A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

在青少年黄斑变性（JMD）（例如Stargardt's，Best's）和常见的干性年龄相关性黄斑变性中， 在视网膜色素变性（dAMD）中，细胞和生化碎片在视网膜色素内和下方积聚 上皮（RPE）。这些疾病部分反映了视杆细胞和视锥细胞自然发生的昼夜脱落 光感受器（PR）外节尖端的细胞毒性，以及RPE细胞的吞噬作用和溶酶体消化。在人类 在dAMD开始的地方，单个RPE细胞位于约30-35个视杆细胞和一些视锥细胞之下。由于RPE 消化限制过量的年龄相关物质称为脂褐素（LF）积累在RPE吞噬溶酶体中。 LF含有蛋白质、脂质和碳水化合物成分，并导致RPE下沉积物（视网膜色素沉着、玻璃疣） 在JMD和dAMD中可见。LF在蓝光激发下具有明亮的自发荧光， 存在有毒的双类维生素A吡啶鎓盐（A2 E）和视黄醇二聚体（RetDi）。这些来源于 共价反应，在PR外节，两个分子的全反式视网膜（ATR），由视觉 色素漂白，与膜氨基脂质磷脂酰乙醇胺（PE）的单分子。 在正常人中，自发荧光LF色素随着年龄的增长而积累，到30岁时，通过眼底检查很容易定量。 自体荧光（FAF）。RPE细胞中A2 E和RetDi的积累反映了A2 E和RetDi的正常每日积累。 来自视觉周期的ATR，具有视杆和视锥视蛋白的漂白和再生，整合了许多 年在JMD和dAMD中，A2 E和RetDi积累速率加快。A2 E和更有效的RetDi 对RPE细胞有多种毒性作用，直接促进细胞凋亡。A2 E/Ret-Di的蓄积 在dAMD/JMD中RPE细胞和重叠PR的空间地理损失之前。A2 E和RetDi都很好 用于治疗dAMD/JMD的有效分子靶标。我们的假设是dAMD/JMD可以治疗 通过减少RPE细胞中A2 E和Ret-Di的时间依赖性积累。基本原理是稳定的- 所述A2 E/RetDi的降低将降低其时间积分毒性并使存活的RPE细胞维持更长时间 融入生活这种效应将起到保护重叠PR、维持中央视觉以及减缓或停止视网膜脱离的出现的作用。 地图样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。所提出的实验的目的是使用锤头状核酶（hhRz）或RNA 干扰（shRNA）作为基因工具来敲低（KD）视杆PR或RPE细胞中关键蛋白的表达 其定量地有助于RPE中A2 E/RetDi的每日积累。这种新的策略在一个新的 dAMD/JMD（ABCR-/-//RDH 8-/-双敲除）小鼠模型，其具有中央下RPE和PR损失 由于A2 E/RetDi积累。策略是：1）减少视紫红质（RHO）以抑制ATR形成， 结果主要来自视杆色素漂白，和2）通过减少 11-顺式-视黄醇脱氢酶（RDH 5/RDH 11）。通过减少杆中形成和漂白的RHO的量， PR，在正常照明下，每日ATR产量将减少。由于ATR是A2 E和RetDi的底物， 形成，预期毒性类维生素A积累的速率降低。在确定安全KD水平后， 通过hhRzs/shRNA的靶点（RHO，RDH 5/RDH 11），预期的结果是这些靶点的减少将 在小鼠模型中挽救A2 E/RetDi介导的视网膜变性，代价是轻微的暗视 敏感度丧失（< -0.3 log）和明视敏感度保留（视锥细胞使用涉及M？ller的类维生素A视觉周期 细胞）。具体目标是：目标1。鉴定并优化先导候选hhRz和shRNA表达 构建体靶向小鼠RHO和RDH 5和RDH 11用于特异性敲低。目标2.确定 通过hhRzs/shRNA的RHO和RHD 5/RDH 11的最大耐受（无毒）敲低水平 在视网膜下递送后通过rAAV载体转导至光感受器或RPE。目标3。抢救试验 通过敲低RHO在JMD和dAMD的ABCR-/-//RDH 8-/-小鼠模型中的视网膜变性 和视网膜下递送rAAV hhRz/shRNA表达构建体后的RHD 5/RDH 11靶向。

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.