Phase 1/2 AAV5-hRKp.RPGR (Botaretigene Sparoparvovec) Gene Therapy: Safety and Efficacy in RPGR-associated X-linked Retinitis Pigmentosa.

PURPOSE: To assess the safety and efficacy of AAV5-hRKp.RPGR in participants with retinitis pigmentosa GTPase regulator (RPGR)-associated X-linked retinitis pigmentosa (XLRP). DESIGN: Open-label, phase 1/2 dose escalation/expansion study (NCT03252847). METHODS: Males (≥5 years old) with XLRP-RPGR were evaluated. In the dose escalation phase, subretinal AAV5-hRKp.RPGR (low: 1.0×1011 vg/ml; intermediate: 2.0×1011 vg/ml; high: 4.0×1011 vg/ml) was administered to the poorer-seeing eye (n = 10). Dose confirmation (intermediate dose) was carried out in 3 pediatric participants. In the dose expansion phase, 36 participants were randomized 1:1:1 to immediate (low or intermediate dose) or deferred (control) treatment. The primary outcome was safety. Secondary efficacy outcomes included static perimetry, microperimetry, vision-guided mobility, best corrected visual acuity, and contrast sensitivity. Safety and efficacy outcomes were assessed for 52 weeks for immediate treatment participants and 26 weeks for control participants. RESULTS: AAV5-hRKp.RPGR was safe and well tolerated, with no reported dose-limiting events. Most adverse events (AEs) were transient and related to the surgical procedure, resolving without intervention. Two serious AEs were reported with immediate treatment (retinal detachment, uveitis). A third serious AE (increased intraocular pressure) was reported outside the reporting period. All ocular inflammation-related AEs responded to corticosteroids. Treatment with AAV5-hRKp.RPGR resulted in improvements in retinal sensitivity and functional vision compared with the deferred group at Week 26; similar trends were observed at Week 52. CONCLUSIONS: AAV5-hRKp.RPGR demonstrated an anticipated and manageable AE profile through 52 weeks. Safety and efficacy findings support investigation in a phase 3 trial.

Glutamine catabolism supports amino acid biosynthesis and suppresses the integrated stress response to promote photoreceptor survival.

Photoreceptor loss results in vision loss in many blinding diseases, and metabolic dysfunction underlies photoreceptor degeneration. So, exploiting photoreceptor metabolism is an attractive strategy to prevent vision loss. Yet, the metabolic pathways that maintain photoreceptor health remain largely unknown. Here, we investigated the dependence of photoreceptors on Gln catabolism. Gln is converted to glutamate via glutaminase (GLS), so mice lacking GLS in rod photoreceptors were generated to inhibit Gln catabolism. Loss of GLS produced rapid rod photoreceptor degeneration. In vivo metabolomic methodologies and metabolic supplementation identified Gln catabolism as critical for glutamate and aspartate biosynthesis. Concordant with this amino acid deprivation, the integrated stress response (ISR) was activated with protein synthesis attenuation, and inhibiting the ISR delayed photoreceptor loss. Furthermore, supplementing asparagine, which is synthesized from aspartate, delayed photoreceptor degeneration. Hence, Gln catabolism is integral to photoreceptor health, and these data reveal a novel metabolic axis in these metabolically-demanding neurons. Impact statement: For the first time, this work reveals the metabolic dependency of photoreceptors on glutamine catabolism in vivo and further demonstrates the flexibility of photoreceptors to utilize fuel sources beyond glucose.

Classification and Growth Rate of Chorioretinal Atrophy after Voretigene Neparvovec-Rzyl for RPE65-Mediated Retinal Degeneration.

PURPOSE: Classify the appearance and quantify the growth rate of chorioretinal atrophy in patients who received voretigene neparvovec-rzyl (VN) for RPE65-mediated retinal degeneration. DESIGN: Multicenter retrospective analysis. SUBJECTS: Patients who underwent subretinal VN injection at 5 institutions and demonstrated posterior-pole chorioretinal atrophy. METHODS: Ultrawidefield scanning laser ophthalmoscopy or color fundus photos were assessed before and after subretinal VN. Atrophy was defined as regions with ≥ 2 of the following: (1) partial or complete retinal pigment epithelial depigmentation; (2) round shape; (3) sharp margins; and (4) increased visibility of choroidal vessels. Atrophy was qualitatively classified into different subtypes. All atrophy was manually segmented. Linear mixed-effects models with random slopes and intercepts were fit using atrophy area and square root of atrophy area. MAIN OUTCOME MEASURES: Number of eyes with each atrophy pattern, and slopes of linear mixed-effects models. RESULTS: Twenty-seven eyes from 14 patients across 5 centers developed chorioretinal atrophy after subretinal VN. A mean of 5.8 ± 2.7 images per eye obtained over 2.2 ± 0.8 years were reviewed, and atrophy was categorized into touchdown (14 eyes), nummular (15 eyes), and perifoveal (12 eyes) subtypes. Fifteen eyes demonstrated > 1 type of atrophy. Thirteen of 14 patients demonstrated bilateral atrophy. The slopes of the mixed-effects models of atrophy area and square root of atrophy area (estimate ± standard error) were 1.7 ± 1.3 mm2/year and 0.6 ± 0.2 mm/year for touchdown atrophy, 5.5 ± 1.3 mm2/year and 1.2 ± 0.2 mm/year for nummular atrophy, and 16.7 ± 1.8 mm2/year and 2.3 ± 0.2 mm/year for perifoveal atrophy. The slopes for each type of atrophy were significantly different in the square root of atrophy model, which best fit the data (P < 0.05). CONCLUSIONS: Chorioretinal atrophy after subretinal VN for RPE65-mediated retinal degeneration developed according to a touchdown, nummular, and/or perifoveal pattern. Perifoveal atrophy grew the most rapidly, while touchdown atrophy grew the least rapidly. Understanding the causes of these findings, which are present in a minority of patients, merits further investigation. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Diabetic Retinopathy Screening Using a Portable Retinal Camera in Vanuatu.

Background and Objective: Proof-of-concept study to test the feasibility of using an all-in-one portable retinal camera for the screening of diabetic retinopathy in the Pacific Island of Vanuatu, which has a high rate of diabetes and its associated complications and a dearth of ophthalmologists. Study Design/Materials and methods: From February 10, 2020, through February 28, 2020, 49 patients with diabetes mellitus from three islands in Vanuatu were recruited to participate in the study. Demographics, basic health data and retinal photography were obtained. A non-mydriatic, handheld camera was used (Volk Pictor Plus). Results: Eleven participants (24%) had referral-warranted diabetic retinopathy. There was moderately high inter-rater reliability for our dependent variables: referral status (κ = 0.62, 95% CI 0.42-0.83), retinopathy severity (κ = 0.76, 95% CI 0.55-0.96), and clinically significant macular edema (κ = 0.50, 95% CI 0.25-0.74). Conclusion: Our study confirms that portable handheld cameras can be used to obtain retinal images of sufficient quality for diabetic retinopathy screening even in resource limited environments like Vanuatu. Among this cohort, a relatively high (24%) prevalence of referral-warranted diabetic retinopathy was found in Vanuatu.

Metabolic Alterations Caused by Simultaneous Loss of HK2 and PKM2 Leads to Photoreceptor Dysfunction and Degeneration.

HK2 and PKM2 are two main regulators of aerobic glycolysis. Photoreceptors (PRs) use aerobic glycolysis to produce the biomass necessary for the daily renewal of their outer segments. Previous work has shown that HK2 and PKM2 are important for the normal function and long-term survival of PRs but are dispensable for PR maturation, and their individual loss has opposing effects on PR survival during acute nutrient deprivation. We generated double conditional (dcKO) mice lacking HK2 and PKM2 expression in rod PRs. Western blotting, immunofluorescence, optical coherence tomography, and electroretinography were used to characterize the phenotype of dcKO animals. Targeted and stable isotope tracing metabolomics, qRT-PCR, and retinal oxygen consumption were performed. We show that dcKO animals displayed early shortening of PR inner/outer segments, followed by loss of PRs with aging, much more rapidly than either knockout alone without functional loss as measured by ERG. Significant alterations to central glucose metabolism were observed without any apparent changes to mitochondrial function, prior to PR degeneration. Finally, PR survival following experimental retinal detachment was unchanged in dcKO animals as compared to wild-type animals. These data suggest that HK2 and PKM2 have differing roles in promoting PR neuroprotection and identifying them has important implications for developing therapeutic options for combating PR loss during retinal disease.

Flow cytometric method for the detection and quantification of retinal cell death and oxidative stress.

Retinal cell death is the major cause of vision loss in many forms of blinding retinal disease. A plethora of research is focused on understanding the mechanisms of retinal cell death to identify potential neuroprotective strategies that prevent vision loss in these diseases. Traditionally, histological techniques have been used to determine the type and extent of cell death in the retina. These techniques, such as TUNEL labeling and immunohistochemistry, are laborious and time consuming, resulting in low throughput and variable results depending on the experimenter. To increase throughput and reduce variability, we developed several flow cytometry-based assays to detect and quantify retinal cell death. The methods and accompanying data presented demonstrate that flow cytometry can readily detect both retinal cell death and oxidative stress and importantly, the efficacy of neuroprotective agents. These methods will be of interest to investigators looking to increase throughput and efficiency without compromising sensitivity as the methods herein reduce analysis time from several months to less than a week. As such, the flow cytometry methods presented have the potential to expedite research efforts focused on developing novel strategies for retinal cell neuroprotection.

Development of Novel Small-Molecule Activators of Pyruvate Kinase Muscle Isozyme 2, PKM2, to Reduce Photoreceptor Apoptosis.

Treatment options are lacking to prevent photoreceptor death and subsequent vision loss. Previously, we demonstrated that reprogramming metabolism via the pharmacologic activation of PKM2 is a novel photoreceptor neuroprotective strategy. However, the features of the tool compound used in those studies, ML-265, preclude its advancement as an intraocular, clinical candidate. This study sought to develop the next generation of small-molecule PKM2 activators, aimed specifically for delivery into the eye. Compounds were developed that replaced the thienopyrrolopyridazinone core of ML-265 and modified the aniline and methyl sulfoxide functional groups. Compound 2 demonstrated that structural changes to the ML-265 scaffold are tolerated from a potency and efficacy standpoint, allow for a similar binding mode to the target, and circumvent apoptosis in models of outer retinal stress. To overcome the low solubility and problematic functional groups of ML-265, compound 2's efficacious and versatile core structure for the incorporation of diverse functional groups was then utilized to develop novel PKM2 activators with improved solubility, lack of structural alerts, and retained potency. No other molecules are in the pharmaceutical pipeline for the metabolic reprogramming of photoreceptors. Thus, this study is the first to cultivate the next generation of novel, structurally diverse, small-molecule PKM2 activators for delivery into the eye.

The impact of the COVID-19 lockdown on retinopathy of prematurity screening and management in the United States: a multicenter study.

PURPOSE: To study the effect of the pandemic-related lockdown (physical distance measures and movement restrictions) on the characteristics and management of retinopathy of prematurity (ROP). METHODS: In this controlled, multicenter cohort study, the medical records of patients born prematurely and screened for ROP in the neonatal intensive care unit during four time periods were reviewed retrospectively: (1) November 1, 2018, to March 15, 2019; (2) March 16, 2019, to August 2, 2019 (lockdown control period); (3) November 1, 2019, to March 15, 2020; and (4) March 16, 2020-August 2, 2020. RESULTS: A total of 1,645 patients met inclusion criteria. Among the 1,633 patients with complete data, mean gestational age (GA) at birth was 28.2, 28.4, 28.0, and 28.3 weeks across time periods 1 to 4, respectively (P = 0.16). The mean birth weight of all patients was 1079.1 ± 378.60 g, with no significant variation across time periods (P = 0.08). There were fewer patients screened during the lockdown period (n = 411) compared with the period immediately before (n = 491) and the same period in the prior year (n = 533). Significantly more patients were screened using indirect ophthalmoscopy, compared to digital imaging (telemedicine), during the lockdown (P < 0.01). There were 11.7%, 7.7%, 9.0%, and 8.8% of patients requiring treatment in each time period, respectively (P = 0.42), with a median postmenstrual age at initial treatment of 37.2, 36.45, 37.1, and 36.3 weeks, respectively (P = 0.32). CONCLUSIONS: We recorded a decrease in the number of infants meeting criteria for ROP screening during the lockdown. The GA at birth and birth weight did not differ. Significantly more infants were screened with indirect ophthalmoscopy, compared to digital imaging, during the lockdown.

First-in-Human Gene Therapy Trial of AAV8-hCARp.hCNGB3 in Adults and Children With CNGB3-associated Achromatopsia.

PURPOSE: To assess the safety and efficacy of AAV8-hCARp.hCNGB3 in participants with CNGB3-associated achromatopsia (ACHM). DESIGN: Prospective, phase 1/2 (NCT03001310), open-label, nonrandomized clinical trial. METHODS: The study enrolled 23 adults and children with CNGB3-associated ACHM. In the dose-escalation phase, adult participants were administered 1 of 3 AAV8-hCARp.hCNGB3 dose levels in the worse-seeing eye (up to 0.5 mL). After a maximum tolerated dose was established in adults, an expansion phase was conducted in children ≥3 years old. All participants received topical and oral corticosteroids. Safety and efficacy parameters, including treatment-related adverse events and visual acuity, retinal sensitivity, color vision, and light sensitivity, were assessed for 6 months. RESULTS: AAV8-hCARp.hCNGB3 (11 adults, 12 children) was safe and generally well tolerated. Intraocular inflammation occurred in 9 of 23 participants and was mainly mild or moderate in severity. Severe cases occurred primarily at the highest dose. Two events were considered serious and dose limiting. All intraocular inflammation resolved following topical and systemic steroids. There was no consistent pattern of change from baseline to week 24 for any efficacy assessment. However, favorable changes were observed for individual participants across several assessments, including color vision (n = 6/23), photoaversion (n = 11/20), and vision-related quality-of-life questionnaires (n = 21/23). CONCLUSIONS: AAV8-hCARp.hCNGB3 for CNGB3-associated ACHM demonstrated an acceptable safety and tolerability profile. Improvements in several efficacy parameters indicate that AAV8-hCARp.hCNGB3 gene therapy may provide benefit. These findings, with the development of additional sensitive and quantitative end points, support continued investigation.

Characteristics of a Three-Generation Family with Stickler Syndrome Type I Carrying Two Different COL2A1 Mutations.

Stickler Syndrome is typically characterized by ophthalmic manifestations including vitreous degeneration and axial lengthening that predispose to retinal detachment. Systemic findings consist of micrognathia, cleft palate, sensorineural hearing loss, and joint abnormalities. COL2A1 mutations are the most common, however, there is a lack of genotype-phenotype correlations. Retrospective, single-center case series of a three-generation family. Clinical features, surgical requirements, systemic manifestations, and genetic evaluations were collected. Eight individuals clinically displayed Stickler Syndrome, seven of whom had genetic confirmation, and two different COL2A1 mutations (c.3641delC and c.3853G>T) were identified. Both mutations affect exon 51, but display distinct phenotypes. The c.3641delC frameshift mutation resulted in high myopia and associated vitreous and retinal findings. Individuals with the c.3853G>T missense mutation exhibited joint abnormalities, but mild ocular manifestations. One individual in the third generation was biallelic heterozygous for both COL2A1 mutations and showed ocular and joint findings in addition to autism and severe developmental delay. These COL2A1 mutations exhibited distinct eye vs. joint manifestations. The molecular basis for these phenotypic differences remains unknown and demonstrates the need for deep phenotyping in patients with Stickler syndrome to correlate COL2A1 gene function and expression with ocular and systemic findings.

Late Vitreoretinal Complications of Regressed Retinopathy of Prematurity: Retinal Break, Vitreous Hemorrhage, and Retinal Detachment.

PURPOSE: To investigate late vitreoretinal complications and visual outcomes in patients with regressed retinopathy of prematurity (ROP) with or without prior treatment. DESIGN: International, multicenter, noncomparative retrospective case series. PARTICIPANTS: We analyzed 264 eyes of 238 patients from 13 centers worldwide who developed vitreoretinal complications (retinal detachment [RD], vitreous hemorrhage [VH], or retinal break) ≥ 2 years after resolution of acute ROP. METHODS: Each participant was assigned to 1 of 3 groups (the RD, VH, and retinal break groups) according to their primary diagnosis. The average age at presentation, visual acuities, refractive error, axial length, gestational age, birth weight, acute ROP classification, prior treatments for acute ROP, postoperative visual acuity (VA), and concomitant eye conditions in the 3 groups were documented and compared. MAIN OUTCOME MEASURES: Clinical features and visual outcomes of late vitreoretinal complications in patients with regressed ROP. RESULTS: A total of 264 eyes of 238 patients were included. The prior acute ROP status was comparable among the 3 groups, except that the VH group had a higher proportion of patients with type 1 ROP (P = 0.03) and prior treatment (P < 0.001) than the other groups. The average age at presentation was earlier in the RD (20.3 ± 15.5 years) and VH (21.4 ± 18.9 years) groups than in the retinal break group (31.9 ± 18.2 years; P < 0.001). The retinal break group had the best presenting best-corrected VA, followed by the RD and VH groups (P < 0.001). Surgical intervention improved VA in both the RD and VH groups (both P < 0.05). The overall trend of VA was the most favorable in the retinal break group, followed by that in the VH and RD groups. Cicatricial changes in the fellow retina were observed in > 90% of patients with unilateral involvement. CONCLUSIONS: Infants with acute ROP remain at a high risk of vision-threatening complications throughout childhood and adulthood. Continual follow-up of patients with ROP is important. When severe complications, such as RD or VH, are detected, timely surgical intervention is necessary to ensure favorable visual outcomes in these patients.

Retinal Neovascularization in Posterior Polar Annular Choroidal Dystrophy.

This case report describes the development of posterior subcapsular cataract and retinal neovascularization in a patient with posterior polar annular choroidal dystrophy.

Quantitative analysis of tear angiogenic factors in retinopathy of prematurity: a pilot biomarker study.

PURPOSE: To determine whether vascular endothelial growth factor (VEGF), angiopoeitin-1 (Ang-1), angiopoetin-2 (Ang-2), and matrix metalloproteinase-9 (MMP-9) can be reliably collected and analyzed from infant tears to aid in the diagnosis of retinopathy of prematurity (ROP) and enhance the ability to objectively monitor its clinical course. METHODS: In this nonrandomized controlled investigation, tear and saliva samples collected from 20 premature infants during serial ophthalmic examination were analyzed using enzyme-linked immunoassay with results analyzed as a function of disease stage and need for treatment. RESULTS: Tear volume was directly correlated with corrected gestational age (P < 0.001). Tear VEGF levels from samples corresponding to stage 3 ROP were 47.9% lower (P = 0.006) than in samples corresponding to stage 0-1 and 49.1% lower (P = 0.01) than in samples corresponding to stage 2 ROP. There were no between-group differences after normalizing tear VEGF by saliva VEGF levels. Tear/saliva ratio for Ang-1 was 200% greater (P = 0.042) and tear/saliva ratio for Ang-2 was 165% greater (P = 0.035) in samples corresponding to stage 2 versus stage 0-1 ROP disease. Ang-1/Ang-2 ratio was lower in samples from infants who developed stage 2 or worse ROP than in samples from infants who never developed worse than stage 1 ROP (P = 0.031). CONCLUSIONS: In this study cohort, cytokines involved in the pathophysiology of ROP could be reliably identified in and analyzed from infant tears, and showed variation with ROP severity.

COMBINED HAMARTOMA OF THE RETINA AND RETINAL PIGMENT EPITHELIUM AT PEDIATRIC AGE: Surgical Versus Conservative Approach.

PURPOSE: To report outcomes of pediatric patients with combined hamartoma of the retina and the retina pigment epithelium followed up conservatively or after pars plana vitrectomy. METHODS: This retrospective multicenter study included 62 eyes of 59 pediatric patients with combined hamartoma of the retina and the retina pigment epithelium from 13 different international centers with an average age of 7.7 ± 4.7 (0.3-17) years at the time of the diagnosis and having undergone pars plana vitrectomy or followed conservatively. At baseline and each visit, visual acuity values, optical coherence tomography for features and central foveal thickness, and tumor location were noted. Lesions were called as Zone 1, if it involves the macular and peripapillary areas, and the others were called as Zone 2 lesions. RESULTS: Twenty-one eyes of 20 patients in the intervention group and 41 eyes of 39 patients in the conservative group were followed for a mean of 36.2 ± 40.4 (6-182) months. Best-corrected visual acuity improved in 11 (68.8%) of 16 eyes in the intervention group and 4 (12.9%) of 31 eyes in the conservative group ( P < 0.001). The mean central foveal thickness decreased from 602.0 ± 164.9 µ m to 451.2 ± 184.3 µ m in the intervention group, while it increased from 709.5 ± 344.2 µ m to 791.0 ± 452.1 µ m in Zone 1 eyes of the conservative group. Posterior location of tumor, irregular configuration of the foveal contour and ellipsoid Zone defect in optical coherence tomography, subretinal exudate and prominent vascular tortuosity were associated with poor visual acuity. CONCLUSION: Vitreoretinal surgery is safe and effective in improving vision and reducing retinal distortion in Zone 1 combined hamartoma of the retina and the retina pigment epithelium in children.

Rod photoreceptor-specific deletion of cytosolic aspartate aminotransferase, GOT1, causes retinal degeneration.

Photoreceptor cell death is the cause of vision loss in many forms of retinal disease. Metabolic dysfunction within the outer retina has been shown to be an underlying factor contributing to photoreceptor loss. Therefore, a comprehensive understanding of the metabolic pathways essential to photoreceptor health and function is key to identifying novel neuroprotective strategies. Glutamic-oxaloacetic transaminase 1 (Got1) encodes for a cytosolic aspartate aminotransferase that reversibly catalyzes the transfer of an amino group between glutamate and aspartate and is an important aspect of the malate-aspartate shuttle (MAS), which transfers reducing equivalents from the cytosol to the mitochondrial matrix. Previous work has demonstrated that the activity of this enzyme is highest in photoreceptor inner segments. Furthermore, ex vivo studies have demonstrated that the retina relies on aspartate aminotransferase for amino acid metabolism. Importantly, aspartate aminotransferase has been suggested to be an early biomarker of retinal degeneration in retinitis pigmentosa and a possible target for neuroprotection. In the present study, we characterized the effect of Got1 deletion on photoreceptor metabolism, function, and survival in vivo by using a rod photoreceptor-specific, Got1 knockout mouse model. Loss of the GOT1 enzyme from rod photoreceptors resulted in age-related photoreceptor degeneration with an accumulation of retinal aspartate and NADH and alterations in the expression of genes involved in the MAS, the tricarboxylic acid (TCA) cycle, and redox balance. Hence, GOT1 is critical to in vivo photoreceptor metabolism, function, and survival.

A Caveat About Financial Incentives for Anti-Vascular Endothelial Growth Factor Therapy for Diabetic Retinopathy.

PURPOSE: To highlight the financial incentive to the physician of choosing an intravitreal anti-vascular endothelial growth factor (VEGF)-based strategy for treating non-proliferative and proliferative diabetic retinopathy, and its possible risks to the patient and costs to the health care system. DESIGN: Perspective with retrospective cost and profit analysis. METHODS: Review and synthesis of selected literature on the treatment of diabetic retinopathy, with interpretation of activity-based and time-based costing of an intravitreal aflibercept strategy for diabetic retinopathy. Data from the DRCR Retina Network Protocols W and AB and PANORAMA trial were used to illustrate the potential financial incentive underlying such a treatment strategy. RESULTS: Physician treatment algorithms for diabetic vitreous hemorrhage and non-proliferative diabetic retinopathy may be influenced by the substantial financial incentives that intravitreal aflibercept strategies present, despite functional equivalence with alternative and less profitable strategies. For example, pursuing an intravitreal aflibercept-based strategy for diabetic vitreous hemorrhage presents a 76% increased profit over pars plana vitrectomy with laser, with equivalent functional outcomes. For non-proliferative diabetic retinopathy, preventative aflibercept injections represent a potential 414% increase in profit over observation and an increased cost of $12,164 to $17,542 over 2 years per patient, with no improvement in visual function. These findings demonstrate that there may be misaligned financial incentives in the management of diabetic retinopathy. CONCLUSIONS: While anti-VEGF therapy is a useful tool in the management of proliferative diabetic retinopathy and diabetic macular edema, it is believed that physicians should avoid overreliance on anti-VEGF injections in the treatment of diabetic retinopathy. Retinal specialists should be cognizant of the limitations, costs, and risks of anti-VEGF monotherapy and prophylactic therapy, and of the imperative to avoid bias towards financially remunerative practice patterns when equally effective alternatives exist.