Role of HIF1α and HIF2α in Cre Recombinase–Induced Retinal Pigment Epithelium Pathology and Its Secondary Effect on Choroidal Neovascularization

CreTrp1 mice are widely used for conditional retinal pigment epithelium (RPE) gene function studies. Like other Cre/LoxP models, phenotypes in CreTrp1 mice can be affected by Cre-mediated cellular toxicity, leading to RPE dysfunction, altered morphology and atrophy, activation of innate immunity, and consequent impairment of photoreceptor function. These effects are common among the age-related alterations of RPE that feature in early/intermediate forms of age-related macular degeneration. This article characterizes Cre-mediated pathology in the CreTrp1 line to elucidate the impact of RPE degeneration on both developmental and pathologic choroidal neovascularization. Nonredundant roles of the two major components of the hypoxia-inducible factor (HIF) family of transcription regulators, HIF1α and HIF2α, were identified. Genetic ablation of Hif1a protected against Cre-induced degeneration of RPE and choroid, whereas ablation of Hif2a exacerbated this degeneration. Furthermore, HIF1α deficiency protected CreTrp1 mice against laser-induced choroidal neovascularization, whereas HIF2α deficiency exacerbated the phenotype. Cre-mediated degeneration of the RPE in CreTrp1 mice offers an opportunity to investigate the impact of hypoxia signaling in the context of RPE degeneration. These findings indicate that HIF1α promotes Cre recombinase–mediated RPE degeneration and laser-induced choroidal neovascularization, whereas HIF2α is protective

Validation of a Vision-Guided Mobility Assessment for RPE65-Associated Retinal Dystrophy

Purpose: To validate a vision-guided mobility assessment for individuals affected by RPE65-associated retinal dystrophy (RPE65-RD). / Methods: In this comparative cross-sectional study, 29 subjects, comprising 19 subjects with RPE65-RD and 10 normally-sighted subjects undertook three assessments of mobility: following a straight line, navigating a simple maze, and stepping over a sidewalk "kerb." Performance was quantified as the time taken to complete each assessment, number of errors made, walking speed, and percent preferred walking speed, for each assessment. Subjects also undertook assessments of visual acuity, contrast sensitivity, full-field static perimetry, and age-appropriate quality of life questionnaires. To identify the most relevant metric to quantify vision-guided mobility, we investigated repeatability, as well as convergent, discriminant, and criterion validity. We also measured the effect of illumination on mobility. / Results: Walking speed through the maze assessment best discriminated between RPE65-RD and normally-sighted subjects, with both convergent and discriminant validity. Walking speed also approached statistical significance when assessed for criterion validity (P = 0.052). Subjects with RPE65-RD had quantifiably poorer mobility at lower illumination levels. A relatively small mean difference (-0.09 m/s) was identified in comparison to a relatively large repeatability coefficient (1.10 m/s). / Conclusions: We describe a novel, quantifiable, repeatable, and valid assessment of mobility designed specifically for subjects with RPE65-RD. The assessment is sensitive to the visual impairment of individuals with RPE65-RD in low illumination, identifies the known phenotypic heterogeneity and will furthermore provide an important outcome measure for RPE65-RD. / Translational Relevance: This assessment of vision-guided mobility, validated in a dedicated cohort of subjects with RPE65-RD, is a relevant and quantifiable outcome measure for RPE65-RD

Severe Loss of Tritan Color Discrimination in RPE65 Associated Leber Congenital Amaurosis

Purpose: RPE65-associated Leber congenital amaurosis (RPE65-LCA) is a progressive severe retinal dystrophy with early profound dysfunction of rod photoreceptors followed by progressive cone photoreceptor degeneration. We aim to provide detailed information about how cone dysfunction affects color discrimination. Methods: Seven adults (aged 16-21) with RPE65-LCA underwent monocular color discrimination assessment using the Trivector and Ellipse versions of three computerized tests: Cambridge Colour Test (CCT), low vision version of the Cambridge Colour Test (lvvCCT), and the Universal Colour Discrimination Test (UCDT). For comparison, subjects were also tested using the American Optical Hardy Rand Rittler (AO-HRR) plates. Each assessment was repeated three times. Results: The Trivector version of the tests demonstrated that color discrimination along the tritan axis was undetectable in four subjects, and severely reduced in three subjects. These findings were confirmed by the Ellipse version of the tests. Color discrimination along the protan and deutan axes was evident but reduced in six of seven subjects. Four of seven subjects were unable to read any of the HRR plates. Conclusions: The computerized color vision tests adopted in this study provide detailed information about color discrimination in adult RPE65-LCA patients. The condition is associated with severe impairment of color discrimination, particularly along the tritan axis indicating possible early involvement of S-cones, with additional protan and deutan loss to a lesser extent. This psychophysical assessment strategy is likely to be valuable in measuring the impact of therapeutic intervention on cone function

Retinal gene therapy

INTRODUCTION: Inherited retinal diseases are the leading cause of sight impairment in people of working age in England and Wales, and the second commonest in childhood. Gene therapy offers the potential for benefit. SOURCES OF DATA: Pubmed and clinicaltrials.gov. Areas of agreement: Gene therapy can improve vision in RPE65-associated Leber Congenital Amaurosis (RPE65-LCA). Potential benefit depends on efficient gene transfer and is limited by the extent of retinal degeneration. AREAS OF CONTROVERSY: The magnitude of vision improvement from RPE65-LCA gene therapy is suboptimal, and its durability may be limited by progressive retinal degeneration. GROWING POINTS: The safety and potential benefit of gene therapy for inherited and acquired retinal diseases is being explored in a rapidly expanding number of trials. Areas timely for developing research: Developments in vector design and delivery will enable greater efficiency and safety of gene transfer. Optimization of trial design will accelerate reliable assessment of outcomes

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-derived Retinal Organoids and Fetal Retinae

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were FAC-sorted using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. This article is protected by copyright. All rights reserved

A Cross-Sectional and Longitudinal Study of Retinal Sensitivity in RPE65-Associated Leber Congenital Amaurosis

Purpose: RPE65-associated Leber congenital amaurosis (RPE65-LCA) is an early-onset severe retinal dystrophy associated with progressive visual field loss. Phase I/II and III gene therapy trials have identified improved retinal sensitivity but little is known about the natural history of retinal sensitivity in RPE65-LCA. Methods: A total of 19 subjects (aged 9 to 23 years) undertook monocular full-field static perimetry of which 13 subjects were monitored longitudinally. Retinal sensitivity was measured as mean sensitivity (MS) and volumetrically quantified (in decibel-steradian) using visual field modeling and analysis software for the total (VTOT), central 30° (V30) and central 15° (V15) visual field. Correlation was evaluated between retinal sensitivity and age, best-corrected visual acuity (BCVA), contrast sensitivity, vision-related quality of life, and genotype. Test-retest reliability was also investigated. Results: V30 was identified to have a strong, weak, and moderate correlation with age, BCVA and contrast sensitivity respectively. Furthermore, V30 was identified as having a weak linear relationship with the mobility and independence domains of the vision-related quality of life questionnaire. Longitudinal analysis demonstrated a slow loss of retinal sensitivity in this cohort. Subjects with at least one RPE65 nonsense variant appeared to show greater progressive loss of retinal sensitivity in the second decade of life than those without. Conclusions: Volumetric assessment of central 30° visual field sensitivity, V30, is a useful independent measure of retinal function and, in our data, represented the best metric to monitor deterioration of retinal sensitivity in RPE65-LCA. Furthermore, functional correlation with genotype may enable more informed prognostic counseling

Retinal Structure in RPE65-Associated Retinal Dystrophy

PURPOSE: RPE65-associated retinal dystrophy (RPE65-RD) is an early onset, progressive, severe retinal dystrophy. We sought to characterize the natural history of retinal degeneration in affected individuals. METHODS: We performed cross-sectional and longitudinal quantitative and qualitative assessments of retinal architecture in RPE65-RD using spectral domain optical coherence tomography (SD-OCT) and fundus autofluorescence (FAF) imaging. Twenty-six subjects (mean age, 14.8 years, range, 5–24 years) with RPE65-RD underwent SD-OCT and FAF imaging, of whom 14 subjects were followed up over time. Foveal thickness (FT), outer nuclear layer thickness (ONLT), ellipsoid zone width (EZW), and ellipsoid zone area (EZA) were calculated where possible. These were correlated with age, best corrected visual acuity (BCVA), and central 30° retinal sensitivity (V_{30}). Intra-observer agreement, test-retest repeatability, and interocular symmetry were also investigated. RESULTS: We identified structural interocular symmetry, the presence of autofluorescence in 46% (12/26) of subjects, and the presence of foveal hypoplasia (associated with significantly worse BCVA) in 50% of subjects. EZW and EZA were measurable in 67% (35/52) and 37% (19/52) of eyes, respectively, with both demonstrating good agreement on repeated measurement. The annual rate of progression using EZW was −300.63 µm/year, and −1.17 mm^{2}/year in EZA. EZW was found to have a statistically significant correlation with BCVA and V_{30}. CONCLUSIONS: We identified the presence of autofluorescence in half of our subjects, with foveal hypoplasia also noted in half of our cohort. EZW, and to a lesser extent EZA, were robust measures of retinal degeneration and represent valuable metrics to determine the impact of intervention. (ClinicalTrials.gov number NCT02714816.

Stabilization of myeloid-derived HIFs promotes vascular regeneration in retinal ischemia

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network

The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration

Restoration of visual function in advanced disease after transplantation of purified human pluripotent stem cell-derived cone photoreceptors

Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision