A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

描述(由申请人提供)： 在幼年型黄斑变性(JMD)(如Stargardt‘s、Best’s)和常见的干性年龄相关性黄斑变性(DAMD)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)。由于有毒的双维A酸类吡啶盐(A2E)和视黄醛二聚体(RetDi)的主要存在，LF在蓝光激发下具有明亮的自发荧光。这些来自PR外段的两个全反式视网膜(ATR)分子的共价反应，该反应是由视觉色素漂白产生的，与膜氨基脂磷脂酰乙醇胺(PE)的单分子发生共价反应。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，眼底自发荧光(FAF)很容易被定量。RPE细胞中A2E和RetDi的积累反映了视觉周期中ATR的正常日常积累，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，A2E和RetDi的积累速度加快。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。A2E和RetDi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RPE细胞中A2E和Ret-Di的时间依赖性积聚来治疗。其基本原理是，稳定减少A2E/RetDi将降低其时间累积毒性，并使RPE细胞存活更长时间。这一作用将保护覆盖的PR，维持中心视力，并减缓或阻止地理性黄斑萎缩的出现。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RPE中A2E/RetDi的每日积累有定量贡献。这一新策略在一种新的dAMD/JMD小鼠模型(ABCR-/-//RDH8-/-双基因敲除)中进行了测试，该模型由于A2E/RetDi的积累而具有中枢性下位RPE和PR丢失。其策略是：1)减少视紫红质(Rho)，以抑制主要由视杆色素漂白引起的ATR的形成；2)通过减少11-顺式视黄醇脱氢酶(RDH5/RDH11)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产量将会减少。由于ATR是A2E和RetDi形成的底物，预计毒性维甲酸积累的速度将会降低。在通过hhRzs/shRNAs确定靶点(Rho，RDH5/RDH11)的安全KD水平后，预期的结果是，这些靶点的减少将挽救A2E/RetDi介导的小鼠模型中的视网膜退化，但代价是轻微的暗视敏感度损失(&lt；-0.3log)和保留的光敏感度(视锥细胞使用涉及M|ler细胞的视黄醇视觉周期)。具体目标是：目的1.确定并优化针对小鼠Rho、RDH5和RDH11的主要候选hhRz和shRNA表达载体，以进行特定的敲除。目的2.通过rAAV载体将hhRZS/shRNAs转导到光感受器或RPE，确定Rho和RHD5/RDH11的最大耐受(无毒)基因敲除水平。目的3.在ABCR-/-//RDH8-/-小鼠模型中，通过视网膜下注射rAAV hhRz/shRNA表达载体后，通过击倒Rho和RHD5/RDH11靶标来挽救视网膜变性。

DESCRIPTION (provided by applicant): 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.