RESUMO
Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.
Assuntos
Defeitos da Visão Cromática , Opsinas de Bastonetes , Defeitos da Visão Cromática/genética , Deleção de Genes , Humanos , Família Multigênica/genética , Células Fotorreceptoras Retinianas Cones , Opsinas de Bastonetes/genéticaRESUMO
INTRODUCTION: The aim of this study was to evaluate the progression of atrophy as determined by spectral-domain optical coherence tomography (SD-OCT) in patients with molecularly confirmed ABCA4-associated Stargardt disease type 1 (STGD1) over a 24-month period in a multicenter prospective cohort study. METHODS: SD-OCT images from 428 eyes of 236 patients were analyzed. Change of mean thickness (MT) and intact area were estimated after semiautomated segmentation for the following individual layers in the central subfield (CS), inner ring (IR), and outer ring (OR) of the ETDRS grid: retinal pigment epithelium (RPE), outer segments (OSs), inner segments (IS), outer nuclear layer (ONL) inner retina (IR), and total retina. RESULTS: Statistically significant decreases of all outer retinal layers (RPE, OS, IS, and ONL) could be observed over a 24-month period both in decline of mean retinal thickness and intact area (p < 0.0001, respectively), whereas the IR showed an increase of retinal thickness in the CS and IR and remained unchanged in the OR. CONCLUSIONS: Significant loss could be detected in outer retinal layers by SD-OCT over a 24-month period in patients with STGD1. Loss of thickness and/or intact area of such layers may serve as potential endpoints for clinical trials that aim to slow down the disease progression of STGD1.
Assuntos
Progressão da Doença , Degeneração Macular , Epitélio Pigmentado da Retina , Doença de Stargardt , Tomografia de Coerência Óptica , Acuidade Visual , Humanos , Tomografia de Coerência Óptica/métodos , Doença de Stargardt/diagnóstico , Masculino , Estudos Prospectivos , Feminino , Adulto , Adulto Jovem , Pessoa de Meia-Idade , Degeneração Macular/diagnóstico , Degeneração Macular/congênito , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/diagnóstico por imagem , Adolescente , Seguimentos , Retina/diagnóstico por imagem , Retina/patologia , CriançaRESUMO
Modern advances in disease genetics have uncovered numerous modifier genes that play a role in the severity of disease expression. One such class of genetic conditions is known as inherited retinal degenerations (IRDs), a collection of retinal degenerative disorders caused by mutations in over 300 genes. A single missense mutation (K42E) in the gene encoding the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which is required for protein N-glycosylation in all cells and tissues, causes DHDDS-IRD (retinitis pigmentosa type 59 (RP59; OMIM #613861)). Apart from a retinal phenotype, however, DHDDS-IRD is surprisingly non-syndromic (i.e., without any systemic manifestations). To explore disease pathology, we selected five glycosylation-related genes for analysis that are suggested to have disease modifier variants. These genes encode glycosyltransferases (ALG6, ALG8), an ER resident protein (DDOST), a high-mannose oligosaccharyl transferase (MPDU1), and a protein N-glycosylation regulatory protein (TNKS). DNA samples from 11 confirmed DHDDS (K42E)-IRD patients were sequenced at the site of each candidate genetic modifier. Quantitative measures of retinal structure and function were performed across five decades of life by evaluating foveal photoreceptor thickness, visual acuity, foveal sensitivity, macular and extramacular rod sensitivity, and kinetic visual field extent. The ALG6 variant, (F304S), was correlated with greater macular cone disease severity and less peripheral rod disease severity. Thus, modifier gene polymorphisms may account for a significant portion of phenotypic variation observed in human genetic disease. However, the consequences of the polymorphisms may be counterintuitively complex in terms of rod and cone populations affected in different regions of the retina.
Assuntos
Alquil e Aril Transferases , Glucosiltransferases , Proteínas de Membrana , Degeneração Retiniana , Humanos , Genes Modificadores , Glucosiltransferases/genética , Proteínas de Membrana/genética , Mutação , Retina , Degeneração Retiniana/genéticaRESUMO
Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.
Assuntos
Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Retinose Pigmentar/genética , Processamento Alternativo/genética , Proteínas de Transporte/genética , Cílios/genética , Cílios/patologia , Éxons/genética , Feminino , Fibroblastos , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Masculino , Mutação/genética , Isoformas de Proteínas/genética , Retina/metabolismo , Retina/patologia , Retinose Pigmentar/patologiaRESUMO
The inherited childhood blindness caused by mutations in NPHP5, a form of Leber congenital amaurosis, results in abnormal development, dysfunction, and degeneration of photoreceptors. A naturally occurring NPHP5 mutation in dogs leads to a phenotype that very nearly duplicates the human retinopathy in terms of the photoreceptors involved, spatial distribution of degeneration, and the natural history of vision loss. We show that adeno-associated virus (AAV)-mediated NPHP5 gene augmentation of mutant canine retinas at the time of active degeneration and peak cell death stably restores photoreceptor structure, function, and vision with either the canine or human NPHP5 transgenes. Mutant cone photoreceptors, which failed to form outer segments during development, reform this structure after treatment. Degenerating rod photoreceptor outer segments are stabilized and develop normal structure. This process begins within 8 weeks after treatment and remains stable throughout the 6-month posttreatment period. In both photoreceptor cell classes mislocalization of rod and cone opsins is minimized or reversed. Retinal function and functional vision are restored. Efficacy of gene therapy in this large animal ciliopathy model of Leber congenital amaurosis provides a path for translation to human treatment.
Assuntos
Proteínas de Ligação a Calmodulina/administração & dosagem , Dependovirus/genética , Amaurose Congênita de Leber/terapia , Células Fotorreceptoras Retinianas Cones/patologia , Animais , Proteínas de Ligação a Calmodulina/farmacologia , Modelos Animais de Doenças , Cães , Eletrorretinografia , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Amaurose Congênita de Leber/genética , Resultado do TratamentoRESUMO
BACKGROUND: Inherited retinal degenerations (IRDs) affect daylight and night vision to different degrees. In the current work, we devise a method to quantify mobility under dark-adapted conditions in patients with severe childhood blindness due to Leber congenital amaurosis (LCA). Mobility thresholds from two different LCA genotypes are compared to dark-adapted vision measurements using the full-field stimulus test (FST), a conventional desktop outcome measure of rod vision. METHODS: A device consisting of vertical LED strips on a plane resembling a beaded curtain was programmed to produce a rectangular pattern target defining a 'door' of varying luminance that could appear at one of three positions. Mobility performance was evaluated by letting the subject walk from a fixed starting position ~ 4 m away from the device with instructions to touch the door. Success was defined as the subject touching within the 'door' area. Ten runs were performed and the process was repeated for different levels of luminance. Tests were performed monocularly in dark-adapted and dilated eyes. Results from LCA patients with the GUCY2D and CEP290 genotypes and normal subjects were analyzed using logistic regression to estimate the mobility threshold for successful navigation. The relation of thresholds for mobility, FST and visual acuity were quantified using linear regression. RESULTS: Normal subjects had mobility thresholds near limits of dark-adapted rod vision. GUCY2D-LCA patients had a wide range of mobility thresholds from within 1 log of normal to greater than 8 log abnormal. CEP290-LCA patients had abnormal mobility thresholds that were between 5 and 6 log from normal. Sensitivity loss estimates using FST related linearly to the mobility thresholds which were not correlated with visual acuity. CONCLUSIONS: The mobility task we developed can quantify functional vision in severely disabled patients with LCA. Taken together with other outcome measures of rod and cone photoreceptor-mediated vision, dark-adapted functional vision should provide a more complete understanding of the natural history and effects of treatment in patients with LCA.
Assuntos
Amaurose Congênita de Leber , Degeneração Retiniana , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Criança , Proteínas do Citoesqueleto/genética , Adaptação à Escuridão , Humanos , Amaurose Congênita de Leber/diagnóstico , Amaurose Congênita de Leber/genética , Mutação , Células Fotorreceptoras Retinianas Cones , Visão OcularRESUMO
The form of hereditary childhood blindness Leber congenital amaurosis (LCA) caused by biallelic RPE65 mutations is considered treatable with a gene therapy product approved in the US and Europe. The resulting vision improvement is well accepted, but long-term outcomes on the natural history of retinal degeneration are controversial. We treated four RPE65-mutant dogs in mid-life (age = 5-6 years) and followed them long-term (4-5 years). At the time of the intervention at mid-life, there were intra-ocular and inter-animal differences in local photoreceptor layer health ranging from near normal to complete degeneration. Treated locations having more than 63% of normal photoreceptors showed robust treatment-related retention of photoreceptors in the long term. Treated regions with less retained photoreceptors at the time of the intervention showed progressive degeneration similar to untreated regions with matched initial stage of disease. Unexpectedly, both treated and untreated regions in study eyes tended to show less degeneration compared to matched locations in untreated control eyes. These results support the hypothesis that successful long-term arrest of progression with RPE65 gene therapy may only occur in retinal regions with relatively retained photoreceptors at the time of the intervention, and there may be heretofore unknown mechanisms causing long-distance partial treatment effects beyond the region of subretinal injection.
Assuntos
Terapia Genética/métodos , Amaurose Congênita de Leber/terapia , Mutação , Degeneração Retiniana/genética , cis-trans-Isomerases/genética , Animais , Modelos Animais de Doenças , Cães , Eletrorretinografia , Feminino , Seguimentos , Amaurose Congênita de Leber/diagnóstico por imagem , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/diagnóstico por imagem , Retina/metabolismo , Degeneração Retiniana/diagnóstico por imagem , Resultado do Tratamento , Visão OcularRESUMO
Inherited retinal degenerations are caused by mutations in >250 genes that affect photoreceptor cells or the retinal pigment epithelium and result in vision loss. For autosomal recessive and X-linked retinal degenerations, significant progress has been achieved in the field of gene therapy as evidenced by the growing number of clinical trials and the recent commercialization of the first gene therapy for a form of congenital blindness. However, despite significant efforts to develop a treatment for the most common form of autosomal dominant retinitis pigmentosa (adRP) caused by >150 mutations in the rhodopsin (RHO) gene, translation to the clinic has stalled. Here, we identified a highly efficient shRNA that targets human (and canine) RHO in a mutation-independent manner. In a single adeno-associated viral (AAV) vector we combined this shRNA with a human RHO replacement cDNA made resistant to RNA interference and tested this construct in a naturally occurring canine model of RHO-adRP. Subretinal vector injections led to nearly complete suppression of endogenous canine RHO RNA, while the human RHO replacement cDNA resulted in up to 30% of normal RHO protein levels. Noninvasive retinal imaging showed photoreceptors in treated areas were completely protected from retinal degeneration. Histopathology confirmed retention of normal photoreceptor structure and RHO expression in rod outer segments. Long-term (>8 mo) follow-up by retinal imaging and electroretinography indicated stable structural and functional preservation. The efficacy of this gene therapy in a clinically relevant large-animal model paves the way for treating patients with RHO-adRP.
Assuntos
Dependovirus , Técnicas de Introdução de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Terapia Genética/métodos , Vetores Genéticos , RNA Catalítico , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar , Rodopsina , Animais , Cães , Células HEK293 , Humanos , RNA Catalítico/biossíntese , RNA Catalítico/genética , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Retinose Pigmentar/patologia , Rodopsina/biossíntese , Rodopsina/genéticaRESUMO
Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies.
Assuntos
Bestrofinas/genética , Oftalmopatias Hereditárias/terapia , Terapia Genética/métodos , Doenças Retinianas/terapia , Animais , Doenças do Cão/terapia , Cães , Oftalmopatias Hereditárias/diagnóstico por imagem , Oftalmopatias Hereditárias/patologia , Oftalmopatias Hereditárias/veterinária , Feminino , Vetores Genéticos/farmacologia , Humanos , Luz , Masculino , Mutação , Descolamento Retiniano/diagnóstico por imagem , Descolamento Retiniano/patologia , Descolamento Retiniano/terapia , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologia , Doenças Retinianas/veterinária , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência ÓpticaRESUMO
Gene augmentation therapy is being planned for GUCY2D-associated Leber congenital amaurosis (LCA). To increase our understanding of the natural history of GUCY2D-LCA, patients were evaluated twice with an interval of 4 to 7 years between visits using safety and efficacy outcome measures previously determined to be useful for monitoring this disorder. In this group of molecularly-identified LCA patients (n = 10; ages 7-37 years at first visit), optical coherence tomography (OCT) was used to measure foveal cone outer nuclear layer (ONL) thickness and rod ONL at a superior retinal locus. Full-field stimulus testing (FST) with chromatic stimuli in dark- and light-adapted states was used to assay rod and cone vision. Changes in OCT and FST over the interval were mostly attributable to inter-visit variability. There were no major negative changes in structure or function across the cohort and over the intervals studied. Variation in severity of disease expression between patients occurs; however, despite difficulties in quantifying structure and function in such seriously visually impaired individuals with nystagmus, the present work supports the use of OCT as a safety outcome and FST as an efficacy outcome in a clinical trial of GUCY2D-LCA. A wide age spectrum for therapy was confirmed, and there was relative stability of structure and function during a typical time interval for clinical trials.
Assuntos
Guanilato Ciclase/genética , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Receptores de Superfície Celular/genética , Retina/patologia , Retina/fisiopatologia , Visão Ocular , Adolescente , Adulto , Criança , Fluorescência , Humanos , Amaurose Congênita de Leber/diagnóstico por imagem , Retina/diagnóstico por imagem , Células Fotorreceptoras Retinianas Cones/metabolismo , Tomografia de Coerência Óptica , Adulto JovemRESUMO
The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca2+ concentrations decline, and decelerates the cyclase in the dark, when Ca2+ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca2+-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca2+ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca2+ concentrations characteristic of dark-adapted photoreceptors. Ca2+-dependent increase in Trp94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca2+ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca2+, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca2+ sensitivity of the cyclase within the physiological range of intracellular Ca2+ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly86, leading to abnormally high affinity for the target enzyme and reduced Ca2+ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca2+ influx in photoreceptors in the dark.
Assuntos
Cálcio/metabolismo , Distrofias de Cones e Bastonetes/genética , Proteínas Ativadoras de Guanilato Ciclase/genética , Proteínas Ativadoras de Guanilato Ciclase/metabolismo , Guanilato Ciclase/metabolismo , Mutação , Retina/enzimologia , Morte Celular/genética , Distrofias de Cones e Bastonetes/enzimologia , Distrofias de Cones e Bastonetes/metabolismo , Distrofias de Cones e Bastonetes/patologia , Proteínas Ativadoras de Guanilato Ciclase/química , Humanos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
In retinal photoreceptors, vectorial transport of cargo is critical for transduction of visual signals, and defects in intracellular trafficking can lead to photoreceptor degeneration and vision impairment. Molecular signatures associated with routing of transport vesicles in photoreceptors are poorly understood. We previously reported the identification of a novel rod photoreceptor specific isoform of Receptor Expression Enhancing Protein (REEP) 6, which belongs to a family of proteins involved in intracellular transport of receptors to the plasma membrane. Here we show that loss of REEP6 in mice (Reep6-/-) results in progressive retinal degeneration. Rod photoreceptor dysfunction is observed in Reep6-/- mice as early as one month of age and associated with aberrant accumulation of vacuole-like structures at the apical inner segment and reduction in selected rod phototransduction proteins. We demonstrate that REEP6 is detected in a subset of Clathrin-coated vesicles and interacts with the t-SNARE, Syntaxin3. In concordance with the rod degeneration phenotype in Reep6-/- mice, whole exome sequencing identified homozygous REEP6-E75K mutation in two retinitis pigmentosa families of different ethnicities. Our studies suggest a critical function of REEP6 in trafficking of cargo via a subset of Clathrin-coated vesicles to selected membrane sites in retinal rod photoreceptors.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Proteínas do Olho/genética , Transdução de Sinal Luminoso , Proteínas de Membrana , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Isoformas de Proteínas/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/metabolismo , Degeneração Retiniana/metabolismo , Retinose Pigmentar/genética , Proteínas SNARE/metabolismoRESUMO
Gene therapy for adRP due to RHO mutations was recently shown to prevent photoreceptor death in a canine model of Class B disease. Among translational steps to be taken, one is to determine a method to detect efficacy in a human clinical trial. The relatively slow progression of adRP becomes a difficulty for clinical trials requiring an answer to whether there is slowed progression of degeneration in response to therapy. We performed a single-center, retrospective observational study of cross-sectional and longitudinal data. The study was prompted by our identification of a pericentral disease distribution in Class B RHO-adRP. Ultrawide optical coherence tomography (OCT) scans were used. Inferior retinal pericentral defects was an early disease feature. Degeneration further inferior in the retina merged with the pericentral defect, which extended into superior retina. In about 70% of patients, there was an asymmetric island of structure with significantly greater superior than inferior ellipsoid zone (EZ) extent. Serial measures of photoreceptor structure by OCT indicated constriction in superior retinal extent within a two-year interval. We conclude that these results should allow early-phase trials of therapy in RHO-adRP to move forward by inclusion of patients with an asymmetric extent of photoreceptor structure and by monitoring therapeutic effects over two years in the superior retina, a reasonable target for subretinal injection.
Assuntos
Mutação , Retinose Pigmentar/diagnóstico por imagem , Rodopsina/genética , Tomografia de Coerência Óptica/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Criança , Estudos Transversais , Feminino , Predisposição Genética para Doença , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Retinose Pigmentar/genética , Estudos Retrospectivos , Adulto JovemRESUMO
Recessively-inherited NR2E3 gene mutations cause an unusual retinopathy with abnormally-increased short-wavelength sensitive cone (S-cone) function, in addition to reduced rod and long/middle-wavelength sensitive cone (L/M-cone) function. Progress toward clinical trials to treat patients with this otherwise incurable retinal degeneration prompted the need to determine efficacy outcome measures. Comparisons were made between three computerized perimeters available in the clinic. These perimeters could deliver short-wavelength stimuli on longer-wavelength adapting backgrounds to measure whether S-cone vision can be quantified. Results from a cohort of normal subjects were compared across the three perimeters to determine S-cone isolation and test-retest variability. S-cone perimetry data from NR2E3-ESCS (enhanced S-cone syndrome) patients were examined and determined to have five stages of disease severity. Using these stages, strategies were proposed for monitoring efficacy of either a focal or retina-wide intervention. This work sets the stage for clinical trials.
Assuntos
Oftalmopatias Hereditárias/diagnóstico , Mutação , Receptores Nucleares Órfãos/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Retiniana/diagnóstico , Transtornos da Visão/diagnóstico , Testes de Campo Visual/métodos , Adolescente , Adulto , Idoso , Criança , Ensaios Clínicos como Assunto , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/fisiopatologia , Humanos , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Transtornos da Visão/metabolismo , Transtornos da Visão/fisiopatologia , Adulto JovemRESUMO
Ciliary defects can result in severe disorders called ciliopathies. Mutations in NPHP5 cause a ciliopathy characterized by severe childhood onset retinal blindness, Leber congenital amaurosis (LCA), and renal disease. Using the canine NPHP5-LCA model we compared human and canine retinal phenotypes, and examined the early stages of photoreceptor development and degeneration, the kinetics of photoreceptor loss, the progression of degeneration and the expression profiles of selected genes. NPHP5-mutant dogs recapitulate the human phenotype of very early loss of rods, and relative retention of the central retinal cone photoreceptors that lack function. In mutant dogs, rod and cone photoreceptors have a sensory cilium, but develop and function abnormally and then rapidly degenerate; L/M cones are more severely affected than S-cones. The lack of outer segments in mutant cones indicates a ciliary dysfunction. Genes expressed in mutant rod or both rod and cone photoreceptors show significant downregulation, while those expressed only in cones are unchanged. Many genes in cell-death and -survival pathways also are downregulated. The canine disease is a non-syndromic LCA-ciliopathy, with normal renal structures and no CNS abnormalities. Our results identify the critical time points in the pathogenesis of the photoreceptor disease, and bring us closer to defining a potential time window for testing novel therapies for translation to patients.
Assuntos
Proteínas de Ligação a Calmodulina/genética , Cílios/genética , Amaurose Congênita de Leber/genética , Células Fotorreceptoras/metabolismo , Retina/patologia , Animais , Cílios/patologia , Modelos Animais de Doenças , Cães , Humanos , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/fisiopatologia , Mutação , Células Fotorreceptoras/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Mutations in the ORF15 exon of the RPGR gene cause a common form of X-linked retinitis pigmentosa, which often results in severe loss of vision. In dogs and mice, gene augmentation therapy has been shown to arrest the progressive degeneration of rod and cone photoreceptors. However, the distribution of potentially treatable photoreceptors across the human retinas and the rate of degeneration are not known. Here, we have defined structural and functional features of the disease in 70 individuals with ORF15 mutations. We also correlated the features observed in patients with those of three Rpgr-mutant (Rpgr-ko, Rd9, and Rpgr-cko) mice. In patients, there was pronounced macular disease. Across the retina, rod and cone dysfunction showed a range of patterns and a spectrum of severity between individuals, but a high symmetry was observed between eyes of each individual. Genotype was not related to disease expression. In the Rpgr-ko mice, there were intra-retinal differences in rhodopsin and cone opsin trafficking. In Rd9 and Rpgr-cko mice, retinal degeneration showed inter-ocular symmetry. Longitudinal results in patients revealed localized rod and cone dysfunction with progression rates of 0.8 to 1.3 log per decade in sensitivity loss. Relatively retained rod and cone photoreceptors in mid- and far-peripheral temporal-inferior and nasal-inferior visual field regions should be good targets for future localized gene therapies in patients.
Assuntos
Proteínas do Olho/genética , Degeneração Retiniana/genética , Retinosquise/genética , Rodopsina/genética , Adolescente , Adulto , Idoso , Animais , Criança , Heterozigoto , Humanos , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinosquise/patologia , Rodopsina/metabolismo , Adulto JovemRESUMO
Retinal gene therapy for Leber's congenital amaurosis, an autosomal recessive childhood blindness, has been widely considered to be safe and efficacious. Three years after therapy, improvement in vision was maintained, but the rate of loss of photoreceptors in the treated retina was the same as that in the untreated retina. Here we describe long-term follow-up data from three treated patients. Topographic maps of visual sensitivity in treated regions, nearly 6 years after therapy for two of the patients and 4.5 years after therapy for the third patient, indicate progressive diminution of the areas of improved vision. (Funded by the National Eye Institute; ClinicalTrials.gov number, NCT00481546.).
Assuntos
Terapia Genética , Amaurose Congênita de Leber/terapia , Células Fotorreceptoras de Vertebrados/patologia , Retina/fisiologia , Adolescente , Progressão da Doença , Seguimentos , Humanos , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Mutação , Visão Ocular , Adulto JovemRESUMO
X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is an early onset and severe cause of blindness. Successful proof-of-concept studies in a canine model have recently shown that development of a corrective gene therapy for RPGR-XLRP may now be an attainable goal. In preparation for a future clinical trial, we have here optimized the therapeutic AAV vector construct by showing that GRK1 (rather than IRBP) is a more efficient promoter for targeting gene expression to both rods and cones in non-human primates. Two transgenes were used in RPGR mutant (XLPRA2) dogs under the control of the GRK1 promoter. First was the previously developed stabilized human RPGR (hRPGRstb). Second was a new full-length stabilized and codon-optimized human RPGR (hRPGRco). Long-term (>2 years) studies with an AAV2/5 vector carrying hRPGRstb under control of the GRK1 promoter showed rescue of rods and cones from degeneration and retention of vision. Shorter term (3 months) studies demonstrated comparable preservation of photoreceptors in canine eyes treated with an AAV2/5 vector carrying either transgene under the control of the GRK1 promoter. These results provide the critical molecular components (GRK1 promoter, hRPGRco transgene) to now construct a therapeutic viral vector optimized for RPGR-XLRP patients.
Assuntos
Proteínas de Transporte/genética , Proteínas do Olho/genética , Genes Ligados ao Cromossomo X , Terapia Genética , Mutação , Retina/metabolismo , Retinose Pigmentar/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Cães , Receptor Quinase 1 Acoplada a Proteína G/genética , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Humanos , Fenótipo , Células Fotorreceptoras de Vertebrados/metabolismo , Primatas , Regiões Promotoras Genéticas , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/terapia , Transdução Genética , Transgenes , Testes VisuaisRESUMO
Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.
Assuntos
Terapia Genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneração Retiniana/terapia , Visão Ocular , Animais , Modelos Animais de Doenças , Cães , Degeneração Retiniana/fisiopatologiaRESUMO
Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.