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.

在少年黄斑变性（JMD）中 变性（DAMD），细胞和生化碎片在残留色素内积聚 上皮（RPE）。这些疾病部分反映了杆和锥的昼夜节律自然发生 RPE细胞的感光受体（PR）外部段，吞噬作用和溶酶体消化。在人类中 DAMD开始的Parafovea，一个RPE单元的基础约为30-35杆和几个锥体。由于RPE 消化局限性超过称为脂肪霉素（LF）的年龄相关材料，这些材料积聚在RPE吞噬体中。 LF含有蛋白质，脂质和碳水化合物成分，并有助于亚RPE沉积物（斑点，drusen） 在JMD和DAMD中看到。由于占主导地位，LF在蓝光兴奋下具有出色的自动荧光 存在有毒的双毒素吡啶核盐（A2E）和视网膜二氢二聚体（RETDI）。这些来自 在视网膜（ATR）的两个分子的PR外段中的共价反应，由视觉造成 色素漂白，带有膜氨基脂磷脂酰乙醇胺（PE）的单个分子。 自动荧光LF颜料随着年龄的年龄而累积，到30岁时，它很容易被眼底定量 自动荧光（FAF）。 A2E和RETDI在RPE细胞中的积累反映了正常的每日积累 视觉周期中的ATR，杆和锥蛋白的漂白和再生，整合了许多 年。在JMD和DAMD A2E中，加速度加速了。 A2E和越来越潜在的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之前。 A2E和RETDI很好 经过验证的DAMD/JMD治疗的分子靶标。我们的假设是可以治疗DAMD/JMD 通过减少RPE细胞中A2E和RET-DI的时间依赖性积累。理由是 A2E/RETDI的状态减少将降低其时间积分的毒性，并保持可行的RPE细胞更长 进入生活。这种效果将采取行动，以保存上覆的PR，保持中央视觉以及缓慢或停止的出现 地理黄斑萎缩。长期目标是开发一种安全有效的基因疗法 DAMD/JMD。提出的实验的目的是使用锤头核酶（HHRZ）或RNA 干扰（shRNA）作为敲低的遗传工具（KD）在ROD PRS或RPE细胞中的键蛋白表达 该定量有助于每天在RPE中A2E/RETDI的每日积累。这种新颖的策略在新的 DAMD/JMD的鼠标模型（ABCR - / - // RDH8 - / - 双敲除），中央较低的RPE和PR损失 由于A2E/RETDI积累。策略是：1）减少视紫红质（Rho）来限制ATR形成 主要来自杆色素漂白，以及2）通过降低来限制视黄素周期再生速率 11-CIS-他醇脱氢酶（RDH5/RDH11）。通过减少杆中形成和漂白的Rho量 PRS，每天的ATR产生将在正常照明下降低。由于ATR是A2E和RETDI的底物 形成，预计有毒性类维生素类似的积累速率降低。确定安全的KD水平之后 HHRZS/SHRNA的目标（RHO，RDH5/RDH11），预期的结果是，这些目标的减少将会 在小鼠模型中拯救A2E/RetDI介导的视网膜变性，以轻微的Scotopic为代价 灵敏度丧失（<-0.3 log）和保留的光波敏感性（锥体使用涉及M ller的类维生素类似的视觉循环 细胞）。具体目的是：目标1。识别和优化铅候选者HHRZ和SHRNA表达 构建针对小鼠RHO和RDH5和RDH11的构造，以进行特定的敲低。目标2。确定 HHRZS/SHRNA的最大可耐受性（无毒）RHO和RHD5/RDH11的敲低水平 视网膜下递送后，通过RAAV向量转导给感光器或RPE。目标3。救援测试 abcr - / - // rdh8 - / - JMD和DAMD小鼠模型的视网膜变性 RAAV HHRZ/SHRNA表达构建体下递送后递送后，RHD5/RDH11靶标。