas conditions in the healthy controls. In the subjects with DR, there were significant differences in VSD between hyperoxic and room air, and between hyperoxic and hypercapnic conditions, but not between hypercapnic and room-air conditions. Similar results were found for VDI. Conclusions: The retinal capillaries, assessed with SS-OCTA, in subjects with DR preferentially reacted to hyperoxia but not hypercapnia, while the healthy controls reacted to both. The difference in the vascular reactivity may be indicative of the underlying pathophysiology of DR.

Novel Technique for Quantifying Retinal Nerve Fiber Bundle Abnormality in the Temporal Raphe.

SIGNIFICANCE: Glaucomatous nasal visual field abnormalities correspond to damage in the temporal raphe-where individual nerve bundles can be visualized. The ability to quantify structural abnormality in the raphe, with a clinically applicable protocol, sets the stage for investigating the raphe as a potential site for assessing early glaucoma. PURPOSE: To develop a clinically applicable imaging and analysis technique for identifying retinal nerve fiber bundle abnormalities in the temporal raphe. METHODS: Spectralis optical coherence tomography scans customized for the temporal raphe were gathered from 30 younger controls, 30 older controls, and 29 patients with glaucoma. An analysis technique was developed based on the reflectance of the nerve fiber bundles. The technique was first developed in the younger controls, and then applied to the older controls to generate normative data for quantifying nerve fiber bundle reflectance abnormalities in the patients with glaucoma. Matrix perimetric data were gathered in the patients with glaucoma to evaluate the reflectance technique's findings. Reflectance abnormality in the patients was defined when the fraction of enface area showing reflectance abnormality was greater than the 95th percentile estimated from controls. Spearman's rho was used to quantify the relation between the total deviation at the perimetric testing locations and the fraction of corresponding enface area showing reflectance abnormality. RESULTS: Twenty-five of the 29 patients had reflectance abnormalities. Eight of these had mild to no perimetric mean deviation abnormality. Similar results were found when perimetric total deviations were compared to reflectance abnormalities in the corresponding enface locations. Spearman's rho comparing the total deviations to reflectance abnormalities found rs(174) = -0.72, P < .001. CONCLUSIONS: The technique typically identified reflectance abnormality when perimetric abnormality was present. It also identified reflectance abnormalities even when perimetric abnormality was mild or absent. The findings support the potential of raphe imaging in detecting early glaucomatous damage.

Evaluating glaucomatous abnormality in peripapillary optical coherence tomography enface visualisation of the retinal nerve fibre layer reflectance.

PURPOSE: Optical coherence tomography (OCT) enface visualisation of the retinal nerve fibre layer (RNFL) reflectance has been found to have some advantages over retinal thickness measures. However, it is not yet clear how abnormalities on enface images relate to findings of abnormalities from other clinical measures such as the circumpapillary retinal nerve fibre layer thickness (cRNFLT). We developed a technique to analyse the RNFL reflectance on the OCT enface images, and to investigate its relation with the cRNFLT. METHODS: Spectralis (www.heidelbergengineering.com) OCT scans of the central retinal ±24° were analysed in the study eye of 31 controls and 33 patients, ages 61 (±9) and 69 (±8) years respectively. Enface slab-images were extracted at 16-24, 24-36, and 24-52 µm from the inner limiting membrane in the temporal raphe, perifoveal and disc regions respectively. Reflectance probability maps were generated for the patients based on the control data. Glaucomatous abnormality was defined on the slab-images when the slab-area with reflectance abnormality was greater than the 95th percentile, and on the cRNFLT when the thickness measure was less than the fifth percentile, of that found in controls. The fraction of slab-image showing reflectance abnormality was compared to cRNFLT in the patient group, using Spearman's rho. Agreement between the findings of abnormality based on cRNFLT and slab-image reflectance was assessed using Cohen's kappa. RESULTS: Slab-image and cRNFLT findings were in agreement for 26/33 eyes; four subjects showed cRNFLT abnormality but not slab-image abnormality, and three subjects showed slab-image abnormality but not cRNFLT abnormality. Spearman's rho found rs (31) = -0.82. The reflectance findings and cRNFLT findings were consistent in 27/33 for both the superior temporal (ST) and inferior temporal (IT) sectors, and Cohen's kappa found 0.53 and 0.61 respectively. CONCLUSION: The surface area of enface slab-images showing RNFL reflectance were strongly related to the cRNFLT measures, and the classification of a subject with glaucoma based on enface reflectance findings and cRNFLT findings had a generally good agreement. The larger retinal area assessed by the enface method preserves the spatial location of the RNFL abnormalities, and makes the technique a useful approach for identifying regions of potential RNFL abnormality for targeted perimetry.

Retinal putative glial alterations: implication for glaucoma care.

PURPOSE: Gliosis-like retinal alterations, presumed to be activated retinal astrocytes and Müller cells (ARAM), have been reported to occur frequently in patients with glaucoma but rarely in controls. We investigated the association between glaucomatous abnormality and the presence, the extent of retinal region, and the spatial distribution, of hyperreflective retinal alterations on optical coherence tomography (OCT) en-face images, presumed to be ARAM. METHODS: Findings of hyperreflective structures, presumed to be ARAM, in the central retinal ±24 degrees of OCT en-face images (acquired with the SPECTRALIS® OCT) were compared between 35 younger controls, 42 older controls and 38 patients with glaucoma. Presumed ARAM was defined as reflective structures on the en-face images other than retinal vasculature and retinal nerve fibre bundles. Chi-square tests were used to compare the proportion of younger controls vs older controls with presumed ARAM to investigate the effect of ageing, and the proportion of patients vs age-similar older controls with presumed ARAM to investigate the effect of disease. We also investigated the effect of glaucoma on the retinal area with presumed ARAM when it was present; we used an analysis of covariance (ancova) to compare the retinal area with hyperreflectivity in patients vs controls, adjusting for the effects of age and axial length. RESULTS: The mean (S.D.) age of the younger controls, older controls, and patients with glaucoma was 26 (3), 62 (10) and 69 (8) years, respectively. The median (25th quartile, 75th quartile) of the retinal region with the hyperreflective structures, presumed to be ARAM, was zero (0,0), 1 (0,6), and 11 (0,43) degrees square in the younger controls, older controls and patients with glaucoma respectively. The chi-square test investigating the effect of ageing found χ2 (1, N = 77) = 24.8, p < 0.001, and that investigating the effect of disease found χ2 (1, N = 80) = 2.3, p = 0.1. The ancova found F(1, 46) = 10.32, P = 0.02. CONCLUSIONS: There was an effect of ageing on the presence of the hyperreflective structures, presumed to be ARAM, on OCT images. Compared to the presence of hyperreflective structures, the extent of retinal region with the hyperreflective structures has a greater potential of being an indicator of glaucomatous degeneration. Further study is needed to investigate the nature of the relation between glaucomatous abnormality and the extent of the retina with the hyperreflective structures, presumed to be ARAM.

Between-Subject Variability in Healthy Eyes as a Primary Source of Structural-Functional Discordance in Patients With Glaucoma.

PURPOSE: To test with an independent data set the finding that between-subject variability in healthy eyes is the primary source of structural-functional discordance in patients with glaucoma. METHODS: Neuroretinal rim area, retinal nerve fiber layer thickness, and perimetric data were analyzed for one eye in each of 55 control subjects and for 245 right eyes of patients in the United Kingdom Glaucoma Treatment Study. Data were gathered with the Heidelberg Retina Tomograph (HRT), Stratus Optical Coherence Tomograph (OCT), and Humphrey Field Analyzer (HFA). Discordance was quantified as width of the limits of agreement from a Bland-Altman analysis of depth of defect. The ratio of variances (F test) for the patient and control groups was computed for comparisons of HFA-OCT, HFA-HRT, and OCT-HRT. Bonferroni adjustment required P less than 0.017 for statistical significance. The discordance in the patients was also quantified as the 95% prediction interval computed from the discordance in controls using the Hood-Kardon model for the HFA-OCT comparison. RESULTS: The F ratio comparing discordance in patients and controls was 0.77, 1.43, and 1.32 for the HFA-OCT, HFA-HRT, and OCT-HRT comparisons with P values 0.88, 0.06, and 0.11, respectively. For the Hood-Kardon model, 4.7% of the patients had discordance outside the 95% prediction interval computed from the discordance in controls. Similar results were obtained when all comparisons were repeated for left eyes of patients. CONCLUSIONS: These results confirm previous findings that between-subject variability in healthy eyes is the primary source of structural-functional discordance in patients with glaucoma, and extends this finding to a structural-structural comparison.