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.

The Arabidopsis D27-LIKE1 is a cis/cis/trans-ß-carotene isomerase that contributes to Strigolactone biosynthesis and negatively impacts ABA level.

The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-ß-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed an unreported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-ß-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed a more pronounced branching phenotype compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely a result of its formation of 9-cis-ß-carotene that was present at higher levels in AtD27-like1 overexpressing lines. By contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of ABA, in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing GUS driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings revealed that AtD27LIKE1 is expressed in different tissues and affects ABA and auxin. Taken together, our work reports a cis/cis-ß-carotene isomerase that affects the content of both cis-carotenoid-derived plant hormones, ABA and SLs.

Rapamycin Inhibits Light-Induced Necrosome Activation Occurring in Wild-Type, but not RPE65-Null, Mouse Retina.

Purpose: Both photodamage and aberrant visual cycle contribute to disease progress of many retinal degenerative disorders, whereas the signaling pathways causing photoreceptor death remain unclear. Here we investigated the effects of intense photo-stress on (1) necrosome activation in wild-type and RPE65-null mice, (2) interaction of p62/Sequestosome-1 with the necrosome proteins, and (3) the effects of rapamycin on photodamage-induced necrosome activation and retinal degeneration in wild-type mice. Methods: Dark-adapted rd12 mice and 129S2/Sv mice with or without rapamycin treatment were exposed to 15,000 lux light for different times. Expression levels and subcellular localization of proteins were determined through immunoblot and immunohistochemical analyses. Cone sheaths were stained with peanut agglutinin. Correlation between photoreceptor degeneration and receptor-interacting protein kinase-1 (RIPK1) expression was assessed with Spearman's correlation analysis. Protein-protein interaction was analyzed by immunoprecipitation. Results: Intense light caused rod and cone degeneration accompanied by a significant increase in RIPK1-RIPK3 expressions, mixed lineage kinase domain-like protein phosphorylation, damage-associated molecular patterns protein release, and inflammatory responses in wild-type mouse retina. The same intense light did not induce the necrosome activation in rd12 retina, but it did in rd12 mice that received 9-cis-retinal supply. RIPK1 expression levels are positively correlated with the degrees of rod and cone degeneration. Photodamage upregulated expression and interaction of the p62 autophagosome cargo protein with the necrosome proteins, whereas rapamycin treatment attenuated the light-induced necrosome activation and photoreceptor degeneration. Conclusions: Necrosome activation contributed to photodamage-induced rod and cone degeneration. The visual cycle and autophagy are the important therapeutic targets to alleviate light-induced retinal necroptosis.

Functional expression of the nitrogenase Fe protein in transgenic rice.

Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N2. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis-trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N2 fixation in planta. Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops.

The novel visual cycle inhibitor (±)-RPE65-61 protects retinal photoreceptors from light-induced degeneration.

The visual cycle refers to a series of biochemical reactions of retinoids in ocular tissues and supports the vision in vertebrates. The visual cycle regenerates visual pigments chromophore, 11-cis-retinal, and eliminates its toxic byproducts from the retina, supporting visual function and retinal neuron survival. Unfortunately, during the visual cycle, when 11-cis-retinal is being regenerated in the retina, toxic byproducts, such as all-trans-retinal and bis-retinoid is N-retinylidene-N-retinylethanolamine (A2E), are produced, which are proposed to contribute to the pathogenesis of the dry form of age-related macular degeneration (AMD). The primary biochemical defect in Stargardt disease (STGD1) is the accelerated synthesis of cytotoxic lipofuscin bisretinoids, such as A2E, in the retinal pigment epithelium (RPE) due to mutations in the ABCA4 gene. To prevent all-trans-retinal-and bisretinoid-mediated retinal degeneration, slowing down the retinoid flow by modulating the visual cycle with a small molecule has been proposed as a therapeutic strategy. The present study describes RPE65-61, a novel, non-retinoid compound, as an inhibitor of RPE65 (a key enzyme in the visual cycle), intended to modulate the excessive activity of the visual cycle to protect the retina from harm degenerative diseases. Our data demonstrated that (±)-RPE65-61 selectively inhibited retinoid isomerase activity of RPE65, with an IC50 of 80 nM. Furthermore, (±)-RPE65-61 inhibited RPE65 via an uncompetitive mechanism. Systemic administration of (±)-RPE65-61 in mice resulted in slower chromophore regeneration after light bleach, confirming in vivo target engagement and visual cycle modulation. Concomitant protection of the mouse retina from high-intensity light damage was also observed. Furthermore, RPE65-61 down-regulated the cyclic GMP-AMP synthase stimulator of interferon genes (cGAS-STING) pathway, decreased the inflammatory factor, and attenuated retinal apoptosis caused by light-induced retinal damage (LIRD), which led to the preservation of the retinal function. Taken together, (±)-RPE65-61 is a potent visual cycle modulator that may provide a neuroprotective therapeutic benefit for patients with STGD and AMD.

RPE65-Associated Retinopathies in the Italian Population: A Longitudinal Natural History Study.

Purpose: To investigate the course of inherited retinal degenerations (IRD) due to mutations in the RPE65 gene. Methods: This longitudinal multicentric retrospective chart-review study was designed to collect best corrected visual acuity (BCVA), Goldman visual field, optical coherence tomography (OCT), and electroretinography (ERG) measurements. The data, including imaging, were collected using an electronic clinical research form and were reviewed at a single center to improve consistency. Results: From an overall cohort of 60 Italian patients with RPE65-associated IRD, 43 patients (mean age, 27.8 ± 19.7 years) were included and showed a mean BCVA of 2.0 ± 1.0 logMAR. Time-to-event analysis revealed a median age of 33.8 years and 41.4 years to reach low vision and blindness based on BCVA, respectively. ERG (available for 34 patients) showed undetectable responses in most patients (26; 76.5%). OCT (available for 31 patients) revealed epiretinal membranes in five patients (16.1%). Central foveal thickness significantly decreased with age at a mean annual rate of -0.6%/y (P = 0.044). We identified 43 different variants in the RPE65 gene in the entire cohort. Nine variants were novel. Finally, to assess genotype-phenotype correlations, patients were stratified according to the number of RPE65 loss-of-function (LoF) alleles. Patients without LoF variants showed significantly (P < 0.05) better BCVA compared to patients with one or two LoF alleles. Conclusions: We described the natural course of RPE65-associated IRD in an Italian cohort showing for the first time a specific genotype-phenotype association. Our findings can contribute to a better management of RPE65-associated IRD patients.

Functional Diversification of the Carotenoid-cis-trans-Isomerases CrtISO, CrtISO-L1, and CrtISO-L2 in Tomato Species (Solanum, Section Lycopersicon).

The expression of the genes of carotenoid-cis-trans-isomerases CrtISO, CrtISO-L1, and CrtISO-L2 was studied in comparison with the content of carotenoids in tomato species with different ripe fruit colors: green (Solanum habrochaites), yellow (S. cheesmaniae), and red (S. pimpinellifolium and S. lycopersicum). More ancient origin of CrtISO-L2 in relation to CrtISO and CrtISO-L1 was shown. A similar content of total carotenoids (leaves) and ß-carotene (ripe fruits) between the samples was found. Unlike the fruits of S. habrochaites and S. cheesmaniae, the red fruits accumulated lycopene and 20-30 times greater total carotenoids. The highest level of transcripts both in leaves and in ripe fruits was detected for CrtISO. The CrtISO-L1 and CrtISO-L2 genes were transcribed at high levels in leaves and at low levels in fruits, except for the high expression of CrtISO-L2 in S. lycopersicum fruits. No correlation between the content of carotenoids and the level of gene expression in the fruit was observed. In the leaves, a positive correlation between the amount of carotenoids and the levels of CrtISO-L1 and CrtISO-L2 transcripts was found.

Histology and clinical imaging lifecycle of black pigment in fibrosis secondary to neovascular age-related macular degeneration.

PURPOSE: Melanotic cells with large spherical melanosomes, thought to originate from retinal pigment epithelium (RPE), are found in eyes with neovascular age-related macular degeneration (nvAMD). To generate hypotheses about RPE participation in fibrosis, we correlate histology to clinical imaging in an eye with prominent black pigment in fibrotic scar secondary to nvAMD. METHODS: Macular findings in a white woman with untreated inactive subretinal fibrosis due to nvAMD in her right eye were documented over 9 years with color fundus photography (CFP), fundus autofluorescence (FAF) imaging, and optical coherence tomography (OCT). After death (age 90 years), this index eye was prepared for light and electron microscopy to analyze 7 discrete zones of pigmentation in the fibrotic scar. In additional donor eyes with nvAMD, we determined the frequency of black pigment (n = 36 eyes) and immuno-labeled for retinoid, immunologic, and microglial markers (RPE65, CD68, Iba1, TMEM119; n = 3 eyes). RESULTS: During follow-up of the index eye, black pigment appeared and expanded within a hypoautofluorescent fibrotic scar. The blackest areas correlated to melanotic cells (containing large spherical melanosomes), some in multiple layers. Pale areas had sparse pigmented cells. Gray areas correlated to cells with RPE organelles entombed in the scar and multinucleate cells containing sparse large spherical melanosomes. In 94% of nvAMD donor eyes, hyperpigmentation was visible. Certain melanotic cells expressed some RPE65 and mostly CD68. Iba1 and TMEM119 immunoreactivity, found both in retina and scar, did not co-localize with melanotic cells. CONCLUSION: Hyperpigmentation in CFP results from both organelle content and optical superimposition effects. Black fundus pigment in nvAMD is common and corresponds to cells containing numerous large spherical melanosomes and superimposition of cells containing sparse large melanosomes, respectively. Melanotic cells are molecularly distinct from RPE, consistent with a process of transdifferentiation. The subcellular source of spherical melanosomes remains to be determined. Detailed histology of nvAMD eyes will inform future studies using technologies for spatially resolved molecular discovery to generate new therapies for fibrosis. The potential of black pigment as a biomarker for fibrosis can be investigated in clinical multimodal imaging datasets.

Retinal pigment epithelium 65 kDa protein (RPE65): An update.

Vertebrate vision critically depends on an 11-cis-retinoid renewal system known as the visual cycle. At the heart of this metabolic pathway is an enzyme known as retinal pigment epithelium 65 kDa protein (RPE65), which catalyzes an unusual, possibly biochemically unique, reaction consisting of a coupled all-trans-retinyl ester hydrolysis and alkene geometric isomerization to produce 11-cis-retinol. Early work on this isomerohydrolase demonstrated its membership to the carotenoid cleavage dioxygenase superfamily and its essentiality for 11-cis-retinal production in the vertebrate retina. Three independent studies published in 2005 established RPE65 as the actual isomerohydrolase instead of a retinoid-binding protein as previously believed. Since the last devoted review of RPE65 enzymology appeared in this journal, major advances have been made in a number of areas including our understanding of the mechanistic details of RPE65 isomerohydrolase activity, its phylogenetic origins, the relationship of its membrane binding affinity to its catalytic activity, its role in visual chromophore production for rods and cones, its modulation by macromolecules and small molecules, and the involvement of RPE65 mutations in the development of retinal diseases. In this article, I will review these areas of progress with the goal of integrating results from the varied experimental approaches to provide a comprehensive picture of RPE65 biochemistry. Key outstanding questions that may prove to be fruitful future research pursuits will also be highlighted.

Perifoveal Chorioretinal Atrophy after Subretinal Voretigene Neparvovec-rzyl for RPE65-Mediated Leber Congenital Amaurosis.

PURPOSE: To report an anatomic change following subretinal injection of voretigene neparvovec-rzyl (VN) for RPE65-mediated Leber congenital amaurosis. DESIGN: Multicenter, retrospective chart review. PARTICIPANTS: Patients who underwent subretinal VN injection at each of 4 participating institutions. METHODS: Patients were identified as having perifoveal chorioretinal atrophy if (1) the areas of atrophy were not directly related to the touch-down site of the subretinal cannula; and (2) the area of atrophy progressively enlarged over time. Demographic data, visual acuity, refractive error, fundus photographs, OCT, visual fields, and full-field stimulus threshold (FST) were analyzed. MAIN OUTCOME MEASURES: Outcome measures included change in visual acuity, FST, visual fields, and location of atrophy relative to subretinal bleb position. RESULTS: A total of 18 eyes of 10 patients who underwent subretinal injection of VN were identified as having developed perifoveal chorioretinal atrophy. Eight of 10 patients (80%) developed bilateral atrophy. The mean age was 11.6 years (range, 5-20 years), and 6 patients (60%) were male. Baseline mean logarithm of the minimum angle of resolution visual acuity and FST were 0.82 (standard deviation [SD], 0.51) and -1.3 log cd.s/m2 (SD, 0.44), respectively. The mean spherical equivalent was -5.7 diopters (D) (range, -11.50 to +1.75 D). Atrophy was identifiable at an average of 4.7 months (SD, 4.3) after surgery and progressively enlarged in all cases up to a mean follow-up period of 11.3 months (range, 4-18 months). Atrophy developed within and outside the area of the subretinal bleb in 10 eyes (55.5%), exclusively within the area of the bleb in 7 eyes (38.9%), and exclusively outside the bleb in 1 eye (5.5%). There was no significant change in visual acuity (P = 0.45). There was a consistent improvement in FST with a mean improvement of -3.21 log cd.s/m2 (P < 0.0001). Additionally, all 13 eyes with reliable Goldmann visual fields demonstrated improvement, but 3 eyes (23.1%) demonstrated paracentral scotomas related to the atrophy. CONCLUSIONS: A subset of patients undergoing subretinal VN injection developed progressive perifoveal chorioretinal atrophy after surgery. Further study is necessary to determine what ocular, surgical delivery, and vector-related factors predispose to this complication.

RPE65-related retinal dystrophy: Mutational and phenotypic spectrum in 45 affected patients.

INTRODUCTION: Biallelic pathogenic RPE65 variants are related to a spectrum of clinically overlapping inherited retinal dystrophies (IRD). Most affected individuals progress to severe disease, with 50% of patients becoming legally blind by 20 years of age. Deeper knowledge of the mutational spectrum and the phenotype-genotype correlation in RPE65-related IRD is needed. PATIENTS AND METHODS: Forty-five affected subjects from 27 unrelated families with a clinical diagnosis of RPE65-related IRD were included. Clinical evaluation consisted of self-reported ophthalmological history and objective ophthalmological examination. Patients' genotype was classified according to variant class (truncating or missense) or to variant location at different protein domains. The main phenotypic outcome measure was age at onset (AAO) of symptomatic disease and a Kaplan-Meier analysis of disease symptom event-free survival was performed. RESULTS: Twenty-nine different RPE65 variants were identified in our cohort, 7 of them novel. Patients carrying two missense alleles showed a later disease onset than those with 1 or 2 truncating variants (log-rank test p <0.05). While 60% of patients carrying a missense/missense genotype presented symptoms before or during the first year of life, almost all patients with at least 1 truncating allele (91%) had an AAO ≤1 year (p <0.05). CONCLUSION: Our findings suggest an association between the type of RPE65 variant carried and AAO. These findings provide useful data on RPE65-associated IRD phenotypes and may help improve clinical and therapeutic management of these patients.

The bacterial protective armor against stress: The cis-trans isomerase of unsaturated fatty acids, a cytochrome-c type enzyme.

Bacteria have evolved several outstanding strategies to resist to compounds or factors that compromise their survival. The first line of defense of the cell against environmental stresses is the membrane with fatty acids as fundamental building blocks of phospholipids. In this review, we focus on a periplasmic heme enzyme that catalyzes the cis-trans isomerization of unsaturated fatty acids to trigger a decrease in the fluidity of the membrane in order to rapidly counteract the danger. We particularly detailed the occurrence of such cis-trans isomerase in Nature, the different stresses that are at the origin of the double bond isomerization, the first steps in the elucidation of the mechanism of this peculiar metalloenzyme and some aspects of its regulation.

Protective effect of rapamycin in models of retinal degeneration.

Age-related macular degeneration (AMD) is a complex retinal disease with no viable treatment strategy. The causative mechanistic pathway for this disease is not yet clear. Therefore, it is highly warranted to screen effective drugs to treat AMD. Rapamycin are known to inhibit inflammation and has been widely used in the clinic as an immunosuppressant. This study aimed to investigate the protective effect of rapamycin on the AMD retinal degeneration model. The AMD models were established by injection of 35 mg/kg sodium iodate (NaIO3) into the tail vein. Then the treated mice intraperitoneally received rapamycin (2 mg/kg) once a day. The histomorphological analysis showed that rapamycin could inhibit retinal structure damage and apoptosis. Experiments revealed that rapamycin significantly attenuated inflammatory response and oxidative stress. Our experimental results demonstrated that rapamycin has protected the retinal against degeneration induced by NaIO3. The therapeutic effect was more significant after 7 days of treatment. Therefore, our study potentially provides a powerful experimental support for the treatment of AMD.

Rational Alteration of Pharmacokinetics of Chiral Fluorinated and Deuterated Derivatives of Emixustat for Retinal Therapy.

Recycling of all-trans-retinal to 11-cis-retinal through the visual cycle is a fundamental metabolic pathway in the eye. A potent retinoid isomerase (RPE65) inhibitor, (R)-emixustat, has been developed and tested in several clinical trials; however, it has not received regulatory approval for use in any specific retinopathy. Rapid clearance of this drug presents challenges to maintaining concentrations in eyes within a therapeutic window. To address this pharmacokinetic inadequacy, we rationally designed and synthesized a series of emixustat derivatives with strategically placed fluorine and deuterium atoms to slow down the key metabolic transformations known for emixustat. Crystal structures and quantum chemical analysis of RPE65 in complex with the most potent emixustat derivatives revealed the structural and electronic bases for how fluoro substituents can be favorably accommodated within the active site pocket of RPE65. We found a close (∼3.0 Å) F-π interaction that is predicted to contribute ∼2.4 kcal/mol to the overall binding energy.

Durability of Voretigene Neparvovec for Biallelic RPE65-Mediated Inherited Retinal Disease: Phase 3 Results at 3 and 4 Years.

PURPOSE: To determine whether functional vision and visual function improvements after voretigene neparvovec (VN; Luxturna [Spark Therapeutics, Inc]) administration in patients with biallelic RPE65 mutation-associated inherited retinal disease are maintained at 3 to 4 years and to review safety outcomes. DESIGN: Open-label, randomized, controlled phase 3 trial. PARTICIPANTS: Thirty-one individuals were enrolled and randomized 2:1 to intervention (n = 21) or control (n = 10). One participant from each group withdrew before, or at, randomization. METHODS: Patients in the original intervention (OI) group received bilateral subretinal VN injections. Delayed intervention (DI) patients served as control participants for 1 year then received VN. MAIN OUTCOME MEASURES: Change from injection baseline in bilateral performance on the multiluminance mobility test (MLMT), a measure of ambulatory navigation, and change from injection baseline in full-field light sensitivity threshold white light, visual field (VF), and visual acuity (VA). RESULTS: Mean bilateral MLMT change scores at year 4 for OI patients and year 3 for DI patients were 1.7 and 2.4, respectively, with 71% of patients with a year 3 visit able to pass MLMT at the lowest light level. Mean change in full-field light sensitivity threshold white light, averaged over both eyes at year 4 for OI patients and year 3 for DI patients, was -1.90 log10(cd.s/m2) and -2.91 log10(cd.s/m2), respectively. Mean change in Goldmann kinetic VF III4e sum total degrees, averaged across both eyes, was 197.7 at year 4 for OI patients and 157.9 at year 3 for DI patients. Mean change in VA (Holladay scale), averaged across both eyes, was -0.003 logarithm of the minimum angle of resolution (logMAR) at year 4 for OI patients and -0.06 logMAR at year 3 for DI patients. One OI patient experienced retinal detachment at approximately year 4 that impacted VA for the OI group. No product-related serious adverse events (AEs) occurred, nor did any deleterious immune responses. CONCLUSIONS: Improvements in ambulatory navigation, light sensitivity, and VF were consistent in both intervention groups. Overall, improvements were maintained up to 3 to 4 years, with ongoing observation. The safety profile of VN was consistent with vitrectomy and the subretinal injection procedure and was similar between intervention groups, with no product-related serious AEs reported.

A vicious cycle of bisretinoid formation and oxidation relevant to recessive Stargardt disease.

The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (•OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (•OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knockout mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in postbleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain, is not affected. Notably, iron levels were elevated in the neural retina and retinal pigment epithelial (RPE) cells of Abca4-/- mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4-/- mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease.

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE.

Induction of Differentiation of Mesenchymal Stem Cells into Retinal Pigment Epithelial Cells for Retinal Regeneration by Using Ciliary Neurotrophic Factor in Diabetic Rats.

Diabetic retinopathy (DR) is a common cause of blindness all over the world. Bone marrow mesenchymal stem cells (BMSCs) have been considered as a promising strategy for retinal regeneration in the treatment of DR. However, the poor viability and low levels of BMSCs engraftment limit the therapeutic potential of BMSCs. The present study aimed to examine the direct induction of BMSCs differentiation into the cell types related to retinal regeneration by using soluble cytokine ciliary neurotrophic factor (CNTF). We observed remarkably increased expression of cellular retinaldehyde-binding protein (CRALBP) and retinoid isomerohydrolase (RPE65) in BMSCs treated with CNTF in vitro, indicating the directional differentiation of BMSCs into the retinal pigment epithelium (RPE) cells, which are crucial for retinal healing. In vivo, the diabetic rat model was established by use of streptozotocin (STZ), and animals treated with BMSCs+CNTF exhibited better viability and higher delivery efficiency of the transplanted cells than those treated with BMSCs injection alone. Similar to the in-vitro result, treatment with BMSCs and CNTF combined led to the differentiation of BMSCs into beneficial cells (RPE cells), and accelerated retinal healing characterized by the activation of rod photoreceptor cells and phagocytosis function of RPE cells. In conclusion, CNTF contributes to the differentiation of BMSCs into RPE cells, which may help overcome the current stem cell therapy limitations in the field of retinal regeneration.

Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.

KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.

A Comprehensive Study of the Retinal Phenotype of Rpe65-Deficient Dogs.

The Rpe65-deficient dog has been important for development of translational therapies of Leber congenital amaurosis type 2 (LCA2). The purpose of this study was to provide a comprehensive report of the natural history of retinal changes in this dog model. Rpe65-deficient dogs from 2 months to 10 years of age were assessed by fundus imaging, electroretinography (ERG) and vision testing (VT). Changes in retinal layer thickness were assessed by optical coherence tomography and on plastic retinal sections. ERG showed marked loss of retinal sensitivity, with amplitudes declining with age. Retinal thinning initially developed in the area centralis, with a slower thinning of the outer retina in other areas starting with the inferior retina. VT showed that dogs of all ages performed well in bright light, while at lower light levels they were blind. Retinal pigment epithelial (RPE) inclusions developed and in younger dogs and increased in size with age. The loss of photoreceptors was mirrored by a decline in ERG amplitudes. The slow degeneration meant that sufficient photoreceptors, albeit very desensitized, remained to allow for residual bright light vision in older dogs. This study shows the natural history of the Rpe65-deficient dog model of LCA2.