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: 8195556
• 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/8195556
• 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

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.

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 变性 (dAMD)，细胞和生化碎片在视网膜色素内部和下方积聚 这些疾病部分反映了视杆细胞和视锥细胞自然发生的昼夜脱落。 人类 RPE 细胞的光感受器 (PR) 外节尖端以及吞噬作用和溶酶体消化。 中央凹是 dAMD 的起始处，由于 RPE，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。 消化限制 过量的称为脂褐质 (LF) 的与年龄相关的物质在 RPE 吞噬溶酶体中积聚。 LF 含有蛋白质、脂质和碳水化合物成分，有助于亚 RPE 沉积（斑点、玻璃疣） 在 JMD 和 dAMD 中，由于占主导地位，在蓝光激发下具有明亮的自发荧光。 有毒的双视黄醇吡啶盐 (A2E) 和视黄醛二聚体 (RetDi) 的存在。 PR 外段中两个全反式视网膜 (ATR) 分子的共价反应，由视觉引起 色素漂白，用单分子膜氨基脂磷脂酰乙醇胺（PE）。 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，可以通过眼底轻松定量 RPE 细胞中 A2E 和 RetDi 的自发荧光 (FAF) 积累反映了 A2E 和 RetDi 的正常每日积累。 来自视觉循环的 ATR，具有杆状和锥状视蛋白的漂白和再生，集成了许多 在 JMD 和 dAMD 中，A2E 和 RetDi 的积累速度加快。 对RPE细胞产生多种毒性作用并直接促进A2E/Ret-Di的积累。 先于 RPE 细胞的空间地理损失，并且 dAMD/JMD 中的 PR 很好。 经验证的 dAMD/JMD 治疗分子靶点 我们的假设是 dAMD/JMD 是可以治疗的。 通过减少 RPE 细胞中 A2E 和 Ret-Di 的时间依赖性积累，其原理是稳定的。 A2E/RetDi 状态的减少将降低其时间积分毒性并更长时间地维持存活的 RPE 细胞 这种效应将起到保护覆盖的 PR、维持中心视力以及减缓或停止出现的作用。 长期目标是开发一种安全有效的基因疗法来治疗地理性黄斑萎缩。 dAMD/JMD 拟议实验的目的是使用锤头核酶 (hhRz) 或 RNA。 干扰（shRNA）作为遗传工具敲低（KD）杆状 PR 或 RPE 细胞中关键蛋白的表达 定量地促进 RPE 中 A2E/RetDi 的每日积累。这种新颖的策略在新的方法中进行了测试。 dAMD/JMD（ABCR-/-//RDH8-/- 双敲除）小鼠模型，具有中枢下 RPE 和 PR 损失 由于 A2E/RetDi 积累，策略是：1) 减少视紫红质 (RHO) 以限制 ATR 形成。 主要是由于杆状色素漂白造成的，并且 2) 通过减少 11-顺式视黄醇脱氢酶 (RDH5/RDH11) 通过减少杆中形成和漂白的 RHO 量。 PRs，在正常光照下，ATR 的每日产量将会减少，因为 ATR 是 A2E 和 RetDi 的底物。 确定 KD 的安全水平后，预计有毒类视黄醇的形成率会降低。 hhRzs/shRNA 的目标（RHO、RDH5/RDH11），预期结果是这些目标的减少将 拯救小鼠模型中 A2E/RetDi 介导的视网膜变性，但以轻微暗视为代价 敏感性损失（< -0.3 log）和保留的明视敏感性（视锥细胞使用涉及 M¿ller 的类维生素A视觉循环 具体目标是： 目标 1. 识别并优化先导候选 hhRz 和 shRNA 表达。 构建靶向小鼠 RHO 和 RDH5 和 RDH11 的特异性敲低目标 2。 hhRzs/shRNA 对 RHO 和 RHD5/RDH11 的最大耐受（无毒）敲低水平 视网膜下递送后通过 rAAV 载体转导至光感受器或 RPE 目标 3. 救援测试。 通过敲低 RHO 对 JMD 和 dAMD 的 ABCR-/-//RDH8-/- 小鼠模型中视网膜变性的影响 rAAV hhRz/shRNA 表达构建体视网膜下递送后，RHD5/RDH11 靶向。