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.).

Visualizing features with wide-field volumetric OCT angiography.

Optical coherence tomography (OCT) and its extension OCT angiography (OCTA) have become essential clinical imaging modalities due to their ability to provide depth-resolved angiographic and tissue structural information non-invasively and at high resolution. Within a field of view, the anatomic detail available is sufficient to identify several structural and vascular pathologies that are clinically relevant for multiple prevalent blinding diseases, including age-related macular degeneration (AMD), diabetic retinopathy (DR), and vein occlusions. The main limitation in contemporary OCT devices is that this field of view is limited due to a fundamental trade-off between system resolution/sensitivity, sampling density, and imaging window dimensions. Here, we describe a swept-source OCT device that can capture up to a 12 × 23-mm field of view in a single shot and show that it can identify conventional pathologic features such as non-perfusion areas outside of conventional fields of view. We also show that our approach maintains sensitivity sufficient to visualize novel features, including choriocapillaris morphology beneath the macula and macrophage-like cells at the inner limiting membrane, both of which may have implications for disease.

Injection pressure levels for creating blebs during subretinal gene therapy.

Retinal damage has been associated with increased injection pressure during subretinal gene therapy delivery in various animal models, yet there are no human clinical data regarding the pressures required to initiate and propagate subretinal blebs. This study characterized the intraoperative pressure levels for subretinal gene therapy delivery across eight retinal conditions. A total of 116 patients with retinal degenerative diseases have been treated with subretinal gene therapy at OHSU-Casey Eye Institute as of June 2020; seventy patients (60.3%) were treated using a pneumatic-assisted subretinal delivery system. All retinal blebs were performed using a 41-gauge injection cannula, and use of a balanced salt solution (BSS) "pre-bleb" prior to gene therapy delivery was performed at the discretion of the surgeon. Patient age and intraoperative data for BSS and vector injections were analyzed in a masked fashion for all patients who received pneumatic-assisted subretinal gene therapy. The median age of the patients was 35 years (range 4-70). No significant differences in injection pressures were found across the eight retinal conditions. In this study, patient age was shown to affect maximum injection pressures required for bleb propagation, and the relationship between age and pressure varied based on retinal condition. These data have important implications in optimizing surgical protocols for subretinal injections.

THREE-DIMENSIONAL QUANTIFICATION OF INTRARETINAL CYSTOID SPACES ASSOCIATED WITH FULL-THICKNESS MACULAR HOLE.

PURPOSE: To evaluate intraretinal cystoid spaces in patients with idiopathic macular hole (MH). METHODS: Retrospective cohort study included consecutive patients with full-thickness MH who underwent successful MH surgery and 12 months of follow-up. Custom software was applied to preoperative optical coherence tomography scans to generate fluid volume. Inner fluid volume was defined as cystoid spaces in the inner nuclear layer, and outer fluid volume was defined as cystoid spaces in Henle fiber layer of the outer nuclear layer. RESULTS: Thirty-nine eyes from 39 participants were included. Postoperative 12-month visual acuity correlated with both inner fluid volume and minimum MH size (both P < 0.05) but not outer fluid volume. Inner fluid volume positively correlated with minimum MH size ( P = 0.0003). After accounting for minimum MH size with multivariable analysis, inner fluid volume effect on VA remained significant ( P = 0.025). After dividing inner fluid volume into tertiles, mean baseline visual acuity was 20/50 in eyes with small inner fluid volume, and was 20/125 in eyes with large inner fluid volume ( P = 0.0039). Mean postoperative 12-month visual acuity was 20/20 in eyes with small inner fluid volume compared with 20/32 in eyes with large inner fluid volume ( P = 0.019). CONCLUSION: Increased inner fluid volume was associated with worse postoperative VA.

DETECTION OF CLINICALLY UNSUSPECTED RETINAL NEOVASCULARIZATION WITH WIDE-FIELD OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To evaluate wide-field optical coherence tomography angiography (OCTA) for detection of clinically unsuspected neovascularization (NV) in diabetic retinopathy (DR). METHODS: This prospective observational single-center study included adult patients with a clinical diagnosis of nonproliferative DR. Participants underwent a clinical examination, standard 7-field color photography, and OCTA with commercial and prototype swept-source devices. The wide-field OCTA was achieved by montaging five 6 × 10-mm scans from a prototype device into a 25 × 10-mm image and three 6 × 6-mm scans from a commercial device into a 15 × 6-mm image. A masked grader determined the retinopathy severity from color photographs. Two trained readers examined conventional and wide-field OCTA images for the presence of NV. RESULTS: Of 27 participants, photographic grading found 13 mild, 7 moderate, and 7 severe nonproliferative DR. Conventional 6 × 6-mm OCTA detected NV in 2 eyes (7%) and none with 3 × 3-mm scans. Both prototype and commercial wide-field OCTA detected NV in two additional eyes. The mean area of NV was 0.38 mm (range 0.17-0.54 mm). All eyes with OCTA-detected NV were photographically graded as severe nonproliferative DR. CONCLUSION: Wide-field OCTA can detect small NV not seen on clinical examination or color photographs and may improve the clinical evaluation of DR.

Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye.

Retinal vascular diseases are important causes of vision loss. A detailed evaluation of the vascular abnormalities facilitates diagnosis and treatment in these diseases. Optical coherence tomography (OCT) angiography using the highly efficient split-spectrum amplitude decorrelation angiography algorithm offers an alternative to conventional dye-based retinal angiography. OCT angiography has several advantages, including 3D visualization of retinal and choroidal circulations (including the choriocapillaris) and avoidance of dye injection-related complications. Results from six illustrative cases are reported. In diabetic retinopathy, OCT angiography can detect neovascularization and quantify ischemia. In age-related macular degeneration, choroidal neovascularization can be observed without the obscuration of details caused by dye leakage in conventional angiography. Choriocapillaris dysfunction can be detected in the nonneovascular form of the disease, furthering our understanding of pathogenesis. In choroideremia, OCT's ability to show choroidal and retinal vascular dysfunction separately may be valuable in predicting progression and assessing treatment response. OCT angiography shows promise as a noninvasive alternative to dye-based angiography for highly detailed, in vivo, 3D, quantitative evaluation of retinal vascular abnormalities.

OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY FEATURES OF DIABETIC RETINOPATHY.

PURPOSE: To describe the optical coherence tomography angiography features of diabetic retinopathy. METHODS: Using a 70 kHz optical coherence tomography and the split-spectrum amplitude decorrelation angiography algorithm, 6 mm × 6 mm 3-dimensional angiograms of the macula of 4 patients with diabetic retinopathy were obtained and compared with fluorescein angiography for features cataloged by the Early Treatment of Diabetic Retinopathy Study. RESULTS: Optical coherence tomography angiography detected enlargement and distortion of the foveal avascular zone, retinal capillary dropout, and pruning of arteriolar branches. Areas of capillary loss obscured by fluorescein leakage on fluorescein angiography were more clearly defined on optical coherence tomography angiography. Some areas of focal leakage on fluorescein angiography that were thought to be microaneurysms were found to be small tufts of neovascularization that extended above the inner limiting membrane. CONCLUSION: Optical coherence tomography angiography does not show leakage but can better delineate areas of capillary dropout and detect early retinal neovascularization. This new noninvasive angiography technology may be useful for routine surveillance of proliferative and ischemic changes in diabetic retinopathy.

DETECTION OF NONEXUDATIVE CHOROIDAL NEOVASCULARIZATION IN AGE-RELATED MACULAR DEGENERATION WITH OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To evaluate eyes with age-related macular degeneration and high-risk characteristics for choroidal neovascularization (CNV) with optical coherence tomographic (OCT) angiography to determine whether earlier detection of CNV is possible. METHODS: Eyes with drusen, pigmentary changes, and with CNV in the fellow eye were scanned with a 70-kHz spectral domain OCT system (Optovue RTVue-XR Avanti). The split-spectrum amplitude-decorrelation angiography (SSADA) algorithm was used to distinguish blood flow from static tissue. Two masked graders reviewed scans for CNV, defined as flow in the outer retinal/sub-RPE slab. Choroidal neovascularization flow area repeatability and between-grader reproducibility were calculated. RESULTS: Of 32 eyes, 2 (6%) were found to have Type 1 CNV with OCT angiography. The lesions were not associated with leakage on fluorescein angiography or fluid on OCT. One case was followed for 8 months without treatment, and the CNV flow area enlarged slightly without fluid buildup on OCT or vision loss. Between-grader reproducibility of the CNV flow area was 9.4% (coefficient of variation) and within-visit repeatability was 5.2% (pooled coefficient of variation). CONCLUSION: Optical coherence tomographic angiography can detect the presence of nonexudative CNV, lesions difficult to identify with fluorescein angiography and OCT. Further study is needed to understand the significance and natural history of these lesions.

Quantitative optical coherence tomography angiography of choroidal neovascularization in age-related macular degeneration.

PURPOSE: To detect and quantify choroidal neovascularization (CNV) in patients with age-related macular degeneration (AMD) using optical coherence tomography (OCT) angiography. DESIGN: Observational, cross-sectional study. PARTICIPANTS: A total of 5 normal subjects and 5 subjects with neovascular AMD were included. METHODS: A total of 5 eyes with neovascular AMD and 5 normal age-matched controls were scanned by a high-speed (100 000 A-scans/seconds) 1050-nm wavelength swept-source OCT. The macular angiography scan covered a 3 × 3-mm area and comprised 200 × 200 × 8 A-scans acquired in 3.5 seconds. Flow was detected using the split-spectrum amplitude-decorrelation angiography (SSADA) algorithm. Motion artifacts were removed by 3-dimensional (3D) orthogonal registration and merging of 4 scans. The 3D angiography was segmented into 3 layers: inner retina (to show retinal vasculature), outer retina (to identify CNV), and choroid. En face maximum projection was used to obtain 2-dimensional angiograms from the 3 layers. The CNV area and flow index were computed from the en face OCT angiogram of the outer retinal layer. Flow (decorrelation) and structural data were combined in composite color angiograms for both en face and cross-sectional views. MAIN OUTCOME MEASURES: The CNV angiogram, CNV area, and CNV flow index. RESULTS: En face OCT angiograms of CNV showed sizes and locations that were confirmed by fluorescein angiography (FA). Optical coherence tomography angiography provided more distinct vascular network patterns that were less obscured by subretinal hemorrhage. The en face angiograms also showed areas of reduced choroidal flow adjacent to the CNV in all cases and significantly reduced retinal flow in 1 case. Cross-sectional angiograms were used to visualize CNV location relative to the retinal pigment epithelium and Bruch's layer and classify type I and type II CNV. A feeder vessel could be identified in 1 case. Higher flow indexes were associated with larger CNV and type II CNV. CONCLUSIONS: Optical coherence tomography angiography provides depth-resolved information and detailed images of CNV in neovascular AMD. Quantitative information regarding CNV flow and area can be obtained. Further studies are needed to assess the role of quantitative OCT angiography in the evaluation and treatment of neovascular AMD.

Interpretable Diabetic Retinopathy Diagnosis Based on Biomarker Activation Map.

OBJECTIVE: Deep learning classifiers provide the most accurate means of automatically diagnosing diabetic retinopathy (DR) based on optical coherence tomography (OCT) and its angiography (OCTA). The power of these models is attributable in part to the inclusion of hidden layers that provide the complexity required to achieve a desired task. However, hidden layers also render algorithm outputs difficult to interpret. Here we introduce a novel biomarker activation map (BAM) framework based on generative adversarial learning that allows clinicians to verify and understand classifiers' decision-making. METHODS: A data set including 456 macular scans were graded as non-referable or referable DR based on current clinical standards. A DR classifier that was used to evaluate our BAM was first trained based on this data set. The BAM generation framework was designed by combing two U-shaped generators to provide meaningful interpretability to this classifier. The main generator was trained to take referable scans as input and produce an output that would be classified by the classifier as non-referable. The BAM is then constructed as the difference image between the output and input of the main generator. To ensure that the BAM only highlights classifier-utilized biomarkers an assistant generator was trained to do the opposite, producing scans that would be classified as referable by the classifier from non-referable scans. RESULTS: The generated BAMs highlighted known pathologic features including nonperfusion area and retinal fluid. CONCLUSION/SIGNIFICANCE: A fully interpretable classifier based on these highlights could help clinicians better utilize and verify automated DR diagnosis.

Multi-Plexus Nonperfusion Area Segmentation in Widefield OCT Angiography Using a Deep Convolutional Neural Network.

Purpose: To train and validate a convolutional neural network to segment nonperfusion areas (NPAs) in multiple retinal vascular plexuses on widefield optical coherence tomography angiography (OCTA). Methods: This cross-sectional study included 202 participants with a full range of diabetic retinopathy (DR) severities (diabetes mellitus without retinopathy, mild to moderate non-proliferative DR, severe non-proliferative DR, and proliferative DR) and 39 healthy participants. Consecutive 6 × 6-mm OCTA scans at the central macula, optic disc, and temporal region in one eye from 202 participants in a clinical DR study were acquired with a 70-kHz OCT commercial system (RTVue-XR). Widefield OCTA en face images were generated by montaging the scans from these three regions. A projection-resolved OCTA algorithm was applied to remove projection artifacts at the voxel scale. A deep convolutional neural network with a parallel U-Net module was designed to detect NPAs and distinguish signal reduction artifacts from flow deficits in the superficial vascular complex (SVC), intermediate capillary plexus (ICP), and deep capillary plexus (DCP). Expert graders manually labeled NPAs and signal reduction artifacts for the ground truth. Sixfold cross-validation was used to evaluate the proposed algorithm on the entire dataset. Results: The proposed algorithm showed high agreement with the manually delineated ground truth for NPA detection in three retinal vascular plexuses on widefield OCTA (mean ± SD F-score: SVC, 0.84 ± 0.05; ICP, 0.87 ± 0.04; DCP, 0.83 ± 0.07). The extrafoveal avascular area in the DCP showed the best sensitivity for differentiating eyes with diabetes but no retinopathy (77%) from healthy controls and for differentiating DR by severity: DR versus no DR, 77%; referable DR (rDR) versus non-referable DR (nrDR), 79%; vision-threatening DR (vtDR) versus non-vision-threatening DR (nvtDR), 60%. The DCP also showed the best area under the receiver operating characteristic curve for distinguishing diabetes from healthy controls (96%), DR versus no DR (95%), and rDR versus nrDR (96%). The three-plexus-combined OCTA achieved the best result in differentiating vtDR and nvtDR (81.0%). Conclusions: A deep learning network can accurately segment NPAs in individual retinal vascular plexuses and improve DR diagnostic accuracy. Translational Relevance: Using a deep learning method to segment nonperfusion areas in widefield OCTA can potentially improve the diagnostic accuracy of diabetic retinopathy by OCT/OCTA systems.