Reduced Retinal Pigment Epithelial Autophagy Due to Loss of Rab12 Prenylation in a Human iPSC-RPE Model of Choroideremia.

Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in CHM, encoding Rab escort protein 1 (REP-1), leading to under-prenylation of Rab GTPases (Rabs). Despite ubiquitous expression of CHM, the phenotype is limited to degeneration of the retina, retinal pigment epithelium (RPE), and choroid, with evidence for primary pathology in RPE cells. However, the spectrum of under-prenylated Rabs in RPE cells and how they contribute to RPE dysfunction remain unknown. A CRISPR/Cas-9-edited CHM-/- iPSC-RPE model was generated with isogenic control cells. Unprenylated Rabs were biotinylated in vitro and identified by tandem mass tag (TMT) spectrometry. Rab12 was one of the least prenylated and has an established role in suppressing mTORC1 signaling and promoting autophagy. CHM-/- iPSC-RPE cells demonstrated increased mTORC1 signaling and reduced autophagic flux, consistent with Rab12 dysfunction. Autophagic flux was rescued in CHM-/- cells by transduction with gene replacement (ShH10-CMV-CHM) and was reduced in control cells by siRNA knockdown of Rab12. This study supports Rab12 under-prenylation as an important cause of RPE cell dysfunction in choroideremia and highlights increased mTORC1 and reduced autophagy as potential disease pathways for further investigation.

AUTOPHAGY IN THE EYE: FROM PHYSIOLOGY TO PATHOPHYSOLOGY.

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

Gene Supplementation in Mice Heterozygous for the D477G RPE65 Variant Implicated in Autosomal Dominant Retinitis Pigmentosa.

The use of AAV-RPE65 vectors for gene supplementation has achieved spectacular success as a treatment for individuals with autosomal recessive retinal disease caused by biallelic mutations in the visual cycle gene RPE65. However, the efficacy of this approach in treating autosomal dominant retinitis pigmentosa (adRP) associated with a monoallelic mutation encoding a rare D477G RPE65 variant has not been studied. Although lacking a severe phenotype, we now find that knock-in mice heterozygous for D477G RPE65 (D477G KI mice) can be used to evaluate outcomes of AAV-RPE65 gene supplementation. Total RPE65 protein levels, which are decreased in heterozygous D477G KI mice, were doubled following subretinal delivery of rAAV2/5.hRPE65p.hRPE65. In addition, rates of recovery of the chromophore 11-cis retinal after bleaching were significantly increased in eyes that received AAV-RPE65, consistent with increased RPE65 isomerase activity. While dark-adapted chromophore levels and a-wave amplitudes were not affected, b-wave recovery rates were modestly improved. The present findings establish that gene supplementation enhances 11-cis retinal synthesis in heterozygous D477G KI mice and complement previous studies showing that chromophore therapy results in improved vision in individuals with adRP associated with D477G RPE65.

Oxidative stress differentially impacts apical and basolateral secretion of angiogenic factors from human iPSC-derived retinal pigment epithelium cells.

The retinal pigment epithelium (RPE) is a polarized monolayer that secretes growth factors and cytokines towards the retina apically and the choroid basolaterally. Numerous RPE secreted proteins have been linked to the pathogenesis of age-related macular degeneration (AMD). The purpose of this study was to determine the differential apical and basolateral secretome of RPE cells, and the effects of oxidative stress on directional secretion of proteins linked to AMD and angiogenesis. Tandem mass tag spectrometry was used to profile proteins in human iPSC-RPE apical and basolateral conditioned media. Changes in secretion after oxidative stress induced by H2O2 or tert-butyl hydroperoxide (tBH) were investigated by ELISA and western analysis. Out of 926 differentially secreted proteins, 890 (96%) were more apical. Oxidative stress altered the secretion of multiple factors implicated in AMD and neovascularization and promoted a pro-angiogenic microenvironment by increasing the secretion of pro-angiogenic molecules (VEGF, PTN, and CRYAB) and decreasing the secretion of anti-angiogenic molecules (PEDF and CFH). Apical secretion was impacted more than basolateral for PEDF, CRYAB and CFH, while basolateral secretion was impacted more for VEGF, which may have implications for choroidal neovascularization. This study lays a foundation for investigations of dysfunctional RPE polarized protein secretion in AMD and other chorioretinal degenerative disorders.

Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration.

Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product ß-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.

Relative Contributions of All-Trans and 11-Cis Retinal to Formation of Lipofuscin and A2E Accumulating in Mouse Retinal Pigment Epithelium.

Purpose: Bis-retinoids are a major component of lipofuscin that accumulates in the retinal pigment epithelium (RPE) in aging and age-related macular degeneration (AMD). Although bis-retinoids are known to originate from retinaldehydes required for the light response of photoreceptor cells, the relative contributions of the chromophore, 11-cis retinal, and photoisomerization product, all-trans retinal, are unknown. In photoreceptor outer segments, all-trans retinal, but not 11-cis retinal, is reduced by retinol dehydrogenase 8 (RDH8). Using Rdh8-/- mice, we evaluated the contribution of increased all-trans retinal to the formation and stability of RPE lipofuscin. Methods: Rdh8-/- mice were reared in cyclic-light or darkness for up to 6 months, with selected light-reared cohorts switched to dark-rearing for the final 1 to 8 weeks. The bis-retinoid A2E was measured from chloroform-methanol extracts of RPE-choroid using HPLC-UV/VIS spectroscopy. Lipofuscin fluorescence was measured from whole flattened eyecups (excitation, 488 nm; emission, 565-725 nm). Results: Cyclic-light-reared Rdh8-/- mice accumulated A2E and RPE lipofuscin approximately 1.5 times and approximately 2 times faster, respectively, than dark-reared mice. Moving Rdh8-/- mice from cyclic-light to darkness resulted in A2E levels less than expected to have accumulated before the move. Conclusions: Our findings establish that elevated levels of all-trans retinal present in cyclic-light-reared Rdh8-/- mice, which remain low in wild-type mice, contribute only modestly to RPE lipofuscin formation and accumulation. Furthermore, decreases in A2E levels occurring after moving cyclic-light-reared Rdh8-/- mice to darkness are consistent with processing of A2E within the RPE and the existence of a mechanism that could be a therapeutic target for controlling A2E cytotoxicity.

Advancing Clinical Trials for Inherited Retinal Diseases: Recommendations from the Second Monaciano Symposium.

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.

Natural History and Genotype-Phenotype Correlations in RDH12-Associated Retinal Degeneration.

Mutations in retinol dehydrogenase 12 (RDH12) cause a severe early-onset retinal degeneration, for which there is no treatment. RDH12 is involved in photoreceptor retinoid metabolism and is a potential target for gene therapy, which has been successful in treating RPE65-associated LCA. RDH12-associated retinal degeneration is particularly devastating due to early macular atrophy, which will likely impact therapeutic outcomes. Defining the unique features and natural history of disease associated with RDH12 mutations is a critical first step in developing treatments. The purpose of this review is to aggregate and summarize the body of literature on phenotypes in RDH12-associated retinal degeneration to help map the natural history of disease and identify phenotypic milestones in disease progression. The results reveal a severe blinding disorder with onset in early childhood and frequent retention of reduced yet useful vision until adolescence. The severity is associated with genotype in some cases. Distinct phenotypic features include macular atrophy followed by bone spicule pigment early in life, in contrast to other forms of LCA which often have a relatively normal fundus appearance in childhood despite severe visual dysfunction. Formal natural history studies are needed to define milestones in disease progression and identify appropriate outcome measures for future therapy trials.

Highly Differentiated Human Fetal RPE Cultures Are Resistant to the Accumulation and Toxicity of Lipofuscin-Like Material.

Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.

Shifting the balance of autophagy and proteasome activation reduces proteotoxic cell death: a novel therapeutic approach for restoring photoreceptor homeostasis.

The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23H mice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23H mice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23H mice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.

Development of a Gene Therapy Vector for RDH12-Associated Retinal Dystrophy.

Early-onset severe retinal dystrophy (EOSRD) is a genetically heterogeneous group of diseases resulting in serious visual disability in children. A significant number of EOSRD cases, often diagnosed as Leber congenital amaurosis (LCA13), are associated with mutations in the gene encoding retinol dehydrogenase 12 (RDH12). RDH12 is a member of the enzyme family of short-chain dehydrogenases/reductases. In the retina, RDH12 plays a critical role in reducing toxic retinaldehydes generated by visual cycle activity that is required for the light response of the photoreceptor cells. Individuals with RDH12 deficiency exhibit widespread retinal degeneration impacting both rods and cones. Although Rdh12-deficient (Rdh12-/-) mice do not exhibit retinal degeneration, functional deficits relevant to visual cycle function can be demonstrated. In the present study, we describe the development and preclinical testing of a recombinant adeno-associated viral (rAAV) vector that has the potential for use in treating EOSRD due to RDH12 mutations. Wild-type and Rdh12-/- mice that received a subretinal injection of rAAV2/5 carrying a human RDH12 cDNA driven by a human rhodopsin-kinase promoter exhibited transgene expression that was stable, correctly localized, and did not cause retinal toxicity. In addition, administration of the vector reconstituted retinal reductase activity in the retinas of Rdh12-/- mice and decreased susceptibility to light damage associated with Rdh12 deficiency, thus demonstrating potential therapeutic efficacy in an animal model that does not exhibit a retinal degeneration phenotype. These findings support further efforts to develop gene replacement therapy for individuals with RDH12 mutations.

Detailed clinical characterisation, unique features and natural history of autosomal recessive RDH12-associated retinal degeneration.

BACKGROUND: Defects in retinol dehydrogenase 12 (RDH12) account for 3.4%-10.5 % of Leber congenital amaurosis and early-onset severe retinal dystrophy (EOSRD) and are a potential target for gene therapy. Clinical trials in inherited retinal diseases have unique challenges, and natural history studies are critical to successful trial design. The purpose of this study was to characterise the natural history of RDH12-associated retinal degeneration. METHODS: A retrospective chart review was performed in individuals with retinal degeneration and two likely disease-causing variants in RDH12. RESULTS: 57 subjects were enrolled from nine countries. 33 subjects had clinical records available from childhood. The data revealed an EOSRD, with average age of onset of 4.1 years. Macular atrophy was a universal clinical finding in all subjects, as young as 2 years of age. Scotopic and photopic electroretinography (ERG) responses were markedly reduced in all subjects, and a non-recordable ERG was documented as young as 1 year of age. Assessment of visual acuity, visual field and optical coherence tomography revealed severe loss of function and structure in the majority of subjects after the age of 10 years. Widefield imaging in 23 subjects revealed a unique, variegated watercolour-like pattern of atrophy in 13 subjects and sparing of the peripapillary area in 18 subjects. CONCLUSIONS: This study includes the largest collection of phenotypic data from children with RDH12-associated EOSRD and provides a comprehensive description of the timeline of vision loss in this severe, early-onset condition. These findings will help identify patients with RDH12-associated retinal degeneration and will inform future design of therapeutic trials.

A platform for assessing outer segment fate in primary human fetal RPE cultures.

The daily shedding and renewal of photoreceptor outer segments (OS) is critical for maintaining vision. This process relies on the efficient uptake, degradation, and sorting of shed OS material by the retinal pigment epithelium (RPE). Poor OS degradation has been linked to retinal degenerations such as Stargardt disease and may contribute to macular degeneration. While primary human fetal RPE cultures have emerged as a valuable model of in vivo human RPE function, surprisingly few studies have utilized the model for tracking the degradation and fate of OS components in the RPE. Here, we establish an improved platform for studying this topic by modifying existing protocols and creating new methods. Our human fetal culture model facilitates studies of RPE secretion in response to OS ingestion, preserves RPE differentiation and polarization during live-cell imaging of OS phagocytosis, and minimizes costs. We optimize Mer tyrosine kinase-dependent OS phagocytosis assays specifically in human fetal cultures and provide a simple and accurate method for measuring total OS consumption by the RPE. Finally, we utilize chemical transfection, dextran labeling, and immunocytochemistry to evaluate key players in OS degradation, including lysosomes and autophagy proteins. To facilitate quantification of autophagy vesicles, we develop customized image analysis macros in the Fiji/ImageJ software environment. These protocols will facilitate a broad range of studies in human fetal RPE cultures aimed at determining the ultimate fate of OS components after ingestion, a critical step in understanding the pathogenesis of numerous retinal diseases.

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding.

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHOP23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.

Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation.

Interphotoreceptor retinoid-binding protein (IRBP) is a specialized lipophilic carrier that binds the all-trans and 11-cis isomers of retinal and retinol, and this facilitates their transport between photoreceptors and cells in the retina. One of these retinoids, all-trans-retinal, is released in the rod outer segment by photoactivated rhodopsin after light excitation. Following its release, all-trans-retinal is reduced by the retinol dehydrogenase RDH8 to all-trans-retinol in an NADPH-dependent reaction. However, all-trans-retinal can also react with outer segment components, sometimes forming lipofuscin precursors, which after conversion to lipofuscin accumulate in the lysosomes of the retinal pigment epithelium and display cytotoxic effects. Here, we have imaged the fluorescence of all-trans-retinol, all-trans-retinal, and lipofuscin precursors in real time in single isolated mouse rod photoreceptors. We found that IRBP removes all-trans-retinol from individual rod photoreceptors in a concentration-dependent manner. The rate constant for retinol removal increased linearly with IRBP concentration with a slope of 0.012 min-1 µm-1 IRBP also removed all-trans-retinal, but with much less efficacy, indicating that the reduction of retinal to retinol promotes faster clearance of the photoisomerized rhodopsin chromophore. The presence of physiological IRBP concentrations in the extracellular medium resulted in lower levels of all-trans-retinal and retinol in rod outer segments following light exposure. It also prevented light-induced lipofuscin precursor formation, but it did not remove precursors that were already present. These findings reveal an important and previously unappreciated role of IRBP in protecting the photoreceptor cells against the cytotoxic effects of accumulated all-trans-retinal.

MERTK signaling in the retinal pigment epithelium regulates the tyrosine phosphorylation of GDP dissociation inhibitor alpha from the GDI/CHM family of RAB GTPase effectors.

Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation.

Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium.

Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related 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.).

Association of Geroprotective Effects of Metformin and Risk of Open-Angle Glaucoma in Persons With Diabetes Mellitus.

IMPORTANCE: Caloric restriction mimetic drugs have geroprotective effects that delay or reduce risks for a variety of age-associated systemic diseases, suggesting that such drugs might also have the potential to reduce risks of blinding ophthalmologic conditions for which age is a major risk factor. OBJECTIVE: To determine whether the caloric restriction mimetic drug metformin hydrochloride is associated with reduced risk of open-angle glaucoma (OAG) in persons with diabetes mellitus. DESIGN, SETTING, AND PATIENTS: Retrospective cohort study of patients aged 40 years or older with diabetes mellitus and no preexisting record of OAG in a large US managed care network from January 1, 2001, through December 31, 2010. EXPOSURES: Quantity of metformin and other prescribed diabetes medications as captured from outpatient pharmacy records. MAIN OUTCOMES AND MEASURES: Risk of developing OAG. RESULTS: Of 150 016 patients with diabetes mellitus, 5893 (3.9%) developed OAG. After adjusting for confounding factors, those prescribed the highest quartile of metformin hydrochloride (>1110 g in 2 years) had a 25% reduced OAG risk relative to those who took no metformin (hazard ratio = 0.75; 95% CI, 0.59-0.95; P = .02). Every 1-g increase in metformin hydrochloride use was associated with a 0.16% reduction in OAG risk (adjusted hazard ratio = 0.99984; 95% CI, 0.99969-0.99999; P = .04), which predicts that taking a standard dose of 2 g of metformin hydrochloride per day for 2 years would result in a 20.8% reduction in risk of OAG. After accounting for potential confounders, including metformin and diabetic medications, the risk of developing OAG was increased by 8% (hazard ratio = 1.08; 95% CI, 1.03-1.13; P = .003) for each unit of increase in glycated hemoglobin level. CONCLUSIONS AND RELEVANCE: Metformin use is associated with reduction in risk of developing OAG, and risk is reduced even when accounting for glycemic control in the form of glycated hemoglobin level. Other diabetes medications did not confer a similar OAG risk reduction. This study suggests that metformin may be affecting OAG risk on multiple levels, some involving improved glycemic control and some involving mechanisms outside glycemic control such as neurogenesis, inflammatory systems, or longevity pathways targeted by caloric restriction mimetic drugs. If confirmed by prospective clinical trials, these findings could lead to novel treatments for this sight-threatening disease.

Advancing therapeutic strategies for inherited retinal degeneration: recommendations from the Monaciano Symposium.

Although rare in the general population, retinal dystrophies occupy a central position in current efforts to develop innovative therapies for blinding diseases. This status derives, in part, from the unique biology, accessibility, and function of the retina, as well as from the synergy between molecular discoveries and transformative advances in functional assessment and retinal imaging. The combination of these factors has fueled remarkable progress in the field, while at the same time creating complex challenges for organizing collective efforts aimed at advancing translational research. The present position paper outlines recent progress in gene therapy and cell therapy for this group of disorders, and presents a set of recommendations for addressing the challenges remaining for the coming decade. It is hoped that the formulation of these recommendations will stimulate discussions among researchers, funding agencies, industry, and policy makers that will accelerate the development of safe and effective treatments for retinal dystrophies and related diseases.