A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

描述（由申请人提供）： 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（dAMD）中，细胞和生物化学碎片在视网膜色素上皮（RPE）内和下方积聚。这些疾病部分反映了视杆和视锥光感受器（PR）外节尖端的自然发生的昼夜节律脱落，以及RPE细胞的吞噬作用和溶酶体消化。在dAMD开始的人类视网膜旁组织中，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，过量的与年龄相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、脂质和碳水化合物成分，并有助于在JMD和dAMD中观察到的RPE下沉积物（视网膜软化、玻璃疣）。LF在蓝光激发下具有明亮的自发荧光，这是由于有毒的双类维生素A吡啶盐（A2 E）和视黄醇二聚体（RetDi）的主要存在。这些衍生自共价反应，在PR外段，两个分子的全反式视网膜（ATR），导致视觉色素漂白，与一个分子的膜氨基脂质磷脂酰乙醇胺（PE）。在正常人中，自发荧光LF色素随着年龄的增长而积累，并且到30岁时，通过眼底自发荧光（FAF）容易定量。RPE细胞中A2 E和RetDi的积累反映了来自视觉周期的ATR的正常每日积累，其中视杆和视锥视蛋白的漂白和再生整合多年。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细胞产生许多毒性作用并直接促进细胞凋亡。A2 E/Ret-Di的积累先于dAMD/JMD中RPE细胞和覆盖PR的空间地理损失。A2 E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A2 E和Ret-Di的时间依赖性积累来治疗。基本原理是A2 E/RetDi的稳态降低将降低其时间积分毒性并将活的RPE细胞维持更长的寿命。这种作用将起到保护覆盖PR、维持中心视力以及减缓或停止地图状黄斑萎缩的出现的作用。长期目标是开发一种安全有效的dAMD/JMD基因治疗方法。所提出的实验的目的是使用锤头状核酶（hhRz）或RNA干扰（shRNA）作为遗传工具来敲低（KD）视杆PR或RPE细胞中的关键蛋白的表达，所述关键蛋白定量地有助于RPE中A2 E/RetDi的每日积累。在dAMD/JMD（ABCR-/-//RDH 8-/-双敲除）的新小鼠模型中测试这种新策略，其具有由于A2 E/RetDi积累而导致的中央下RPE和PR损失。战略是：1）减少视紫红质（RHO）以抑制主要由视杆色素漂白引起的ATR形成，和2）通过减少11-顺式-视黄醇脱氢酶（RDH 5/RDH 11）来抑制类维生素A循环再生速率。通过减少在棒状PR中形成和漂白的RHO的量，在正常照明下将减少每日ATR产生。由于ATR是A2 E和RetDi形成的底物，预期毒性类维生素A蓄积速率降低。在通过hhRzs/shRNAs确定靶标（RHO，RDH 5/RDH 11）的安全KD水平后，预期的结果是这些靶标的减少将挽救小鼠模型中A2 E/RetD 1介导的视网膜变性，代价是轻微的暗视敏感性损失（<-0.3log）和保留的明视敏感性（视锥细胞使用涉及M| Ller细胞）。具体目标是：目标1。鉴定和优化前导候选hhRz和shRNA表达构建体，以靶向小鼠RHO和RDH 5和RDH 11进行特异性敲低。目标二。确定视网膜下递送后通过rAAV载体转导至光感受器或RPE的hhRzs/shRNA对RHO和RHD 5/RDH 11的最大耐受（无毒）敲低水平。目标3。在视网膜下递送rAAV hhRz/shRNA表达构建体后，通过敲低RHO和RHD 5/RDH 11靶标，测试JMD和dAMD的ABCR-/-//RDH 8-/-小鼠模型中视网膜变性的拯救。 公共卫生相关性： 对退伍军人健康的潜在影响：AMD是老年人最常见的视力残疾形式，是一种医疗危机。美国有400万人因AMD而患有严重的视力残疾，预计到2020年这一数字将增加两倍。还有1000万至1500万人患有这种疾病的早期形式。AMD患病率在55岁以上呈指数级增长，随着我们的老龄化社会，有数百万美国退伍军人将因AMD而遭受视力丧失。我在我的视网膜和黄斑变性诊所看到退伍军人，可以证明AMD造成的痛苦。非特异性维生素支持是唯一批准的治疗干性AMD，最常见的形式（80-85%）。到目前为止，还没有针对干性AMD的特异性治疗方法，干性AMD会导致约20%的人出现严重的视力障碍。这项拟议的研究是对干性AMD的新基因治疗策略的概念验证测试。小鼠模型的成功是人类临床试验的第一个重要步骤，可以在VA系统中进行。

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. PUBLIC HEALTH RELEVANCE: Potential Impact on Veterans Health: AMD is the most common form of visual disability in the elderly and is a health care crisis. There are 4 million people in the US with severe visual disability due to AMD, and this number is expected to triple by the year 2020. There are 10-15 million others who have early forms of the disease. AMD prevalence increases exponentially over the age of 55 and, with our aging society, there are many millions of American Veterans who will suffer from visual loss due to AMD. I see Veterans in my Retinal and Macular Degeneration clinic and can attest to the suffering that AMD causes. Nonspecific vitamin support is the only approved therapy for dry AMD, the most common form (80-85%). As yet, there is no specific therapy for dry AMD, which promotes profound visual disability in about 20% of people. This proposed study is a proof-of-concept test of a novel gene therapy strategy for dry AMD. Success in a mouse model is a first essential step toward human clinical trials, which could be conducted in the VA system.