AAV-RPGR Gene Therapy Rescues Opsin Mislocalisation in a Human Retinal Organoid Model of RPGR-Associated X-Linked Retinitis Pigmentosa.

Variants within the Retinitis Pigmentosa GTPase regulator (RPGR) gene are the predominant cause of X-Linked Retinitis Pigmentosa (XLRP), a common and severe form of inherited retinal disease. XLRP is characterised by the progressive degeneration and loss of photoreceptors, leading to visual loss and, ultimately, bilateral blindness. Unfortunately, there are no effective approved treatments for RPGR-associated XLRP. We sought to investigate the efficacy of RPGRORF15 gene supplementation using a clinically relevant construct in human RPGR-deficient retinal organoids (ROs). Isogenic RPGR knockout (KO)-induced pluripotent stem cells (IPSCs) were generated using established CRISPR/Cas9 gene editing methods targeting RPGR. RPGR-KO and isogenic wild-type IPSCs were differentiated into ROs and utilised to test the adeno associated virus (AAV) RPGR (AAV-RPGR) clinical vector construct. The transduction of RPGR-KO ROs using AAV-RPGR successfully restored RPGR mRNA and protein expression and localisation to the photoreceptor connecting cilium in rod and cone photoreceptors. Vector-derived RPGR demonstrated equivalent levels of glutamylation to WT ROs. In addition, treatment with AAV-RPGR restored rhodopsin localisation within RPGR-KO ROs, reducing mislocalisation to the photoreceptor outer nuclear layer. These data provide mechanistic insights into RPGRORF15 gene supplementation functional potency in human photoreceptor cells and support the previously reported Phase I/II trial positive results using this vector construct in patients with RPGR-associated XLRP, which is currently being tested in a Phase III clinical trial.

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

[This corrects the article DOI: 10.1371/journal.pone.0015730.].

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.

Genetic treatment for autosomal dominant inherited retinal dystrophies: approaches, challenges and targeted genotypes.

Inherited retinal diseases (IRDs) have been in the front line of gene therapy development for the last decade, providing a useful platform to test novel therapeutic approaches. More than 40 clinical trials have been completed or are ongoing, tackling autosomal recessive and X-linked conditions, mostly through adeno-associated viral vector delivery of a normal copy of the disease-causing gene. However, only recently has autosomal dominant (ad) disease been targeted, with the commencement of a trial for rhodopsin (RHO)-associated retinitis pigmentosa (RP), implementing antisense oligonucleotide (AON) therapy, with promising preliminary results (NCT04123626).Autosomal dominant RP represents 15%-25% of all RP, with RHO accounting for 20%-30% of these cases. Autosomal dominant macular and cone-rod dystrophies (MD/CORD) correspond to approximately 7.5% of all IRDs, and approximately 35% of all MD/CORD cases, with the main causative gene being BEST1 Autosomal dominant IRDs are not only less frequent than recessive, but also tend to be less severe and have later onset; for example, an individual with RHO-adRP would typically become severely visually impaired at an age 2-3 times older than in X-linked RPGR-RP.Gain-of-function and dominant negative aetiologies are frequently seen in the prevalent adRP genes RHO, RP1 and PRPF31 among others, which would not be effectively addressed by gene supplementation alone and need creative, novel approaches. Zinc fingers, RNA interference, AON, translational read-through therapy, and gene editing by clustered regularly interspaced short palindromic repeats/Cas are some of the strategies that are currently under investigation and will be discussed here.

Spontaneous abortion is associated with differentially expressed angiogenic chemokines in placenta and decidua.

PURPOSE: Miscarriage is one of the most common complications of pregnancy. Although chromosomal abnormalities of the embryo is a well-known cause of miscarriage, a lot of cases remain unexplained, with immunologic and vascular growth alterations being considered as probable causes. Chemokines are produced by a variety of cells and exhibit several functions including both pro and anti-angiogenic properties. In this study, we investigated the role of the angiogenic and angiostatic chemokines in placenta and decidua tissues from spontaneous and induced abortions. METHODS: Total RNA was extracted from the placenta and decidua tissues, which was then purified and converted into cDNA. Real-time PCR was then performed for the expression of the angiogenic CCL2, CCL5, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 and CXCL4, and the angiostatic CXCL9, CXCL10, CXCL11, CXCL12 and CXCL14 and results were then statistically analyzed. RESULTS: Regarding the placenta, CXCL7 (2.29-fold, 2.16-2.38, p < 0.05), CXCL4 (1.01-fold, 0.74-4.447, p < 0.05), CXCL9 (0.87-fold, 0.43-1.34, p < 0.05) and CXCL11 (0.31-fold, 0.22-0.45, p < 0.05) were altered in spontaneous abortions. CCL2, CCL5, CXCL2-3, CXCL8, CXCL10, CXCL12 and CXCL14 were not statistically significant altered. Regarding the decidua, CXCL7 (7.13-fold, 6.32-7.54, p < 0.01), CXCL8 (11.02-fold, 8.58-13.45, p < 0.05), CCL20 (1.21-fold, 0.29-1.89, p < 0.05) and CXCL9 (5.49-fold, 3.67-6.39, p < 0.05) were overexpressed in spontaneous abortions. CXCL2-4, CCL2, CCL5, CXCL10-12 and CXCL14 did not show any differences. The expression of the chemokines CXCL1, CXCL5-6 was absent in either tissue or group. CONCLUSION: Our results show that the overexpression of angiostatic and diminished expression of angiogenic chemokines takes place in the placenta and decidua of spontaneous abortions, suggesting that dysregulation of angiogenesis could be a contributive factor to the pathogenesis of miscarriage.

Spatiotemporal control of actomyosin contractility by MRCKß signaling drives phagocytosis.

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids.

The generation of retinal organoids from human pluripotent stem cells (hPSC) is now a well-established process that in part recapitulates retinal development. However, hPSC-derived photoreceptors that exhibit well-organized outer segment structures have yet to be observed. To facilitate improved inherited retinal disease modeling, we determined conditions that would support outer segment development in maturing hPSC-derived photoreceptors. We established that the use of antioxidants and BSA-bound fatty acids promotes the formation of membranous outer segment-like structures. Using new protocols for hPSC-derived retinal organoid culture, we demonstrated improved outer segment formation for both rod and cone photoreceptors, including organized stacked discs. Using these enhanced conditions to generate iPSC-derived retinal organoids from patients with X-linked retinitis pigmentosa, we established robust cellular phenotypes that could be ameliorated following adeno-associated viral vector-mediated gene augmentation. These findings should aid both disease modeling and the development of therapeutic approaches for the treatment of photoreceptor disorders.

RNAi-mediated suppression of vimentin or glial fibrillary acidic protein prevents the establishment of Müller glial cell hypertrophy in progressive retinal degeneration.

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer-A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts.

Purpose: Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. Methods: HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. Results: LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. Conclusions: ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.

Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.

BACKGROUND: The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. METHODS: We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. RESULTS: Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. CONCLUSIONS: Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.

Prevention of Photoreceptor Cell Loss in a Cln6nclf Mouse Model of Batten Disease Requires CLN6 Gene Transfer to Bipolar Cells.

The neuronal ceroid lipofuscinoses (NCLs) are inherited lysosomal storage disorders characterized by general neurodegeneration and premature death. Sight loss is also a major symptom in NCLs, severely affecting the quality of life of patients, but it is not targeted effectively by brain-directed therapies. Here we set out to explore the therapeutic potential of an ocular gene therapy to treat sight loss in NCL due to a deficiency in the transmembrane protein CLN6. We found that, although Cln6nclf mice presented mainly with photoreceptor degeneration, supplementation of CLN6 in photoreceptors was not beneficial. Because the level of CLN6 is low in photoreceptors but high in bipolar cells (retinal interneurons that are only lost in Cln6-deficient mice at late disease stages), we explored the therapeutic effects of delivering CLN6 to bipolar cells using adeno-associated virus (AAV) serotype 7m8. Bipolar cell-specific expression of CLN6 slowed significantly the loss of photoreceptor function and photoreceptor cells. This study shows that the deficiency of a gene normally expressed in bipolar cells can cause the loss of photoreceptors and that this can be prevented by bipolar cell-directed treatment.

Harnessing the Potential of Human Pluripotent Stem Cells and Gene Editing for the Treatment of Retinal Degeneration.

PURPOSE OF REVIEW: A major cause of visual disorders is dysfunction and/or loss of the light-sensitive cells of the retina, the photoreceptors. To develop better treatments for patients, we need to understand how inherited retinal disease mutations result in the dysfunction of photoreceptors. New advances in the field of stem cell and gene editing research offer novel ways to model retinal dystrophies in vitro and present opportunities to translate basic biological insights into therapies. This brief review will discuss some of the issues that should be taken into account when carrying out disease modelling and gene editing of retinal cells. We will discuss (i) the use of human induced pluripotent stem cells (iPSCs) for disease modelling and cell therapy; (ii) the importance of using isogenic iPSC lines as controls; (iii) CRISPR/Cas9 gene editing of iPSCs; and (iv) in vivo gene editing using AAV vectors. RECENT FINDINGS: Ground-breaking advances in differentiation of iPSCs into retinal organoids and methods to derive mature light sensitive photoreceptors from iPSCs. Furthermore, single AAV systems for in vivo gene editing have been developed which makes retinal in vivo gene editing therapy a real prospect. SUMMARY: Genome editing is becoming a valuable tool for disease modelling and in vivo gene editing in the retina.

Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration.

The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs). Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor ß2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1-/- mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

Rescue of mutant rhodopsin traffic by metformin-induced AMPK activation accelerates photoreceptor degeneration.

Protein misfolding caused by inherited mutations leads to loss of protein function and potentially toxic 'gain of function', such as the dominant P23H rhodopsin mutation that causes retinitis pigmentosa (RP). Here, we tested whether the AMPK activator metformin could affect the P23H rhodopsin synthesis and folding. In cell models, metformin treatment improved P23H rhodopsin folding and traffic. In animal models of P23H RP, metformin treatment successfully enhanced P23H traffic to the rod outer segment, but this led to reduced photoreceptor function and increased photoreceptor cell death. The metformin-rescued P23H rhodopsin was still intrinsically unstable and led to increased structural instability of the rod outer segments. These data suggest that improving the traffic of misfolding rhodopsin mutants is unlikely to be a practical therapy, because of their intrinsic instability and long half-life in the outer segment, but also highlights the potential of altering translation through AMPK to improve protein function in other protein misfolding diseases.

Enhanced Ccl2-Ccr2 signaling drives more severe choroidal neovascularization with aging.

The impact of many inflammatory diseases is influenced by age-related changes in the activation of resident and circulating myeloid cells. In the eye, a major sight-threatening consequence of age-related macular degeneration is the development of severe choroidal neovascularization (CNV). To identify the molecular pathways and myeloid cell populations involved in this increased neovascular response, we characterized the immune status of murine choroid and retina during aging and in the context of experimental CNV. In the choroid, but not in the retina, advancing age is associated with proinflammatory upregulation of CCL2-CCR2 signaling. Genetic excision of CCL2 diminishes age-related inflammatory changes in the choroid, with reduced recruitment of proinflammatory myeloid cells and attenuation of CNV. These findings indicate that CCL2-driven recruitment of myeloid cells contributes to increased severity of CNV with age. Similar mechanisms may be involved in other age-related inflammatory diseases.

IL-4 regulates specific Arg-1(+) macrophage sFlt-1-mediated inhibition of angiogenesis.

One of the main drivers for neovascularization in age-related macular degeneration is activation of innate immunity in the presence of macrophages. Here, we demonstrate that T helper cell type 2 cytokines and, in particular, IL-4 condition human and murine monocyte phenotype toward Arg-1(+), and their subsequent behavior limits angiogenesis by increasing soluble fms-like tyrosine kinase 1 (sFlt-1) gene expression. We document that T helper cell type 2 cytokine-conditioned murine macrophages neutralize vascular endothelial growth factor-mediated endothelial cell proliferation (human umbilical vein endothelial cell and choroidal vasculature) in a sFlt-1-dependent manner. We demonstrate that in vivo intravitreal administration of IL-4 attenuates laser-induced choroidal neovascularization (L-CNV) due to specific IL-4 conditioning of macrophages. IL-4 induces the expression of sFlt-1 by resident CD11b(+) retinal microglia and infiltrating myeloid cells but not from retinal pigment epithelium. IL-4-induced suppression of L-CNV is not prevented when sFlt-1 expression is attenuated in retinal pigment epithelium. IL-4-mediated suppression of L-CNV was abrogated in IL-4R-deficient mice and in bone marrow chimeras reconstituted with myeloid cells that had undergone lentiviral-mediated shRNA silencing of sFlt-1, demonstrating the critical role of this cell population. Together, these data establish how lL-4 directly drives macrophage sFlt-1 production expressing an Arg-1(+) phenotype and support the therapeutic potential of targeted IL-4 conditioning within the tissue to regulate disease conditions such as neovascular age-related macular degeneration.

Long-term effect of gene therapy on Leber's congenital amaurosis.

BACKGROUND: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited. METHODS: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings. RESULTS: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG. CONCLUSIONS: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.