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)(如Stargardt‘s，Best’s)和常见的干性年龄相关性黄斑 变性(DAMD)，细胞和生化碎片在视网膜色素内和下方堆积 上皮(RPE)。这些疾病在一定程度上反映了节律性的杆状和圆锥体脱落。 光感受器(PR)外节末端，以及RPE细胞的吞噬和溶酶体消化。在人类中 DAMD的起始部位是视网膜区，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 消化受限过多的与年龄有关的物质称为脂褐素(LF)，积聚在RPE吞噬溶酶体中。 LF含有蛋白质、脂肪和碳水化合物成分，并有助于RPE下的沉积(斑点、玻璃体) 可见于JMD和DAMD。Lf在蓝光激发下具有明亮的自体荧光，这是由于 存在有毒的双维A酸吡啶盐(A2E)和视黄醛二聚体(RetDi)。这些起源于 由视觉引起的两个全反式视网膜(ATR)分子在PR外段的共价反应 颜料漂白，用单分子氨基脂磷脂酰乙醇胺(PE)。 在正常人中，自发荧光的LF色素随着年龄的增长而积累，到30岁时，眼底很容易定量 自体荧光(FAF)。RPE细胞中A2E和RetDi的积累反映了正常的日常积累 来自视觉周期的ATR，带有杆状和锥状视蛋白的漂白和再生，整合了许多 好几年了。在JMD和DAMD中，A2E和RetDi的积累速度加快。A2E和更强大的RetDi 对RPE细胞有多种毒性作用，直接促进细胞凋亡。A2E/Ret-Di的积累 先于DAMD/JMD的RPE细胞和覆盖的PR的空间地理丢失。A2E和RetDi都很好 验证了治疗DAMD/JMD的分子靶点。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的时间依赖积累。理由是这种稳定- A2E/RetDi的状态降低将降低其时间累积毒性，并使RPE细胞存活更长时间 走进生活。这一效果将保护覆盖的PR，维持中央视力，并减缓或阻止出现 地理性黄斑萎缩。长期目标是开发一种安全有效的基因治疗方法 DAMD/JMD。建议的实验目的是使用锤头状核酶(Hhrz)或rna。 干扰(ShRNA)作为基因工具敲除(KD)杆状PR或RPE细胞中关键蛋白的表达 这在数量上有助于RPE中A2E/RetDi的每日积累。这一新战略在一项新的 中枢下位RPE和PR缺失的dAMD/JMD(ABCR-/-//RDH8-/-双基因敲除)小鼠模型 由于A2E/RetDi的积累。其策略是：1)减少视紫红质(Rho)以抑制ATR的形成 结果主要来自杆状色素漂白，以及2)通过减少 11-顺式视黄醇脱氢酶(RDH5/RDH11)。通过减少在杆中形成和漂白的Rho的量 在正常光照下，日ATR产量将减少。因为ATR是A2E和RetDi的底物 随着维甲酸的形成，有毒的维甲酸积聚的速度有望降低。在确定安全的Kd水平之后 HhRzs/shRNAs的目标(Rho，RDH5/RDH11)，预期结果是这些目标的减少将 挽救A2E/RetDi介导的小鼠视网膜变性模型，代价是轻微的暗视 敏感度损失(&lt；-0.3log)和保留的明视敏感度(视锥细胞使用涉及M？ler的视黄醇视觉周期 单元格)。具体目标是：目标1.确定和优化主要候选hhRz和shRNA表达 针对特定击倒的鼠标Rho和RDH5和RDH11的构造。目标2.确定 HhRzs/shRNAs对Rho和RHD5/RDH11的最大耐受(无毒)击倒水平 视网膜下递送后通过rAAV载体转导到光感受器或RPE。目标3.救援测试 Rho基因敲除对ABCR-/-//RDH8-/-JMD和DAMD小鼠模型视网膜变性的影响 RHD5/RDH11靶向视网膜下输送rAAV hhRz/shRNA表达载体。