Therapeutic adenine base editing corrects nonsense mutation and improves visual function in a mouse model of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is an inherited retinal degeneration that causes severe visual dysfunction in children and adolescents. In patients with LCA, pathogenic variants are evident in specific genes, such as RPE65, which are related to the functions of retinal pigment epithelium and photoreceptors. Base editing confers a way to correct pathogenic substitutions without double-stranded breaks in contrast to the original Cas9. In this study, we prepared dual adeno-associated virus vectors containing the split adenine base editors with trans-splicing intein (AAV-ABE) for in vivo adenine base editing in retinal degeneration 12 (rd12) mice, an animal model of LCA, which possess a nonsense mutation of C to T transition in the Rpe65 gene (p.R44X). AAV-ABE induced an A to G transition in retinal pigment epithelial cells of rd12 mice when injected into the subretinal space. The on-target editing was sufficient to recover wild-type mRNA, RPE65 protein, and light-induced electrical responses of retinal tissues. We suggest adenine base editing to correct pathogenic variants in the treatment of LCA.

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Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport

AJP Cell Physiology ◽

10.1152/ajpcell.00201.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. C132-C140 ◽

Cited By ~ 75

Author(s):

David Reigada ◽

Claire H. Mitchell

Keyword(s):

Epithelial Cells ◽

Apical Membrane ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Vesicular Transport ◽

Atp Release ◽

Retinal Pigment Epithelial Cells ◽

Subretinal Space ◽

Transport Pathways ◽

Rpe Cells

The retinal pigment epithelium (RPE) faces the photoreceptor outer segments and regulates the composition of the interstitial subretinal space. ATP enhances fluid movement from the subretinal space across the RPE. RPE cells can themselves release ATP, but the mechanisms and polarity of this release are unknown. The RPE expresses the cystic fibrosis transmembrane conductance regulator (CFTR), and CFTR is associated with ATP release in other epithelial cells. However, an increasing number of reports have suggested that the exocytotic pathway contributes to release. In the present study, we examined the involvement of CFTR and the vesicular pathway in ATP release from RPE cells. Release from cultured human ARPE-19 cells and across the apical membrane of fresh bovine RPE cells in an eyecup was studied. A cAMP cocktail to activate CFTR triggered ATP release from fresh and cultured RPE cells. Release from both RPE preparations was largely prevented by the broad-acting blocker glibenclamide and the specific thiazolidinone CFTR inhibitor CFTR-172. The block by CFTR-172 was enhanced by preincubation and prevented ATP release with 3.5 μM IC50. The rise in intracellular Ca2+ accompanying hypotonic challenge was prevented by CFTR-172. The vesicular transport inhibitor brefeldin A prevented ATP release after stimulation with both hypotonic and cAMP conditions, suggesting vesicular insertion was also involved. These results show an intimate involvement of CFTR in ATP release from RPE cells which can autostimulate receptors on the apical membrane to modify Ca2+ signaling. The requirement for both CFTR and vesicular transport pathways suggests vesicular insertion of CFTR may underlie the release of ATP.

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Genetic analysis of 10 pedigrees with inherited retinal degeneration by exome sequencing and phenotype-genotype association

Physiological Genomics ◽

10.1152/physiolgenomics.00096.2016 ◽

2017 ◽

Vol 49 (4) ◽

pp. 216-229 ◽

Cited By ~ 11

Author(s):

Pooja Biswas ◽

Jacque L. Duncan ◽

Bruno Maranhao ◽

Igor Kozak ◽

Kari Branham ◽

...

Keyword(s):

Genetic Analysis ◽

Retinal Degeneration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Control Sample ◽

Compound Heterozygous ◽

Illumina Hiseq ◽

Retinal Degenerations ◽

Pathogenic Variants ◽

Causal Variants

Our purpose was to identify causative mutations and characterize the phenotype associated with the genotype in 10 unrelated families with autosomal recessive retinal degeneration. Ophthalmic evaluation and DNA isolation were carried out in 10 pedigrees with inherited retinal degenerations (IRD). Exomes of probands from eight pedigrees were captured using Nimblegen V2/V3 or Agilent V5+UTR kits, and sequencing was performed on Illumina HiSeq. The DHDDS gene was screened for mutations in the remaining two pedigrees with Ashkenazi Jewish ancestry. Exome variants were filtered to detect candidate causal variants using exomeSuite software. Segregation and ethnicity-matched control sample analysis were performed by dideoxy sequencing. Retinal histology of a patient with DHDDS mutation was studied by microscopy. Genetic analysis identified six known mutations in ABCA4 (p.Gly1961Glu, p.Ala1773Val, c.5461–10T>C), RPE65 (p.Tyr249Cys, p.Gly484Asp), PDE6B (p.Lys706Ter) and DHDDS (p.Lys42Glu) and ten novel potentially pathogenic variants in CERKL (p.Met323Val fsX20), RPE65 (p.Phe252Ser, Thr454Leu fsX31), ARL6 (p.Arg121His), USH2A (p.Gly3142Ter, p.Cys3294Trp), PDE6B (p.Gln652Ter), and DHDDS (p.Thr206Ala) genes. Among these, variants/mutations in two separate genes were observed to segregate with IRD in two pedigrees. Retinal histopathology of a patient with a DHDDS mutation showed severe degeneration of retinal layers with relative preservation of the retinal pigment epithelium. Analysis of exome variants in ten pedigrees revealed nine novel potential disease-causing variants and nine previously reported homozygous or compound heterozygous mutations in the CERKL, ABCA4, RPE65, ARL6, USH2A, PDE6B, and DHDDS genes. Mutations that could be sufficient to cause pathology were observed in more than one gene in one pedigree.

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Pigment Epithelium-Derived Factor: A Novel Therapeutic Target for Cardiometabolic Diseases and Related Complications

Current Medicinal Chemistry ◽

10.2174/0929867324666170608103140 ◽

2018 ◽

Vol 25 (13) ◽

pp. 1480-1500 ◽

Cited By ~ 7

Author(s):

Sho-ichi Yamagishi ◽

Takanori Matsui

Keyword(s):

Cardiovascular Disease ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Visceral Obesity ◽

Retinal Pigment Epithelial Cells ◽

Atherosclerotic Cardiovascular Disease ◽

Cardiometabolic Diseases ◽

The Metabolic Syndrome ◽

Pigment Epithelium Derived Factor ◽

Cardiometabolic Disorders

Pigment epithelium-derived factor (PEDF) is a glycoprotein that belongs to the superfamily of serine protease inhibitors, serpins. It was first identified as a neuronal differentiating factor secreted by human retinal pigment epithelial cells, and then found to be the most potent inhibitor of pathological angiogenesis in mammalian eyes. Recently, PEDF has been shown not only to suppress oxidative stress and inflammatory reactions in vascular wall cells, T cells and macrophages, and adipocytes, but also to exert antithrombotic and anti-fibrotic properties, thereby protecting against the development and progression of various cardiometabolic diseases and related complications. Furthermore, accumulating evidence has suggested that circulating PEDF levels may be a biomarker of severity and prognosis of these devastating disorders. Number of subjects with visceral obesity and insulin resistance is increasing, and the metabolic syndrome and its related complications, such as diabetes, nonalcoholic fatty liver disease/non-alcoholic steatohepatits, and atherosclerotic cardiovascular disease are a growing health challenge. Therefore, in this study, we review the pathophysiological role of PEDF in obesity and metabolic disorders, cardiovascular disease, diabetic eye and kidney complications, liver diseases, and reproductive system disorders, and discuss the potential clinical utility of modulating the expression and actions of PEDF for preventing these cardiometabolic disorders. We also refer to the clinical value of PEDF as a biomarker in cardiometabolic complications.

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A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells

Cells ◽

10.3390/cells10010179 ◽

2021 ◽

Vol 10 (1) ◽

pp. 179

Author(s):

Laurence Klipfel ◽

Marie Cordonnier ◽

Léa Thiébault ◽

Emmanuelle Clérin ◽

Frédéric Blond ◽

...

Keyword(s):

Genome Wide Association Study ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Lactate Transport ◽

Ips Cell ◽

Risk Alleles ◽

Age Related ◽

A Genome

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

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The Role of Imaging in Planning Treatment for Central Serous Chorioretinopathy

Pharmaceuticals ◽

10.3390/ph14020105 ◽

2021 ◽

Vol 14 (2) ◽

pp. 105

Author(s):

Stefano Da Pozzo ◽

Pierluigi Iacono ◽

Alessandro Arrigo ◽

Maurizio Battaglia Parodi

Keyword(s):

Central Serous Chorioretinopathy ◽

Therapeutic Strategy ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Subretinal Fluid ◽

Subretinal Space ◽

Planning Treatment ◽

Multiple Biomarkers ◽

Effective Procedures

Central serous chorioretinopathy (CSC) is a controversial disease both in terms of clinical classification and choice of therapeutic strategy. Choroidal layers, retinal pigment epithelium (RPE), photoreceptors, and retina are involved to varying degrees. Beyond well-known symptoms raising the clinical suspect of CSC and slit-lamp fundus examination, multimodal imaging plays a key role in assessing the extent of chorioretinal structural involvement. Subretinal fluid (SRF) originating from the choroid leaks through one or multiple RPE defects and spreads into the subretinal space. Spontaneous fluid reabsorption is quite common, but in some eyes, resolution can be obtained only after treatment. Multiple therapeutic strategies are available, and extensive research identified the most effective procedures. Imaging has carved a significant role in guiding the choice of the most appropriate strategy for each single CSC eye. Multiple biomarkers have been identified, and all of them represent a diagnostic and prognostic reference point. This review aims to provide an updated and comprehensive analysis of the current scientific knowledge about the role of imaging in planning the treatment in eyes affected by CSC.

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OCT imaging of rod mitochondrial respiration in vivo

Experimental Biology and Medicine ◽

10.1177/15353702211013799 ◽

2021 ◽

pp. 153537022110137

Author(s):

Bruce A Berkowitz ◽

Haohua Qian

Keyword(s):

Signaling Pathway ◽

Mitochondrial Respiration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

The Body ◽

Subretinal Space ◽

Outer Retina ◽

Water Efflux ◽

Resolution Imaging

There remains a need for high spatial resolution imaging indices of mitochondrial respiration in the outer retina that probe normal physiology and measure pathogenic and reversible conditions underlying loss of vision. Mitochondria are involved in a critical, but somewhat underappreciated, support system that maintains the health of the outer retina involving stimulus-evoked changes in subretinal space hydration. The subretinal space hydration light–dark response is important because it controls the distribution of vision-critical interphotoreceptor matrix components, including anti-oxidants, pro-survival factors, ions, and metabolites. The underlying signaling pathway controlling subretinal space water management has been worked out over the past 30 years and involves cGMP/mitochondria respiration/pH/RPE water efflux. This signaling pathway has also been shown to be modified by disease-generating conditions, such as hypoxia or oxidative stress. Here, we review recent advances in MRI and commercially available OCT technologies that can measure stimulus-evoked changes in subretinal space water content based on changes in the external limiting membrane-retinal pigment epithelium region. Each step within the above signaling pathway can also be interrogated with FDA-approved pharmaceuticals. A highlight of these studies is the demonstration of first-in-kind in vivo imaging of mitochondria respiration of any cell in the body. Future examinations of subretinal space hydration are expected to be useful for diagnosing threats to sight in aging and disease, and improving the success rate when translating treatments from bench-to-bedside.

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Potential Treatment of Retinal Diseases with Iron Chelators

Pharmaceuticals ◽

10.3390/ph11040112 ◽

2018 ◽

Vol 11 (4) ◽

pp. 112 ◽

Cited By ~ 13

Author(s):

Wanting Shu ◽

Joshua Dunaief

Keyword(s):

Retinal Degeneration ◽

Therapeutic Potential ◽

Pigment Epithelium ◽

Retinal Pigment ◽

The Body ◽

Age Related Macular Degeneration ◽

Hereditary Diseases ◽

Iron Chelators ◽

Excess Iron ◽

Age Related

Iron is essential for life, while excess iron can be toxic. Iron generates hydroxyl radical, which is the most reactive free radical, causing oxidative stress. Since iron is absorbed through the diet but not excreted from the body, it accumulates with age in tissues, including the retina, consequently leading to age-related toxicity. This accumulation is further promoted by inflammation. Hereditary diseases such as aceruloplasminemia, Friedreich’s ataxia, pantothenate kinase-associated neurodegeneration, and posterior column ataxia with retinitis pigmentosa involve retinal degeneration associated with iron dysregulation. In addition to hereditary causes, dietary or parenteral iron supplementation has been recently reported to elevate iron levels in the retinal pigment epithelium (RPE) and promote retinal degeneration. Ocular siderosis from intraocular foreign bodies or subretinal hemorrhage can also lead to retinopathy. Evidence from mice and humans suggests that iron toxicity may contribute to age-related macular degeneration pathogenesis. Iron chelators can protect photoreceptors and RPE in various mouse models. The therapeutic potential for iron chelators is under investigation.

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Calcium gradients and light-evoked calcium changes outside rods in the intact cat retina

Visual Neuroscience ◽

10.1017/s0952523800003059 ◽

1994 ◽

Vol 11 (4) ◽

pp. 753-761 ◽

Cited By ~ 24

Author(s):

Ron P. Gallemore ◽

Jian-Dong Li ◽

Victor I. Govardovskii ◽

Roy H. Steinberg

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Plexiform Layer ◽

Rapid Onset ◽

Subretinal Space ◽

Inner Plexiform Layer ◽

Rod Outer Segments ◽

Volume Increase ◽

Cat Retina ◽

Dependent Sources

AbstractWe have studied light-evoked changes in extracellular Ca2+ concentration in the intact cat eye using ion-sensitive double-barreled microelectrodes. Two prominent changes in Ca2+ concentration were observed that differed in retinal location. There was a light-evoked increase in accompanied by brief ON and OFF transients, which was maximal in the inner plexiform layer and was not further studied. There was an unexpected sustained light-evoked decrease in of relatively rapid onset and offset, which was maximal in the distalmost region of the subretinal space (SRS). in the SRS was 1.0 mM higher than in the vitreous humor during dark adaptation and this transretinal gradient disappeared during rod-saturating illumination. After correcting for the light-evoked increase in the volume of the SRS, an increase in the total Ca2+ content of the SRS during illumination was revealed, which presumably represents the Ca2+ released by rods. To explain the light-evoked changes, we used the diffusion model described in the accompanying paper (Li et al., 1994b), with the addition of light-dependent sources of Ca2+ at the retina/retinal pigment epithelium (RPE) border and rod outer segments. We conclude that a drop in around photoreceptors, which persists during illumination and reduces a transretinal Ca2+ gradient, is the combined effect of the light-evoked SRS volume increase, Ca2+ release from photoreceptors, and an unidentified mechanism(s), which is presumably Ca2+ transport by the RPE. The relatively rapid onset and offset of the decrease remains unexplained. These steady-state shifts in should have significant effects on photoreceptor function, especially adaptation.

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Degradation of extracellular ATP by the retinal pigment epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.00542.2004 ◽

2005 ◽

Vol 289 (3) ◽

pp. C617-C624 ◽

Cited By ~ 32

Author(s):

David Reigada ◽

Wennan Lu ◽

Xiulan Zhang ◽

Constantin Friedman ◽

Klara Pendrak ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Adenosine Diphosphate ◽

Cell Types ◽

P2y Receptors ◽

Extracellular Atp ◽

Pcr Analysis ◽

Subretinal Space ◽

Rpe Cells ◽

Mrs 2179

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and βγ-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

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