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、Best）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，这是由于主要存在有毒的双维甲酸（TBR），如吡啶盐（A2 E）和视黄醇二聚体（RetDi）。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，与一个单一分子的膜氨基脂质磷脂酰乙醇胺。LF色素在正常人中随着年龄的增长而积累，到30岁时通过眼底自发荧光定量。视网膜色素上皮细胞中TBRs的积累反映了视觉周期中ATR的正常每日积累，视杆和视锥视蛋白的漂白和再生，多年来整合。在JMD和dAMD中，TBR累积速率加快。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。TBRs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PR和RPE细胞中时间依赖性的TBRs积累来治疗。其基本原理是，TBRs的稳态降低将降低时间积分（年龄相关）毒性，并将活的RPE细胞维持更长的寿命，这将起到保护覆盖PR的作用，维持视锥细胞引起的中心视力，并减缓或停止黄斑地图状萎缩的出现。长期目标是开发一种安全有效的dAMD/JMD基因治疗方法。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细胞中的关键蛋白质的表达，所述关键蛋白质定量地有助于TBRs的每日积累。在dAMD/JMD（ABCR-/-//RDH 8-/-双敲除）小鼠模型中测试了这种新策略，该模型具有与TBR积累相关的外部视网膜变性。战略是：1）减少PR中的视紫红质（RHO）以限制主要由视杆色素漂白引起的ATR形成，并减少施加在RPE上以消化视杆外节材料（90%RHO）的应力，和2）通过减少RPE中的11-顺式-视黄醇异构酶（RPE 65）来限制类维生素A循环再生速率。通过减少在杆PR中形成和漂白的RHO的量，在正常照明下减少了每日ATR产生。由于ATR是直接底物，预期TBR累积速率降低。预期的结果是，这些靶点的减少将挽救小鼠模型中的TLR介导的视网膜变性，同时保留 明视敏感度（视锥细胞使用Müller细胞类维生素A视觉周期），代价是轻微的暗视敏感度损失（< -0.3 log）。具体目标是：目标1。进一步优化RHO的先导候选hhRz，并为小鼠RPE 65开发更强的hhRz和shRNA表达构建体。目标二。在针对正常RHO人源化的小鼠模型中视网膜下或玻璃体内rAAV递送后测试候选基因治疗剂的毒性。目标3。在dAMD/JMD的双敲除（ABCR-/-//RDH 8-/-）小鼠模型中，通过RHO或RPE 65的敲低，在视网膜下或玻璃体内rAAV递送治疗候选物后，测试视网膜变性的拯救，测量为内源性大鼠视网膜变性的变化。

 DESCRIPTION (provided by applicant): In juvenile macular degeneration (JMD) (e.g. Stargardt, Best) 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-retinoids (TBR) such as 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. LF pigments accumulate with age in normals, and by age 30 are quantitated by fundus autofluorescence. Accumulation of TBRs 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 TBR 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. TBRs 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 TBRs in PR and RPE cells. The rationale is that steady-state reductions in TBRs would decrease time-integrated (agerelated) toxicity and maintain viable RPE cells longer into life, which would act to preserve overlying PRs, maintain central vision due to cones, and slow or halt emergence of macular geographic 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 PR or RPE cells that quantitatively contribute to daily accumulation of TBRs. This novel strategy is tested in a mouse model of dAMD/JMD (ABCR-/-//RDH8-/- double knockout), which has an outer retinal degeneration related to TBR accumulation. The strategy is: 1) reduce rhodopsin (RHO) in PRs to constrain ATR formation that results mostly from rod pigment bleaching, and reduce the stress imposed on the RPE to digest rod outer segment material (90% RHO), and 2) constrain retinoid cycle regeneration rates by reduction of 11-cis-retinol isomerase (RPE65) in RPE. By reducing the amount of RHO that forms and bleaches in rod PRs, daily ATR production is reduced under normal lighting. As ATR is a direct substrate, reduction in the rate of TBR accumulation is expected. The expected outcome is that reduction of these targets will rescue TBR-mediated retinal degeneration in the mouse model, with preserved photopic sensitivity (cones use a Müller cell retinoid visual cycle) at the expense of slight scotopic sensitivity loss (< -0.3 log). Specific Aims are: Aim 1. Further optimize the lead candidate hhRz for RHO and develop stronger hhRz and shRNA expression constructs for mouse RPE65. Aim 2. Test for toxicity of candidate gene therapeutic agents after subretinal or intravitreal rAAV delivery in a mouse model humanized for normal RHO. Aim 3. Test for rescue of retinal degeneration after subretinal or intravitreal rAAV delivery of therapeutic candidates in the double knockout (ABCR-/-//RDH8-/-) mouse model of dAMD/JMD, through knockdown of RHO or RPE65, measured as changes in the intrinsic rat