A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

干性年龄相关性黄斑变性的核酶救援策略

• 批准号: 7797912
• 负责人: JOHN M. SULLIVAN
• 金额: --
• 依托单位: VA WESTERN NEW YORK HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7797912
• 关键词: Address; Adolescent; Affect; Age; Age related macular degeneration; Aging; American; Apoptosis; Atrophic; Biochemical; Biomass; Blindness; Carbohydrates; Catalytic RNA; Cell Death; Cells; Cessation of life; Chemicals; Circadian Rhythms; Clinic; Clinical Trials; Deposition; Digestion; Disease; Drusen; Elderly; Electroretinography; Elements; Engineering; Ethanolamines; Flecks; Fundus; Genetic; Health; Healthcare; High Pressure Liquid Chromatography; Histology; Human; Individual; Inferior; Knock-out; Lead; Life; Light; Lighting; Lipids; Lipofuscin; Macular degeneration; Measures; Mediating; Membrane; Messenger RNA; Metabolic; Minor; Molecular; Molecular Target; Mus; Natural regeneration; Neuroglia; Nonexudative age-related macular degeneration; Opsin; Outcome; Oxidative Stress; Performance; Phagocytosis; Phagolysosome; Phase; Photoreceptors; Phototoxicity; Pigments; Prevalence; Production; Proteins; RNA Interference; Reaction; Recombinant adeno-associated virus (rAAV); Retinal; Retinal Cone; Retinal Degeneration; Retinal Pigments; Retinaldehyde; Retinoids; Retinol dehydrogenase; Rhodopsin; Rod Outer Segments; Salts; Site; Societies; Specificity; Structure; Structure of retinal pigment epithelium; System; Testing; Therapeutic; Time; Toxic effect; Vertebrate Photoreceptors; Veterans; Visible Radiation; Vision; Visual; Vitamins; adeno-associated viral vector; age related; base; design; dimer; disability; gene therapy; hammerhead ribozyme; in vivo; macula; mouse model; normal aging; novel; novel strategies; pre-clinical; public health relevance; research study; retinal rods; safety study; single molecule; small hairpin RNA; small molecule; success; tool; visual cycle

DESCRIPTION (provided by applicant): In juvenile macular degeneration (JMD) (e.g Stargardt's, Best's) and common dry age-related macular degeneration (dAMD), cellular and biochemical debris accumulates within and beneath the retinal pigment epithelium (RPE). These diseases reflect, in part, naturally occurring circadian shedding of rod and cone photoreceptor (PR) outer segment tips, and phagocytosis and lysosomal digestion by RPE cells. In the human parafovea, where dAMD starts, a single RPE cell underlies about 30-35 rods and a few cones. Due to RPE digestive limitations excess age-related materials called lipofuscin (LF) accumulate in RPE phagolysosomes. LF contains protein, lipid and carbohydrate components and contributes to sub-RPE deposits (flecks, drusen) seen in JMD and dAMD. LF has a brilliant autofluorescence under blue light excitation due to the dominant presence of toxic bis-retinoid pyridinium salts (A2E) and retinaldehyde dimers (RetDi). These derive from covalent reaction, in PR outer segments, of two molecules of all-trans-retinal (ATR), resulting from visual pigment bleaching, with a single molecule of the membrane aminolipid phosphatidyl-ethanolamine (PE). Autofluorescent LF pigments accumulate with age in normals, and by age 30 are readily quantitated by fundus autofluorescence (FAF). Accumulation of A2E and RetDi in RPE cells reflects the normal daily accumulation of ATR from the visual cycle, with bleaching and regeneration of rod and cone opsins, integrated over many years. In JMD and dAMD A2E and RetDi accumulation rates are accelerated. A2E and the more potent RetDi exert many toxic effects on the RPE cells and directly promote apoptosis. Accumulation of A2E/Ret-Di precedes spatial geographic loss of RPE cells and overlying PRs in dAMD/JMD. A2E and RetDi are well validated molecular targets for therapy of dAMD/JMD. Our hypothesis is that dAMD/JMD can be treated by reducing time-dependent accumulation of A2E and Ret-Di in RPE cells. The rationale is that steady- state reductions in A2E/RetDi would decrease its time-integrated toxicity and maintain viable RPE cells longer into life. This effect would act to preserve overlying PRs, maintain central vision, and slow or halt emergence of geographic macular atrophy. The long term objective is to develop a safe and effective gene therapy for dAMD/JMD. The objective of the proposed experiments is to use hammerhead ribozymes (hhRz) or RNA interference (shRNA) as genetic tools to knockdown (KD) expression of key proteins in rod PRs or RPE cells that quantitatively contribute to daily accumulation of A2E/RetDi in RPE. This novel strategy is tested in a new mouse model of dAMD/JMD (ABCR-/-//RDH8-/- double knockout), which has central inferior RPE and PR loss due to A2E/RetDi accumulation. The strategy is: 1) reduce rhodopsin (RHO) to constrain ATR formation that results mostly from rod pigment bleaching, and 2) constrain retinoid cycle regeneration rates by reduction of 11-cis-retinol dehydrogenases (RDH5/RDH11). By reducing the amount of RHO that forms and bleaches in rod PRs, daily ATR production will be reduced under normal lighting. As ATR is a substrate of A2E and RetDi formation, reduction in the rate of toxic retinoid accumulation is expected. After determining safe KD levels of targets (RHO, RDH5/RDH11) by hhRzs/shRNAs, the expected outcome is that reduction of these targets will rescue A2E/RetDi-mediated retinal degeneration in the mouse model, at the expense of slight scotopic sensitivity loss (< -0.3 log) and preserved photopic sensitivity (cones use a retinoid visual cycle involving M|ller cells). Specific Aims are: Aim 1. Identify and optimize lead candidate hhRz and shRNA expression constructs to target mouse RHO and RDH5 and RDH11 for specific knockdown. Aim 2. Determine maximum tolerable (nontoxic) knockdown levels of RHO and RHD5/RDH11 by hhRzs/shRNAs transduced to photoreceptors or RPE by rAAV vectors after subretinal delivery. Aim 3. Test for rescue of retinal degeneration in the ABCR-/-//RDH8-/- mouse model of JMD and dAMD by knockdown of RHO and RHD5/RDH11 targets following subretinal delivery of rAAV hhRz/shRNA expression constructs. PUBLIC HEALTH RELEVANCE: Potential Impact on Veterans Health: AMD is the most common form of visual disability in the elderly and is a health care crisis. There are 4 million people in the US with severe visual disability due to AMD, and this number is expected to triple by the year 2020. There are 10-15 million others who have early forms of the disease. AMD prevalence increases exponentially over the age of 55 and, with our aging society, there are many millions of American Veterans who will suffer from visual loss due to AMD. I see Veterans in my Retinal and Macular Degeneration clinic and can attest to the suffering that AMD causes. Nonspecific vitamin support is the only approved therapy for dry AMD, the most common form (80-85%). As yet, there is no specific therapy for dry AMD, which promotes profound visual disability in about 20% of people. This proposed study is a proof-of-concept test of a novel gene therapy strategy for dry AMD. Success in a mouse model is a first essential step toward human clinical trials, which could be conducted in the VA system.

描述（由申请人提供）： 在青少年黄斑变性 (JMD)（例如 Stargardt's、Best's）和常见的干性年龄相关性黄斑变性 (dAMD) 中，细胞和生化碎片在视网膜色素上皮 (RPE) 内部和下方积聚。这些疾病部分反映了视杆细胞和视锥细胞光感受器 (PR) 外节尖端自然发生的昼夜节律脱落，以及 RPE 细胞的吞噬作用和溶酶体消化。在 dAMD 起始的人类中央凹中，单个 RPE 细胞位于大约 30-35 个视杆细胞和一些视锥细胞下方。由于 RPE 消化限制，过量的与年龄相关的物质（称为脂褐素 (LF)）会积聚在 RPE 吞噬溶酶体中。 LF 含有蛋白质、脂质和碳水化合物成分，有助于形成 JMD 和 dAMD 中可见的亚 RPE 沉积物（斑点、玻璃疣）。由于有毒的双维甲酸吡啶盐 (A2E) 和视黄醛二聚体 (RetDi) 的主要存在，LF 在蓝光激发下具有明亮的自发荧光。这些源自视色素漂白产生的全反式视网膜 (ATR) 的两个分子在 PR 外段与膜氨基脂磷脂酰乙醇胺 (PE) 的单分子的共价反应。正常情况下，自发荧光 LF 色素会随着年龄的增长而积累，到 30 岁时，可以通过眼底自发荧光 (FAF) 轻松定量。 RPE 细胞中 A2E 和 RetDi 的积累反映了视觉周期中 ATR 的正常每日积累，以及视杆细胞和视锥细胞视蛋白的漂白和再生，经过多年的整合。在 JMD 和 dAMD 中，A2E 和 RetDi 积累速率加快。 A2E和更有效的RetDi对RPE细胞产生许多毒性作用并直接促进细胞凋亡。 A2E/Ret-Di 的积累先于 dAMD/JMD 中 RPE 细胞和​​上覆 PR 的空间地理损失。 A2E 和 RetDi 是经过充分验证的 dAMD/JMD 治疗分子靶点。我们的假设是，可以通过减少 RPE 细胞中 A2E 和 Ret-Di 的时间依赖性积累来治疗 dAMD/JMD。其基本原理是，A2E/RetDi 的稳态减少将降低其时间积分毒性并维持 RPE 细胞的存活时间更长。这种效应将起到保护覆盖的 PR、维持中心视力并减缓或阻止地理性黄斑萎缩的出现的作用。长期目标是开发一种安全有效的 dAMD/JMD 基因疗法。拟议实验的目的是使用锤头核酶 (hhRz) 或 RNA 干扰 (shRNA) 作为遗传工具来敲低 (KD) 视杆 PR 或 RPE 细胞中关键蛋白的表达，这些蛋白定量地促进 RPE 中 A2E/RetDi 的日常积累。这种新颖的策略在新的 dAMD/JMD（ABCR-/-//RDH8-/- 双敲除）小鼠模型中进行了测试，该模型由于 A2E/RetDi 积累而具有中枢下 RPE 和 PR 损失。该策略是：1) 减少视紫红质 (RHO) 以限制 ATR 形成，ATR 形成主要由视杆色素漂白引起，2) 通过减少 11-顺式视黄醇脱氢酶 (RDH5/RDH11) 来限制类视黄醇循环再生率。通过减少 PR 杆中形成和漂白的 RHO 量，正常照明下每日 ATR 产量将会减少。由于 ATR 是 A2E 和 RetDi 形成的底物，因此预计会降低有毒类视黄醇积累的速率。通过 hhRzs/shRNA 确定目标（RHO、RDH5/RDH11）的安全 KD 水平后，预期结果是这些目标的减少将挽救小鼠模型中 A2E/RetDi 介导的视网膜变性，但代价是轻微的暗视敏感性损失（< -0.3 log）和保留的明视觉敏感性（视锥细胞使用涉及 M|ller 的类维生素A视觉循环） 细胞）。具体目标是： 目标 1. 识别并优化主要候选 hhRz 和 shRNA 表达构建体，以靶向小鼠 RHO、RDH5 和 RDH11 进行特异性敲低。目标 2. 确定视网膜下递送后通过 rAAV 载体转导至光感受器或 RPE 的 hhRzs/shRNA 实现的 RHO 和 RHD5/RDH11 的最大耐受（无毒）敲低水平。目标 3. 在 JMD 和 dAMD 的 ABCR-/-//RDH8-/- 小鼠模型中测试通过在视网膜下递送 rAAV hhRz/shRNA 表达构建体后敲低 RHO 和 RHD5/RDH11 靶标来挽救视网膜变性。 公共卫生相关性： 对退伍军人健康的潜在影响：AMD 是老年人最常见的视力障碍形式，也是一场医疗保健危机。在美国，有 400 万人因 AMD 导致严重视力障碍，预计到 2020 年这一数字将增加两倍。还有 10-1500 万人患有这种疾病的早期形式。 AMD 患病率在 55 岁以上呈指数级增长，并且随着社会老龄化，数以百万计的美国退伍军人将因 AMD 而遭受视力丧失。我在我的视网膜和黄斑变性诊所见过退伍军人，可以证明 AMD 造成的痛苦。非特异性维生素支持是唯一被批准治疗干性 AMD 的疗法，干性 AMD 是最常见的形式 (80-85%)。干性 AMD 会导致约 20% 的人出现严重的视力障碍，迄今为止，还没有具体的治疗方法。这项拟议的研究是对干性 AMD 的新型基因治疗策略的概念验证测试。小鼠模型的成功是人类临床试验的第一步，该试验可以在 VA 系统中进行。