Among the more than 750 carotenoids identified in nature, only lutein, zeaxanthin, meso-zeaxanthin, and their oxidative metabolites are selectively accumulated in the macula lutea region of the human retina. These retinal carotenoids are collectively referred to as the macular pigment (MP) and are obtained only through dietary sources such as green leafy vegetables and yellow and orange fruits and vegetables. Lutein- and zeaxanthin-specific binding proteins (StARD3 and GSTP1, respectively) mediate the highly selective uptake of MP into the retina. Meso-zeaxanthin is rarely present in the diet, and its unique presence in the human eye results from metabolic conversion from dietary lutein by the RPE65 enzyme. The MP carotenoids filter high-intensity, short-wavelength visible light and are powerful antioxidants in a region vulnerable to light-induced oxidative stress. This review focuses on MP chemistry, absorption, metabolism, transport, and distribution with special emphasis on animal models used for MP study. This article is part of a Special Issue entitled Carotenoids recent advances in cell and molecular biology edited by Johannes von Lintig and Loredana Quadro.

Leber congenital amaurosis (LCA) is a part of the spectrum of early-onset retinal dystrophy (EORD). It usually presents in the first few years of life, most often before the age of 1 year. The prevalence is about 1:80,000. Also known as congenital retinitis pigmentosa (RP), patients have wandering nystagmus, with reduced vision from birth. The fundus is almost normal to start with. Later, pigmentary disturbances develop. The best corrected visual acuity (BCVA) ranges from no light perception (in nearly one third of cases) to no better than 20/400, often with a hyperopic refraction (≥5.0D). Patients have absent pupillary reflex and some of them have keratoconus (CRB1 and AIPL1). Oculodigital reflex (eye rubbing or poking) is a common association. Electroretinography (ERG) responses are almost undetectable. Patients may have normal intelligence, but some studies suggest that as many as 20% of children with LCA without associated anomalies develop intellectual disability. Patients with GUCY2D mutations have a stable clinical course; those with RPE65 mutations show a period of limited improvement followed by progressive deterioration; and those with AIPL1, CRB1, CEP290, and NMNAT1 mutations show gradual progression over several decades.

Stem cell-based therapies are attraction approaches for regenerative medicine for treating retinal diseases. One of the limitations in cell therapy is cell death following post-injection whit preventing functional integration with retinal tissue. Fibrin gel, a bio-polymeric material with excellent biocompatibility, provides numerous advantages as a tissue engineering scaffold and a stem cell carrier. Therefore, current research is focusing on developing fibrin hydrogel scaffolds to protect stem cells during delivery and to stimulate endogenous regeneration through interactions of transplanted stem cells and retinal tissue. In this study fibrin gel was used as hydrogel scaffold for immobilization of cells. The structural characteristics of fibrin gel scaffold were examined with SEM. Rheological properties of fibrin gel were measured by rheometer and biodegradation rate of fibrin were assayed for 2 weeks. After isolation of stem cells CJMSCs, the cells were differentiated into photoreceptor-like cells by exposing with taurin for 14 days in tissue culture plate (TCP group) and fibrin hydrogel (3 D group). The attachment of cells was analyzed with SEM and MTT. The expression of rhodopsin, PKC, CRX, recoverin, peripherin, nestin and RPE65 as photoreceptor-like cell markers was evaluated by immunocytochemistry and quantitative real-time PCR (RT-PCR) in TCP and 3 D groups. The results of SEM analysis showed CJMSCs were well attached in fibrin gels and there were good integrity between cells and scaffold. The elastic modulus and constant degradation of the gel contributes to the growth and proliferation of cells. There was no toxicity effect of fibrin hydrogel on cells and the viability of cultured cells was higher in 3 D fibrin gels in comparison with TCP groups. After 2 weeks, the expression of rhodopsin, PKC, CRX, peripherin, recoverin, nestin and RPE65 as special markers of photoreceptor cells were detected by Real time PCR and immunofluorescence that these expressions in 3 D groups were higher than TCP groups. In conclusion, our findings showed that application of readily available sources of adult stem cells like human conjunctiva stem cells encapsulated in fibrin gel could be interesting strategy to enhance photoreceptor progenitor cell numbers for repair and regeneration of retina disease such as photoreceptor injury.

Leber's congenital amaurosis (LCA) and recent gene therapy advancement for treating inherited retinopathies were extensive literature reviewed using MEDLINE, PubMed and EMBASE. Adeno-associated viral vectors were the most utilised vectors for ocular gene therapy. Cone photoreceptor cells might use an alternate pathway which was not reliant of the retinal pigment epithelium (RPE) derived retinoid isomerohydrolase (RPE65) to access the 11-cis retinal dehydechromophore. Research efforts dedicated on the progression of a gene-based therapy for the treatment of LCA2. Such gene therapy approaches were extremely successful in canine, porcine and rodent LCA2 models. The recombinant AAV2.hRPE65v2 adeno-associated vector contained the RPE65 cDNA and was replication deficient. Its in vitro injection in target cells induced RPE65 protein production. The gene therapy trials that were so far conducted for inherited retinopathies have generated promising results. Phase I clinical trials to cure LCA and choroideremia demonstrated that adeno-associated viral vectors containing RPE genes and photoreceptors respectively, could be successfully administered to inherited retinopathy patients. A phase III trial is presently ongoing and if successful, it will lead the way to additional gene therapy attempts to cure monogenic, inherited retinopathies.

A protein called RPE65 performs a key role in the trans-cis isomerization of retinol in the retinal pigment epithelium of the eye. The palmitoylation of RPE65 serves to switch off the visual cycle in darkness and to switch it on in the light.

The eye is a small compartment separated from the systemic circulation by the blood-ocular barriers, providing advantages for intraocular gene transfer - an approach which is being investigated for several types of retinal and choroidal diseases. A compelling application is gene replacement for homozygous loss-of-function mutations in genes differentially expressed in photoreceptors or retinal pigmented epithelial (RPE) cells that result in retinal degeneration. Considerable progress has been made in this area, including demonstration of return of visual function in RPE65 (-/-) dogs after subretinal injection of adeno-associated viral vectors encoding RPE65, providing groundwork for a clinical trial in patients with Leber's Congenital Amaurosis. Proof of principle has been provided for intraocular gene transfer of ribozymes for dominantly inherited retinal degenerations. Survival factor gene therapy shows promise for treatments that may be used in multiple retinal degenerations. Transduction of intraocular and/or periocular cells with constructs that encode antiangiogenic proteins provides a new approach for sustained local delivery treatment of retinal and choroidal neovascularisation. While considerable investigation remains to work out critical details, there is substantial evidence suggesting that in the near future, gene therapy-based treatments will be an important addition to what is currently offered to patients with retinal and/or choroidal diseases.

The absorption of light by rhodopsin leads to the cis-to-trans isomerization of the chromophore to generate all-trans-retinal. In the visual cycle, the resultant all-trans-retinal is converted back into the 11-cis-retinal. In the mammalian eye, the retinal pigment epithelium (RPE) plays an essential role in the visual cycle. We have identified cDNA clones encoding three putative visual cycle proteins, homologs of mammalian retinal G-protein-coupled receptor (RGR), cellular retinaldehyde-binding protein (CRALBP) and beta-carotene 15,15'-monooxygenase (BCO)/RPE65 in a primitive chordate, ascidian Ciona intestinalis. The mRNAs for these proteins are specifically expressed in the central nervous system during embryonic development. In the larva, the transcripts were widely distributed in the brain vesicle and visceral ganglion. Since visual pigment, Ci-opsin1, is solely expressed in photoreceptor cells, the visual cycle in this primitive chordate may take place in two compartments, which are coupled into a cycle by the direct flow of retinoids though the intercellular matrix. The Ci-opsin3, an ascidian homolog of mammalian RGR, was expressed in HEK 293S cells and purified after binding of retinal. The chromophore of Ci-opsin3 is in an all-trans-retinal and it is isomerized to an 11-cis-form upon absorption of light. Mammalian CRALBP and BCO/RPE65 are believed to play critical roles in the process of reisomerization of all-trans-retinoid to 11-cis-retinoid in RPE. The present data suggest that isomerization of all-trans-retinoid to 11-cis-retinoid occurs in the brain vesicle and visceral ganglion of a primitive chordate.

Genesis and progression of diabetic retinopathy are due to glucotoxicity-induced changes in intracellular milieu in the retina. This study investigated effects of trans-resveratrol on type 1 diabetes-induced changes in gene expressions and retinoic acid metabolism pathway in the RPE (retinal pigment epithelium) of Dark Agouti rats. Microarray analysis showed differential expressions of 833 genes in the RPE of 14 day-long diabetic rats, which increased to 1249 after they received 5 mg/kg trans-resveratrol. Diabetes inhibited the expression of retinoic acid metabolism pathway genes- Lpl, Lrat, RPE65, Rdh5, Rdh10, Rdh12, Rlbp1 and Rbp1 and increased Crabp1. Trans-resveratrol further downregulated the expression of these genes except Lpl, Rdh5, and Rdh12 but upregulated Cyp26b1. RT-PCR showed inhibition of Lrat, Rdh5, and Rdh10 in diabetic rats supplemented with or without trans-resveratrol on 14d. Trans-resveratrol normalized Rdh5 and increased Lrat and Rdh10 transcriptions compared to control and diabetic rats. Trans-resveratrol amplified diabetes-induced inhibition of RPE65, but it inhibited the induced increase in Crabp1 transcription on 30d. Trans-resveratrol reversed the diabetes-induced decrease in Cyp26b1 transcription on 14d and 30d and normalized Cyp3a9 transcription on 30d. Trans-resveratrol normalized the diabetes-induced increase in Rdh5, Rdh10, and Cyp3a9 protein levels, but it further increased Cyp26b1 protein level. In conclusion, diabetes differentially regulates numerous genes in the RPE, including that of retinoic acid metabolism pathway. Trans-resveratrol supplementation is beneficial to normalize long-term effects, but not short-term effects, of diabetes on retinoic acid metabolism pathway in the RPE.

Metabolism inside cells differs between cancer and normal cells. Because disturbance of vitamin A metabolism might be important, we investigated expression of the enzymes lecithin retinol acyltransferase (LRAT) and RPE65 by immunohistochemistry in melanoma metastases and melanocytic nevi. Semiquantitative evaluation of this expression revealed downregulated expression of RPE65 in malignant melanoma compared with benign melanocytic nevi (P < 0.001). In contrast, expression of LRAT was not significantly different (P = 0.339). High LRAT expression in melanoma metastases was inversely correlated with patient survival; Kaplan-Meier analysis revealed earlier melanoma-related death (P = 0.003). Expression of LRAT might, therefore, be a prognostic marker of the clinical course of melanoma.

The efficient recycling of the chromophore of visual pigments, 11-cis-retinal, through the retinoid visual cycle is an essential process for maintaining normal vision. RPE65 is the isomerohydrolase in retinal pigment epithelium and generates predominantly 11-cis-retinol (11cROL) and a minor amount of 13-cis-retinol (13cROL), from all-trans-retinyl ester (atRE). We recently identified and characterized novel homologues of RPE65, RPE65c, and 13-cis-isomerohydrolase (13cIMH), which are expressed in the zebrafish inner retina and brain, respectively. Although these two homologues have 97% identical amino acid sequences, they exhibit distinct product specificities. Under the same assay conditions, RPE65c generated predominantly 11cROL, similar to RPE65, while 13cIMH generated exclusively 13cROL from atRE substrate. To study the impacts of the key residues determining the isomerization product specificity of RPE65, we replaced candidate residues by site-directed mutagenesis in RPE65c and 13cIMH. Point mutations at residues Tyr58, Phe103, and Leu133 in RPE65c resulted in significantly altered isomerization product specificities. In particular, our results showed that residue 58 is a primary determinant of isomerization specificity, because the Y58N mutation in RPE65c and its reciprocal N58Y mutation in 13cIMH completely reversed the respective enzyme isomerization product specificities. These findings will contribute to the elucidation of molecular mechanisms underlying the isomerization reaction catalyzed by RPE65.

In previous studies, we demonstrated that recombinant adeno-associated virus (rAAV)-mediated gene transfer of the doxycycline (Dox)-regulatable system allows for the regulation of erythropoietin (EPO) expression in the retina of nonhuman primates after intravenous or oral administration of Dox. In addition, it was shown that administrating different amounts of Dox resulted in a dose-response dynamic of transgene expression. Adeno-associated viral gene therapy has raised hope for the treatment of patients with Leber congenital amaurosis, caused by mutations in the retinal pigment epithelium (RPE)-specific gene RPE65. The preliminary results of three clinical trials suggest some improvement in visual function. However, further improvements might be necessary to optimize vision recovery and this means developing vectors able to generate transgene expression at physiological levels. The purpose of this study was to investigate the ability of the Dox-regulatable system to regulate retinal function in RPE65(-/-) Briard dogs. rAAV vectors expressing RPE65 under the control of either the TetOff and TetOn Dox-regulated promoters or the cytomegalovirus (CMV) constitutive promoter were generated and administered subretinally to seven RPE65-deficient dogs. We demonstrate that the induction and deinduction of retinal function, as assessed by electroretinography (ERG), can be achieved using a Dox-regulatable system, but do not lead to any recovery of vision.

OBJECTIVE

Recent success of rescuing vision by photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to identify approaches to generate photoreceptors cells for future replacement therapies. We explored the possibility of whether in the mouse eye photoreceptor-like cells could arise from the RPE experimentally manipulated to express a regulatory gene participating in transcriptional networks leading to photoreceptor genesis during retinal development.

METHODS

Transgenic mice were generated with a DNA construct that would express neurogenin1 from RPE bestrophin-1 promoter or neurogenin3 from RPE65 promoter. Transgenic mice were examined with histology and immunohistology for the presence of photoreceptor-like cells and for the presence of cells that might represent transitional stages in RPE-to-photoreceptor reprogramming. Explant culture of "sclera+choroid+RPE" eyecup was used to examine whether cells with photoreceptor traits could arise from the eyecup derived from transgenic mice.

RESULTS

Transgenic animals showed varied degrees of phenotype manifestation. Approximately 60% of offspring from ∼50% of founders contained photoreceptor-like cells in the subretinal space. These cells expressed photoreceptor proteins recoverin, red opsin, and rhodopsin, and displayed morphologic similarities to photoreceptors. In these eyes, the RPE was maintained. Cells seemingly amid RPE-to-photoreceptor transformation were observed in young and aged mice, suggesting old animals were responsive to the reprogramming scheme. De novo generation of photoreceptor-like cells was detected in "sclera+choroid+RPE" eyecup explants derived from adult animals.

CONCLUSIONS

Our results point to a potential way to generate photoreceptor cells in situ in adult mammalian eyes.

The RPE65 protein appears late during the retinal development. To study the basis for this regulation, the rat RPE65 cDNA was sequenced and the mRNA subsequently quantitated at various stages by competitive RT-PCR. RPE65 mRNA was detected as early as E18 (36 copies/ng of whole eye total RNA). It gradually accumulates up to P12 (27000 copies/ng) at which point it reaches a steady state level. This increase is interrupted for 3 days (P2-P4) during which the levels of mRNA remain stable. This timing and rate of accumulation parallels that of rat and mouse opsin mRNA and suggests that common factors may control the activation of genes in photoreceptors and retinal pigment epithelium cells.

Clinical trials treating inherited retinal dystrophy caused by RPE65 mutations had put retinal gene therapy at the forefront of gene therapy. Both successes and limitations in these clinical trials have fueled developments in gene vectors, which continue to further advance the field. These novel gene vectors aim to more safely and efficiently transduce retinal cells, expand the gene packaging capacity of AAV, and utilize new strategies to correct the varying mechanisms of dysfunction found with inherited retinal dystrophies. With recent clinical trials and numerous pre-clinical studies utilizing these novel vectors, the future of ocular gene therapy continues to hold vast potential.

Whether we are driving to work or spending time with loved ones, we depend on our sense of vision to interact with the world around us. Therefore, it is understandable why blindness for many is feared above death itself. Heritable diseases of the retina, such as glaucoma, age-related macular degeneration, and retinitis pigmentosa, are major causes of blindness worldwide. The recent success of gene augmentation trials for the treatment of RPE65-associated Leber congenital amaurosis has underscored the need for model systems that accurately recapitulate disease. With the advent of patient-specific induced pluripotent stem cells (iPSCs), researchers are now able to obtain disease-specific cell types that would otherwise be unavailable for molecular analysis. In the present review, we discuss how the iPSC technology is being used to confirm the pathogenesis of novel genetic variants, interrogate the pathophysiology of disease, and accelerate the development of patient-centered treatments. Significance: Stem cell technology has created the opportunity to advance treatments for multiple forms of blindness. Researchers are now able to use a person's cells to generate tissues found in the eye. This technology can be used to elucidate the genetic causes of disease and develop treatment strategies. In the present review, how stem cell technology is being used to interrogate the pathophysiology of eye disease and accelerate the development of patient-centered treatments is discussed.

OBJECTIVE

Thinning of the RPE and the underlying vascular layer, the choroid, is observed with age in many human eye disorders. The reasons for this thinning are ill-defined. Here, we highlight the possible role of T lymphocyte recruitment in choroidoretinal thinning in aged and light-challenged mice.

METHODS

In age and light challenge models, we measured chemokine concentrations using enzyme-linked immunosorbent assay and used flow cytometry to characterize lymphocyte populations. We quantified thinning in eye immunosections and RPE65 expression using quantitative PCR.

RESULTS

Age and light challenge led to increased levels of the lymphotactic protein CXCL10 alone (aging) or in conjunction with CXCL9 (light challenge). Increased numbers of CD3+ T lymphocytes, most of them CD8+ cytotoxic T lymphocytes, were also observed in the choroid and retina of old mice and following light challenge. Influx of T lymphocytes was associated with RPE and choroidal thinning and diminished expression of RPE65 mRNA, an essential enzyme of the visual cycle.

CONCLUSIONS

The observations from this study suggest that cytotoxic CD8(+) T lymphocytes might participate in choroidal and RPE degeneration and that modulation of T lymphocyte recruitment might be a novel strategy to reduce choroidoretinal dysfunctions observed with age and following photo-oxidative stress.

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

BACKGROUND

Culture of retinal pigment epithelium (RPE) cells might be a future option in the therapy of various degenerative retinal diseases. However, the molecular changes which occur during in vitro expansion of RPE cells during culture are not fully elucidated. The aim of this study was to evaluate molecular changes in the RPE cell line ARPE-19 after stimulation with different growth factors.

METHODS

Cultured ARPE-19 cells were stimulated for 72 hours with rh-EGF, rh-IGF-1, rh-VEGF or rh-bFGF, and transcriptional changes of the differentiation markers cytokeratin 18 and RPE65 and of the key molecules of the wnt pathway, beta-catenin, and glycogen synthase kinase-3 (GSK-3) were evaluated by real time RT-PCR.

RESULTS

We found a significant decrease of cytokeratin 18 and RPE65 transcription after stimulation with rh-EGF (0.47 +/- 0.42 and 0.32 +/- 0.57-fold, respectively; p < 0.05). A significant reduction of beta-catenin and GSK-3 mRNA was found in ARPE-19 cells stimulated with rh-IGF-1 (0.61 +/- 0.25 and 0.52 +/- 0.02-fold, respectively) or rh-EGF (0.55 +/- 0.19 and 0.76 +/- 0.26-fold, respectively). No changes of beta-catenin mRNA were observed after stimulation with rh-VEGF or bFGF.

CONCLUSIONS

Our data suggest an inhibition of the beta-catenin-pathway in ARPE-19 cells by IGF-1 and EGF, suggesting that ARPE-19 cell proliferation is, at least in part, driven by the beta-catenin pathway. Furthermore, induction of proliferation by EGF results in a loss of differentiation markers in these cells. Maintaining the RPE phenotype is still one of the main problems for RPE- transplantation.

OBJECTIVE

To characterize visual losses associated with genetic mutations in the RPE65 gene that cause defects in the RPE-specific isomerase, RPE65. RPE65 is an important component of the retinoid cycle that restores 11-cis-retinal after its photoisomerization to its all-trans form. The defects investigated here cause Leber's congenital amaurosis (LCA2), an autosomal, recessively-inherited, severe, congenital-onset rod-cone dystrophy.

METHODS

Vision was assessed in nine patients and 10 normal controls by measuring: (1) long-wavelength sensitive (L-) cone temporal acuity (critical flicker fusion frequency or cff) as a function of target illuminance, and (2) L-cone temporal contrast sensitivity as a function of temporal frequency at a fixed-target illuminance. Measurements were made by modulating either a 650-nm light superimposed on a 480-nm background or the red phosphor of a color monitor on a background produced by the monitor's blue phosphor.

RESULTS

RPE65-mutant observers have severely reduced cffs with shallower cff versus log illuminance functions that rise with a mean slope of 4.53 Hz per decade of illuminance compared with 8.69 Hz in normal controls. Consistent with the cff differences, RPE65-mutant observers show losses in temporal contrast sensitivity that increase rapidly with temporal frequency.

CONCLUSIONS

All RPE65-mutant observers have consistent and substantial losses in temporal acuity and sensitivity compared with normal observers. The losses can be characterized by the addition of two sluggish filters within the mutant visual pathway, both filters with a time constant of 29.5 ms (i.e., low-pass filters with cut-off frequencies of 5.40 Hz).

The purpose of our study was to evaluate the transduction profiles of herpes simplex virus type 1 (HSV-1)-based amplicon vectors following subretinal injection in the rat. Two amplicon vectors were tested, pHy-CMVGFP and pHy-RPEGFP, both carrying the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) ubiquitous promoter or the RPE65-specific promoter, respectively. For the two amplicon vectors, the GFP reporter gene was efficiently expressed in retinal pigment epithelial (RPE) cells but not in the adjacent photoreceptors. GFP expression was maximum as early as 2 days post-administration but decreased over time to become almost undetectable at 6 weeks postinjection. Super-transduction with a second amplicon vector, pHSVlac, reactivated expression of GFP in approximately 10% of the cells initially transduced at 2 days postinjection of pHy-CMVGFP or pHy-RPEGFP. Reactivation of transgene expression was transient, no GFP signal was detected 8 days after pHSVlac injection. In conclusion, HSV-1 amplicon vectors allow rapid and efficient, but transient, gene transfer in RPE cells following subretinal injection.

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

In rare diseases, health-related quality of life (HRQL) data can be difficult to capture. Given the ultrarare nature of RPE65-mediated inherited retinal disease (IRD), it was not feasible to recruit a patient sample and collect HRQL data prospectively. The objectives of this study were to develop health state descriptions of RPE65-mediated IRD, and to estimate associated patient utilities.

Vignette descriptions of IRD states were developed and then assessed to elicit utilities. The vignettes ranged from moderate vision loss through to hand motion to no light perception (NLP). Six retina specialists with additional expertise in IRDs provided a proxy valuation of the vignettes using generic measures of health-the 5-level version of EQ-5D-5L and Health Utility Index 3 (HUI3). The data were then scored using standard methods for each instrument.Weights from both HRQL measures revealed a large decline in scores with vision loss. The EQ-5D-5L weights ranged from 0.709 for moderate vision loss to 0.152 for hand motion to NLP. The HUI3 weights ranged from 0.519 to - 0.039, respectively. A decline was seen on both measures, and the degree of decline from moderate vision loss to NLP was identical on both (-0.56).

CONCLUSION

This is the first study to report HRQL weights (or utilities) for health states describing different levels of vision loss in patients with IRD, specifically those with RPE65-mediated disease. The parallel decline in scores from the EQ-5D and HUI3 corroborates the substantial impact of progressive vision loss on HRQL.