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）： 在少年黄斑变性（JMD）中（例如Stargardt，最佳）和常见的与年龄相关的黄斑变性（DAMD），细胞和生化碎屑在视网膜色素上皮（RPE）内部积累。这些疾病部分反映了RPE细胞的棒状和锥形感受器（PR）外部段尖端（PR）外部段尖端（PR）外部片段尖端（PR）的昼夜节律脱落和RPE细胞的吞噬细胞消化。在Damd开始的人类Parafovea中，一个RPE细胞的基础约为30-35杆和几个锥体。由于RPE消化局限性超过了与年龄相关的材料，称为脂肪霉素（LF）在RPE吞噬体中积聚。 LF含有蛋白质，脂质和碳水化合物成分，并有助于在JMD和DAMD中看到的子RPE沉积物（斑点，drusen）。在蓝光兴奋下，LF具有出色的自动荧光，这是由于有毒的双毒素（TBR）的主要存在，例如吡啶盐盐（A2E）和视网膜二醛二聚体（RetDi）。这些源自在PR外段的共价反应，由视觉色素漂白产生的两个分子的全反视网膜（ATR）分子，其单个分子的膜氨基酰磷脂酰乙醇胺的单个分子。 LF颜料随着正常的年龄而累积，到30岁时，通过眼底自动荧光来定量。 TBR在RPE细胞中的积累反映了视觉周期中ATR的正常每日积累，杆和锥体OPS的漂白和再生多年来整合。在JMD和DAMD TBR加速度中，加速率是加速的。 A2E和较大的RETDI对RPE细胞产生许多毒性作用，并直接促进凋亡。 A2E/RET-DI的积累在DAMD/JMD中的RPE细胞的空间地理损失和上覆的PR。 TBR是良好的DAMD/JMD治疗的分子靶标。我们的假设是，可以通过减少PR和RPE细胞中TBR的时间依赖性积累来治疗DAMD/JMD。理由是，TBR中的稳态减少将减少时间融合（与年龄相关的）毒性，并使可行的RPE细胞更长地进入寿命，这将起到保留上覆的PR，由于锥体而维持中心视力，并缓慢或停止黄斑地理萎缩。长期目标是为DAMD/JMD开发安全有效的基因疗法。所提出的实验的目的是使用锤头核酶（HHRZ）或RNA干扰（SHRNA）作为在ROD PR或RPE细胞中敲低的遗传工具（KD）表达，以定量地促进TBR的每日积累。这种新颖的策略是在DAMD/JMD的小鼠模型（ABCR - / - / - // RDH8 - / - 双基因敲除）中测试的，该模型具有与TBR累积有关的外部视网膜变性。该策略是：1）在PR中减少视紫红质（Rho），以限制主要是由杆色素漂白导致的ATR形成，并减少RPE对消化杆外部段（90％RHO）的压力（90％RHO）和2）约束类黄性素周期周期再生速率，这是通过降低11- cis-retinol osomerase（RPE）（RPE）（RPE）中的RPE）。通过减少杆PR中形成和漂白的RHO量，在正常照明下每天的ATR产生减少。由于ATR是直接底物，因此预计TBR累积速率的降低。预期的结果是，这些目标的减少将挽救小鼠模型中的TBR介导的残留变性，并保留 光波敏感性（锥体使用müller细胞类维生素的视觉循环），以轻微的Scotopic灵敏度损失（<-0.3 log）为代价。具体目的是：目标1。进一步优化RHO的铅候选HHRZ​​，并为小鼠RPE65开发更强的HHRZ和SHRNA表达构建体。 AIM 2。测试候选基因治疗剂的毒性在视网膜下或玻璃室内RAAV递送中的小鼠模型中用于正常RHO的小鼠模型。 AIM 3。测试在双敲除（ABCR-/ - / - / - / - / - //- //- //- // rdh8 - / - ）DAMD/JMD的鼠标中拯救视网膜下或玻璃体内RAAV递送治疗候选者的救助，通过RHO或RPE65的敲除，在内在大鼠的变化中测量