Retinitis Pigmentosa: From Pathomolecular Mechanisms to Therapeutic Strategies.

Retinitis pigmentosa is an inherited disease, in which mutations in different types of genes lead to the death of photoreceptors and the loss of visual function. Although retinitis pigmentosa is the most common type of inherited retinal dystrophy, a clear line of therapy has not yet been defined. In this review, we will focus on the therapeutic aspect and attempt to define the advantages and disadvantages of the protocols of different therapies. The role of some therapies, such as antioxidant agents or gene therapy, has been established for years now. Many clinical trials on different genes and mutations causing RP have been conducted, and the approval of voretigene nepavorec by the FDA has been an important step forward. Nonetheless, even if gene therapy is the most promising type of treatment for these patients, other innovative strategies, such as stem cell transplantation or hyperbaric oxygen therapy, have been shown to be safe and improve visual quality during clinical trials. The treatment of this disease remains a challenge, to which we hope to find a solution as soon as possible.

Atypical retinopathy in ataxia with vitamin E deficiency: report of a sibship.

Typical retinitis pigmentosa (RP) may not be the only retinal phenotype encountered in ataxia with vitamin E deficiency (AVED). The following short case series describes a novel form of retinopathy in AVED. We describe two patients with AVED belonging to the same consanguineous sibship. Both presented an unusual retinopathy consisting of scattered, multifocal, nummular, hyperautofluorescent atrophic retinal patches. The retinopathy remained stable under vitamin E supplementation. We hypothesize these changes to be the result of arrested AVED-related RP following early supplementation with α-tocopherol acetate.

Natural history of retinitis pigmentosa based on genotype, vitamin A/E supplementation, and an electroretinogram biomarker.

BACKGROUNDA randomized clinical trial from 1984 to 1992 indicated that vitamin A supplementation had a beneficial effect on the progression of retinitis pigmentosa (RP), while vitamin E had an adverse effect.METHODSSequencing of banked DNA samples from that trial provided the opportunity to determine whether certain genotypes responded preferentially to vitamin supplementation.RESULTSThe genetic solution rate was 587 out of 765 (77%) of sequenced samples. Combining genetic solutions with electroretinogram outcomes showed that there were systematic differences in severity and progression seen among different genetic subtypes of RP, extending findings made for USH2A, RHO, RPGR, PRPF31, and EYS. Baseline electroretinogram 30-Hz flicker implicit time was an independent, strong predictor of progression rate. Using additional data and baseline implicit time as a predictor, the deleterious effect of vitamin E was still present. Surprisingly, the effect of vitamin A progression in the cohort as a whole was not detectable, with or without data from subsequent trials. Subgroup analyses are also discussed.CONCLUSIONOverall, genetic subtype and implicit time have significant predictive power for a patient's rate of progression, which is useful prognostically. While vitamin E supplementation should still be avoided, these data do not support a generalized neuroprotective effect of vitamin A for all types of RP.TRIAL REGISTRATIONClinicalTrials.gov NCT00000114, NCT00000116, and NCT00346333.FUNDINGFoundation Fighting Blindness and the National Eye Institute: R01 EY012910, R01 EY031036, R01 EY026904, and P30 EY014104.

Dysregulation of Spliceosomes Complex Induces Retinitis Pigmentosa-Like Characteristics in sf3b4-Depleted Zebrafish.

The SF3B4 gene encodes a highly conserved protein that plays a critical role in mRNA splicing. Mutations in this gene are known to cause Nager syndrome, a rare craniofacial disorder. Although SF3B4 expression is detected in the optic vesicle before it is detected in the limb and somite, the role of SF3B4 in the eye is not well understood. This study investigated the function of sf3b4 in the retina by performing transcriptome profiles, immunostaining, and behavioral analysis of sf3b4-/- mutant zebrafish. Results from this study suggest that dysregulation of the spliceosome complex affects not only craniofacial development but also retinogenesis. Zebrafish lacking functional sf3b4 displayed characteristics similar to retinitis pigmentosa (RP), marked by severe retinal pigment epithelium defects and rod degeneration. Pathway analysis revealed altered retinol metabolism and retinoic acid signaling in the sf3b4-/- mutants. Supplementation of retinoic acid rescued key cellular phenotypes observed in the sf3b4-/- mutants, offering potential therapeutic strategies for RP in the future. In conclusion, this study sheds light on the previously unknown role of SF3B4 in retinogenesis and provides insights into the underlying mechanisms of RP.

Protective effect of ZYMT, a traditional Chinese patent medicine in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common genetic disorder that causes blindness. At present, there exists no remedy for the disease. The aim of the current research was to investigate the protective effect of Zhangyanming Tablets (ZYMT) in a mouse model of RP, and explore the underlying mechanism. Eighty RP mice were randomly divided into two groups. The mice in ZYMT group were administered with ZYMT suspension(0.0378 g/mL), while the mice in model group were given the same volume of distilled water. At day 7 and day 14 after intervention, electroretinogram (ERG), fundus photography, and histological examination were used to assess the retinal function and structure. TUNEL, immunofluorescence and qPCR were used to evaluate cell apoptosis and expressions of Sirt1, Iba1, Bcl-2, Bax and Caspase-3. A significantly shortened latency of ERG waves was observed in ZYMT-treated mice, in comparison to those in the model group (P < 0.05). Histologically, ultrastructure of the retina was better preserved, and the outer nuclear layer (ONL) exhibited marked increase in thickness and cell count in ZYMP group (P < 0.05). The apoptosis rate was decreased markedly in ZYMT group. Immunofluorescence analysis showed that the expressions of Iba1 and Bcl-2 in the retina were increased, Bax and Caspase-3 were decreased after ZYMT intervention, while the qPCR revealed that the expressions of Iba1 and Sirt1 were significantly increased (P < 0.05). This study indicated that ZYMT has protective effect on retinal function and morphology of inherited RP mice in the early stage, possibly mediated via the regulation of antioxidant and anti-/pro-apoptotic factors expressions.

Correlation between the Serum Concentration of Vitamin A and Disease Severity in Patients Carrying p.G90D in RHO, the Most Frequent Gene Associated with Dominant Retinitis Pigmentosa: Implications for Therapy with Vitamin A.

The pathogenic variant p.G90D in RHO is believed to be responsible for a spectrum of phenotypes, including congenital stationary blindness (for the purpose of this study termed night blindness without degeneration; NBWD), Sector RP, Pericentral RP, and Classic RP. We present a correlation between the serum concentration of vitamin A and disease severity in patients with this variant. This prospective study involved 30 patients from 7 families (17 male; median age 46 years, range 8−73). Full ophthalmological examination including visual acuity, Goldmann perimetry, slit-lamp exam, optical coherence tomography, fundus autofluorescence, and electrophysiology was performed to determine the presenting phenotype. The serum concentration of vitamin A was determined from a fasting blood sample taken on the day of the exam, where it was found that 23.3% (7/30) of patients had NBWD, 13.3% (4/30) had Sector RP, 3.3% (1/30) had Pericentral RP, and 60% (18/30) had Classic RP. Multiple logistic regression revealed a significantly higher probability of having a milder phenotype (NBWD or Sector RP) in association with younger age (p < 0.05) and a higher concentration of vitamin A (p < 0.05). We hypothesize that vitamin A in its 11-cis-retinal form plays a role in stabilizing the constitutively active p.G90D rhodopsin and its supplementation could be a potential treatment strategy for p.G90D RHO patients.

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.

Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Drug screening with zebrafish visual behavior identifies carvedilol as a potential treatment for an autosomal dominant form of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a mostly incurable inherited retinal degeneration affecting approximately 1 in 4000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish autosomal dominant RP model. This model expresses a truncated human rhodopsin transgene (Q344X) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol may directly on rods as it affected the adrenergic pathway in the photoreceptor-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat autosomal dominant RP patients.

Properties and Therapeutic Implications of an Enigmatic D477G RPE65 Variant Associated with Autosomal Dominant Retinitis Pigmentosa.

RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.

Vitamin A and fish oils for preventing the progression of retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) comprises a group of hereditary eye diseases characterized by progressive degeneration of retinal photoreceptors. It results in severe visual loss that may lead to blindness. Symptoms may become manifest during childhood or adulthood which include poor night vision (nyctalopia) and constriction of peripheral vision (visual field loss). Visual field loss is progressive and affects central vision later in the disease course. The worldwide prevalence of RP is approximately 1 in 4000, with 100,000 individuals affected in the USA. At this time, there is no proven therapy for RP. OBJECTIVES: The objective of this review was to synthesize the best available evidence regarding the effectiveness and safety of vitamin A and fish oils (docosahexaenoic acid (DHA)) in preventing the progression of RP. SEARCH METHODS: We searched the Cochrane Central Register of Controlled Trials (CENTRAL), which contains the Cochrane Eyes and Vision Trials Register (2020, Issue 2); Ovid MEDLINE; Embase.com; PubMed; Latin American and Caribbean Health Sciences Literature Database (LILACS); ClinicalTrials.gov; the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP); and OpenGrey. We did not use any date or language restrictions in the electronic searches for trials. We last searched the electronic databases on 7 February 2020. SELECTION CRITERIA: We included randomized controlled trials that enrolled participants of any age diagnosed with any degree of severity or type of RP, and evaluated the effectiveness of vitamin A, fish oils (DHA), or both compared to placebo, vitamins (other than vitamin A), or no therapy, as a treatment for RP. We excluded cluster-randomized trials and cross-over trials. DATA COLLECTION AND ANALYSIS: We prespecified the following outcomes: mean change from baseline visual field, mean change from baseline electroretinogram (ERG) amplitudes, and anatomic changes as measured by optical coherence tomography (OCT), at one-year follow-up, and mean change in visual acuity, at five-year follow-up. Two review authors independently extracted data and evaluated risk of bias for all included trials. We also contacted study investigators for further information when necessary. MAIN RESULTS: In addition to three trials from the previous version of this review, we included a total of four trials with 944 participants aged 4 to 55 years. Two trials included only participants with X-linked RP and the other two included participants with RP of all forms of genetic predisposition. Two trials evaluated the effect of DHA alone; one trial evaluated vitamin A alone; and one trial evaluated DHA and vitamin A versus vitamin A alone. Two trials recruited participants from the USA, and the other two recruited from the USA and Canada. All trials were at low risk of bias for most domains. We did not perform meta-analysis due to clinical heterogeneity. Four trials assessed visual field sensitivity. Investigators found no evidence of a difference in mean values between the groups. However, one trial found that the annual rate of change of visual field sensitivity over four years favored the DHA group in foveal (-0.02 ± 0.55 (standard error (SE)) dB versus -0.47 ± 0.03 dB, P = 0.039), macular (-0.42 ± 0.05 dB versus -0.85 ± 0.03 dB, P = 0.031), peripheral (-0.39 ± 0.02 versus -0.86 ± 0.02 dB, P < 0.001), and total visual field sensitivity (-0.39 ± 0.02 versus -0.86 ± 0.02 dB, P < 0.001). The certainty of the evidence was very low. The four trials evaluated visual acuity (LogMAR scale) at a follow-up of four to six years. In one trial (208 participants), investigators found no evidence of a difference between the two groups, as both groups lost 0.7 letters of the Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity per year. In another trial (41 participants), DHA showed no evidence of effect on visual acuity (mean difference -0.01 logMAR units (95% confidence interval -0.14 to 0.12; one letter difference between the two groups; very low-certainty evidence). In the third trial (60 participants), annual change in mean number of letters correct was -0.8 (DHA) and 1.4 letters (placebo), with no evidence of between-group difference. In the fourth trial (572 participants), which evaluated (vitamin A + vitamin E trace) compared with (vitamin A trace + vitamin E trace), decline in ETDRS visual acuity was 1.1 versus 0.9 letters per year, respectively. All four trials reported electroretinography (ERG). Investigators of two trials found no evidence of a difference between the DHA and placebo group in yearly rates of change in 31 Hz cone ERG amplitude (mean ± SE) (-0.028 ± 0.001 log µV versus -0.022 ± 0.002 log µV; P = 0.30); rod ERG amplitude (mean ± SE) (-0.010 ± 0.001 log µV versus -0.023 ± 0.001 log µV; P = 0.27); and maximal ERG amplitude (mean ± SE) (-0.042 ± 0.001 log µV versus -0.036 ± 0.001 log µV; P = 0.65). In another trial, a slight difference (6.1% versus 7.1%) in decline of ERG per year favored vitamin A (P = 0.01). The certainty of the evidence was very low. One trial (51 participants) that assessed optical coherence tomography found no evidence of a difference in ellipsoid zone constriction (P = 0.87) over two years, with very low-certainty evidence. The other three trials did not report this outcome. Only one trial reported adverse events, which found that 27/60 participants experienced 42 treatment-related emergent adverse events (22 in DHA group, 20 in placebo group). The certainty of evidence was very low. The rest of the trials reported no adverse events, and no study reported any evidence of benefit of vitamin supplementation on the progression of visual acuity loss. AUTHORS' CONCLUSIONS: Based on the results of four studies, it is uncertain if there is a benefit of treatment with vitamin A or DHA, or both for people with RP. Future trials should also take into account the changes observed in ERG amplitudes and other outcome measures from trials included in this review.

Rescuing cones and daylight vision in retinitis pigmentosa mice.

Hallmark of retinitis pigmentosa (RP) is the primary, genetic degeneration of rods followed by secondary loss of cones, caused by still elusive biologic mechanisms. We previously shown that exposure of rd10 mutant mice, modeling autosomal recessive RP, to environmental enrichment (EE), with enhanced motor, sensorial and social stimuli, results into a sensible delay of retinal degeneration and vision loss. Searching for effectors of EE-mediated retinal protection, we performed transcriptome analysis of the retina of rd10 enriched and control mice and found that gene expression at the peaks of rod and cone degeneration is characterized by a strong inflammatory/immune response, which is however measurably lower in enrichment conditions. Treating rd10 mice with dexamethasone during the period of maximum photoreceptors death lowered retinal inflammation and caused a preservation of cones and cone-mediated vision. Our findings indicate a link between retinal inflammation and bystander cone degeneration, reinforcing the notion that cone vision in RP can be preserved using anti-inflammatory approaches.-Guadagni, V., Biagioni, M., Novelli, E., Aretini, P., Mazzanti, C. M., Strettoi, E. Rescuing cones and daylight vision in retinitis pigmentosa mice.

Filtration of Short-Wavelength Light Provides Therapeutic Benefit in Retinitis Pigmentosa Caused by a Common Rhodopsin Mutation.

Purpose: The role of light exposure in accelerating retinitis pigmentosa (RP) remains controversial. Faster degeneration has however been observed in the inferior than superior retina in several forms ("sector" RP), including those caused by the rhodopsin P23H mutation, suggesting a modifying role of incident light exposure in such cases. Rearing of equivalent animal models in complete darkness has been shown to slow the degeneration. Here we investigate the use of red filters as a potential treatment strategy, with the hypothesis that minimizing retinal exposure to light <600 nm to which rods are maximally sensitive may provide therapeutic benefit. Methods: Knockin mice heterozygous for the P23H dominant rhodopsin mutation (RhoP23H/+) housed in red-tinted plastic cages were divided at weaning into either untinted or red-tinted cages. Subsequently, photoreceptor layer (PRL) thickness was measured by spectral-domain ocular coherence tomography, retinal function quantified by ERG, and cone morphology determined by immunohistochemical analysis (IHC) of retinal flatmounts. Results: Mice remaining in red-tinted cages had a significantly greater PRL thickness than those housed in untinted cages at all time points. Red housing also led to a highly significant rescue of retinal function as determined by both dark- and light-adapted ERG responses. IHC further revealed a dramatic benefit on cone morphology and number in the red- as compared with the clear-housed group. Conclusions: Limitation of short-wavelength light exposure significantly slows degeneration in the RhoP23H/+ mouse model. Red filters may represent a cost-effective and low-risk treatment for patients with rod-cone dystrophy in whom a sectoral phenotype is noted.

(3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one ameliorates retinal degeneration in Pde6brd10 mice.

As a severe photoreceptor-degenerative disease, retinitis pigmentosa (RP) is currently incurable and eventually leads to partial or complete blindness. (3R)-5,6,7-trihydroxy-3-isopropyl-3-methylisochroman-1-one (TIM) is a novel antioxidant isolated from the plant of Alpinia katsumadai Hayata, with protective effects on photoreceptor cells against lipoteichoic acid-induced damage through inhibiting oxidative stress. The present study was to further demonstrate whether TIM could ameliorate retinal degeneration of Pde6brd10 (rd10) mice, a mouse model of RP. rd10 mice were treated with TIM by intraperitoneal injection daily from postnatal Day 10 (P10) to P26. Retinal function was tested by electroretinography. Histology was evaluated by toluidine blue staining and TUNEL assay. Oxidative stress markers were measured by ELISA. Immunohistochemistry, real-time PCR, and western blotting were applied to explore the protective mechanism. Results showed TIM significantly improved the retinal function and decreased photoreceptor cell apoptosis in rd10 mice through reducing oxidative stress. For the first time, this study demonstrated the protective effects of TIM against retinal degeneration in rd10 mice, providing scientific rationale to use TIM treating the RP.

Docosahexaenoic Acid Slows Visual Field Progression in X-Linked Retinitis Pigmentosa: Ancillary Outcomes of the DHAX Trial.

PURPOSE: Docosahexaenoic acid (DHA) was supplemented in a single-site, placebo-controlled, randomized clinical trial designed to slow vision loss associated with X-linked retinitis pigmentosa (XLRP); the DHAX Trial. We previously reported no significant differences between supplemented and placebo groups in intent-to-treat analysis of primary ERG outcomes. Assessed herein are hypothesis-generating measures of ancillary visual function outcomes in participants fully adhering to trial protocol. METHODS: Male participants with XLRP (range, 7-31 years) received 30 mg DHA/kg/d (n = 29) or placebo (n = 22) for 4 years. Visual outcomes were measured annually and red blood cell (RBC) DHA determined every 6 months. RESULTS: Oral DHA supplementation increased mean RBC-DHA levels by 4-fold (P < 0.0001) over placebo. No group differences in progression were found for visual acuity (P = 0.11), shape discrimination (P = 0.18), or fundus appearance (P = 0.70). Optical coherence tomography (OCT) became available during year 2 of the trial; no group differences were seen in ellipsoid zone constriction (P = 0.87) over 2 years. Yearly rates of progression were reduced for dark-adapted thresholds (P = 0.06) and visual field sensitivity for foveal, macular, peripheral, total, and ellipsoid zone regions by DHA supplementation (P = 0.039, P = 0.031, P < 0.0001, P < 0.0001, and P = 0.033). Rates of visual field sensitivity decline were dependent on RBC-DHA (P = 0.046 to <0.0001). CONCLUSIONS: Supplementation of DHA significantly elevated blood DHA levels and reduced the rate of progression in final dark-adapted thresholds and visual field sensitivity. From the relationship between RBC-DHA and the rate of field sensitivity loss, we can extrapolate that an RBC-DHA level of 17% could minimize the decline in field sensitivity. (ClinicalTrials.gov number, NCT00100230.)

A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.

PURPOSE: Inherent instability of the P23H mutant opsin accounts for approximately 10% of autosomal dominant retinitis pigmentosa cases. Our purpose was to develop an overall set of reliable screening strategies to assess if either stabilization or enhanced degradation of mutant rhodopsin could rescue rod photoreceptors expressing this mutant protein. These strategies promise to reveal active compounds and clarify molecular mechanisms of biologically important processes, such as inhibition of target degradation or enhanced target folding. METHODS: Cell-based bioluminescence reporter assays were developed and validated for high-throughput screening (HTS) of compounds that promote either stabilization or degradation of P23H mutant opsin. Such assays were further complemented by immunoblotting and image-based analyses. RESULTS: Two stabilization assays of P23H mutant opsin were developed and validated, one based on ß-galactosidase complementarity and a second assay involving bioluminescence resonance energy transfer (BRET) technology. Moreover, two additional assays evaluating mutant protein degradation also were employed, one based on the disappearance of luminescence and another employing the ALPHA immunoassay. Imaging of cells revealed the cellular localization of mutant rhodopsin, whereas immunoblots identified changes in the aggregation and glycosylation of P23H mutant opsin. CONCLUSIONS: Our findings indicate that these initial HTS and following assays can identify active therapeutic compounds, even for difficult targets such as mutant rhodopsin. The assays are readily scalable and their function has been proven with model compounds. High-throughput screening, supported by automated imaging and classic immunoassays, can further characterize multiple steps and pathways in the biosynthesis and degradation of this essential visual system protein.

Recent advances in treatment of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by the progressive photoreceptors and pigment epithelial cells dysfunction. It is the most common retinal degeneration, responsible for loss of vision of most people worldwide. Until now its exact pathogenesis and etiology are not clear. So far there is no approved therapy. New approaches for RP therapy include cell transplantation, gene therapy, cytokine therapy, nutrition therapy, and hyperbaric oxygen therapy. Such therapies for retinal degenerative diseases are limited in their efficacy. This paper reviews the relevant documents, especially recent researches, and reviews advances in the treatment of RP.

Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.

Safety assessment of docosahexaenoic acid in X-linked retinitis pigmentosa: the 4-year DHAX trial.

PURPOSE: Docosahexaenoic acid (DHA) continues to be evaluated and recommended as treatment and prophylaxis for various diseases. We recently assessed efficacy of high-dose DHA supplementation to slow vision loss in patients with X-linked retinitis pigmentosa (XLRP) in a randomized clinical trial. Because DHA is a highly unsaturated fatty acid, it could serve as a target for free-radical induced oxidation, resulting in increased oxidative stress. Biosafety was monitored during the 4-year trial to determine whether DHA supplementation was associated with identifiable risks. METHODS: Males (n = 78; 7-31 years) meeting entry criteria were enrolled. The modified intent-to-treat cohort (DHA = 33; placebo = 27) adhered to the protocol ≥ 1 year. Participants were randomized to an oral dose of 30 mg/kg/d DHA or placebo plus a daily multivitamin. Comprehensive metabolic analyses were assessed for group differences. Treatment-emergent adverse events including blood chemistry metabolites were recorded. RESULTS: By year 4, supplementation elevated plasma and red blood cell-DHA 4.4- and 3.6-fold, respectively, compared with the placebo group (P < 0.00001). Over the trial duration, no significant differences between DHA and placebo groups were found for vitamin A, vitamin E, platelet aggregation, antioxidant activity, lipoprotein cholesterol, or oxidized LDL levels (all P > 0.14). Adverse events were transient and not considered severe (e.g., gastrointestinal [GI] irritability, blood chemistry alterations). One participant was unable to tolerate persistent GI discomfort. CONCLUSIONS: Long-term, high-dose DHA supplementation to patients with XLRP was associated with limited safety risks in this 4-year trial. Nevertheless, GI symptoms should be monitored in all patients taking high dose DHA especially those with personal or family history of GI disturbances. (ClinicalTrials.gov number, NCT00100230.).

The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is an inherited human retinal disorder that causes progressive photoreceptor cell loss, leading to severe vision impairment or blindness. However, no effective therapy has been established to date. Although genetic mutations have been identified, the available clinical data are not always sufficient to elucidate the roles of these mutations in disease pathogenesis, a situation that is partially due to differences in genetic backgrounds. RESULTS: We generated induced pluripotent stem cells (iPSCs) from an RP patient carrying a rhodopsin mutation (E181K). Using helper-dependent adenoviral vector (HDAdV) gene transfer, the mutation was corrected in the patient's iPSCs and also introduced into control iPSCs. The cells were then subjected to retinal differentiation; the resulting rod photoreceptor cells were labeled with an Nrl promoter-driven enhanced green fluorescent protein (EGFP)-carrying adenovirus and purified using flow cytometry after 5 weeks of culture. Using this approach, we found a reduced survival rate in the photoreceptor cells with the E181K mutation, which was correlated with the increased expression of endoplasmic reticulum (ER) stress and apoptotic markers. The screening of therapeutic reagents showed that rapamycin, PP242, AICAR, NQDI-1, and salubrinal promoted the survival of the patient's iPSC-derived photoreceptor cells, with a concomitant reduction in markers of ER stress and apoptosis. Additionally, autophagy markers were found to be correlated with ER stress, suggesting that autophagy was reduced by suppressing ER stress-induced apoptotic changes. CONCLUSION: The use of RP patient-derived iPSCs combined with genome editing provided a versatile cellular system with which to define the roles of genetic mutations in isogenic iPSCs with or without mutation and also provided a system that can be used to explore candidate therapeutic approaches.