A RIBOZYME RESCUE STRATEGY FOR DRY AGE-RELATED MACULAR DEGENERATION

干龄相关黄斑变性的核酶拯救策略

• 批准号: 8923383
• 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 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8923383
• 关键词: 11-cis-Retinol; Adolescent; Age; Age related macular degeneration; Aging; American; Apoptosis; Atrophic; Biochemical; Blindness; Candidate Disease Gene; Carbohydrates; Catalytic RNA; Cells; Circadian Rhythms; Clinic; Clinical Trials; Cone; Cultured Cells; Deposition; Digestion; Disease; Drusen; Elderly; Ethanolamines; Flecks; Fundus; Genes; Genetic; Health; Healthcare; Healthcare Systems; Human; Knock-out; Knockout Mice; Lead; Life; Light; Lighting; Lipids; Lipofuscin; Macular degeneration; Measures; Mediating; Membrane; Messenger RNA; Metabolism; Molecular Target; Mus; Natural regeneration; Nonexudative age-related macular degeneration; Opsin; Outcome; Outcome Study; Phagocytosis; Phagolysosome; Photoreceptors; Pigments; Population; Preclinical Testing; Prevalence; Process; Production; Proteins; RNA Interference; RPE65 protein; Rattus; Reaction; Research Infrastructure; Retinal; Retinal Cone; Retinal Degeneration; Retinal Pigments; Retinaldehyde; Retinoids; Rhodopsin; Rod Outer Segments; Salts; Societies; Stress; Structure of retinal pigment epithelium; System; Testing; Therapeutic; Therapeutic Agents; Time; Toxic effect; Toxicity Tests; United States Department of Veterans Affairs; Vertebrate Photoreceptors; Veterans; Vision; Visual; Vitamins; age related; base; dimer; disability; gene therapy; geographic atrophy; hammerhead ribozyme; macula; mouse model; normal aging; novel; novel strategies; pre-clinical; research study; retinal rods; retinol isomerase; single molecule; small hairpin RNA; success; targeted treatment; tool; visual cycle

 DESCRIPTION (provided by applicant): In juvenile macular degeneration (JMD) (e.g. Stargardt, Best) 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-retinoids (TBR) such as 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. LF pigments accumulate with age in normals, and by age 30 are quantitated by fundus autofluorescence. Accumulation of TBRs 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 TBR 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. TBRs 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 TBRs in PR and RPE cells. The rationale is that steady-state reductions in TBRs would decrease time-integrated (agerelated) toxicity and maintain viable RPE cells longer into life, which would act to preserve overlying PRs, maintain central vision due to cones, and slow or halt emergence of macular geographic 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 PR or RPE cells that quantitatively contribute to daily accumulation of TBRs. This novel strategy is tested in a mouse model of dAMD/JMD (ABCR-/-//RDH8-/- double knockout), which has an outer retinal degeneration related to TBR accumulation. The strategy is: 1) reduce rhodopsin (RHO) in PRs to constrain ATR formation that results mostly from rod pigment bleaching, and reduce the stress imposed on the RPE to digest rod outer segment material (90% RHO), and 2) constrain retinoid cycle regeneration rates by reduction of 11-cis-retinol isomerase (RPE65) in RPE. By reducing the amount of RHO that forms and bleaches in rod PRs, daily ATR production is reduced under normal lighting. As ATR is a direct substrate, reduction in the rate of TBR accumulation is expected. The expected outcome is that reduction of these targets will rescue TBR-mediated retinal degeneration in the mouse model, with preserved photopic sensitivity (cones use a Müller cell retinoid visual cycle) at the expense of slight scotopic sensitivity loss (< -0.3 log). Specific Aims are: Aim 1. Further optimize the lead candidate hhRz for RHO and develop stronger hhRz and shRNA expression constructs for mouse RPE65. Aim 2. Test for toxicity of candidate gene therapeutic agents after subretinal or intravitreal rAAV delivery in a mouse model humanized for normal RHO. Aim 3. Test for rescue of retinal degeneration after subretinal or intravitreal rAAV delivery of therapeutic candidates in the double knockout (ABCR-/-//RDH8-/-) mouse model of dAMD/JMD, through knockdown of RHO or RPE65, measured as changes in the intrinsic rat

 描述(由申请人提供)： 在幼年型黄斑变性(JMD)(如Stargardt，Best)和常见的干性老年性黄斑变性(DAMD)中，细胞和生化碎片聚集在视网膜色素上皮(RPE)内和下方。这些疾病在一定程度上反映了视杆和视锥感光器(PR)外节末端自然发生的昼夜节律脱落，以及RPE细胞的吞噬和溶酶体消化。在人的视盘旁，即dAMD的起始处，单个RPE细胞位于大约30-35个视杆和几个视锥的下方。由于RPE消化的限制，过量的与年龄有关的物质，称为脂褐素(LF)，会在RPE吞噬溶酶体中积累。LF含有蛋白质、脂肪和碳水化合物成分，并有助于在JMD和DAMD中看到的RPE亚沉积(斑点、玻璃体)。在蓝光激发下，LF具有明亮的自发荧光，这是由于有毒的双维A酸(TBR)，如吡啶盐(A2E)和视黄醛二聚体(RetDi)的主要存在。这些来自PR外段的两个全反式视网膜(ATR)分子与膜氨基脂磷脂酰乙醇胺的共价反应，该反应是由视觉色素漂白产生的。在正常人中，LF色素随着年龄的增长而积累，到30岁时，通过眼底自发荧光进行定量。视网膜色素上皮细胞中TBRs的积累反映了视觉周期中ATR的正常每日积累，伴随着视杆和视锥视蛋白的漂白和再生，多年整合。在JMD和DAMD中，TBR累积速率加快。A2E和更强的RetDi对RPE细胞有许多毒性作用，并直接促进细胞凋亡。在DAMD/JMD中，A2E/Ret-Di的积累先于RPE细胞和覆盖的PR的空间地理丢失。TBRs是治疗DAMD/JMD的有效分子靶点。我们的假设是，dAMD/JMD可以通过减少PR和RPE细胞中TBR的时间依赖积累来治疗。其基本原理是，稳态减少视网膜色素上皮损伤可以减少与时间相关的(与年龄相关的)毒性，并使RPE细胞存活更长时间，这将有助于保护覆盖的PR，由于视锥细胞而维持中心视力，并延缓或阻止黄斑地理性萎缩的出现。长期目标是开发一种安全有效的DAMD/JMD基因治疗方法。本实验的目的是使用锤头状核酶(HhRz)或RNA干扰(ShRNA)作为遗传工具来敲除杆状PR或RPE细胞中关键蛋白的表达，这些蛋白对TBRs的每日积累有定量贡献。这一新策略在dAMD/JMD(ABCR-/-//RDH8-/-双基因敲除)小鼠模型上进行了测试，该小鼠的视网膜外部退化与TBR积累有关。其策略是：1)减少PR中的视紫红质(Rho)，以抑制主要由视杆色素漂白引起的ATR的形成，并减少施加在RPE上以消化视杆外段物质(90%Rho)的压力，以及2)通过降低RPE中的11-顺式视黄醇异构酶(RPE65)来限制视黄醇循环的再生速度。通过减少在棒状PR中形成和漂白的RHO的量，在正常照明下每天的ATR产量减少。由于ATR是一种直接底物，因此预计TBR积累的速度将会降低。预期的结果是，这些靶点的减少将挽救TBR介导的小鼠模型中的视网膜退化，并保留 光敏感度(视锥细胞使用米勒细胞视黄醇视觉周期)，但暗视敏感度略有下降(&lt；-0.3log)。具体目标是：目的1.进一步优化Rho的主要候选基因hhRz，开发更强的针对小鼠RPE65的hhRz和shRNA表达载体。目的2.在人源化的正常Rho小鼠模型上，测试视网膜下或玻璃体内注射rAAV后候选基因治疗剂的毒性。目的3.在双基因敲除(ABCR-/-/RDH8-/-)的dAMD/JMD小鼠模型中，通过敲除Rho或RPE65来检测视网膜变性在视网膜下或玻璃体内注射rAAV后的挽救试验，测量作为固有大鼠的变化