A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

常见干性年龄相关性黄斑变性（dAMD）和孤儿青少年黄斑变性（JMD） (e.g., Stargardt）细胞和生化碎片积聚在视网膜色素上皮内和下方 （RPE）。这些疾病反映了自然发生的昼夜脱落的杆和锥光感受器（PR）外 节尖端，以及RPE细胞的吞噬作用和溶酶体消化。在人类的paraflora，其中dAMD 当JMD开始时，单个RPE细胞位于大约30-35个视杆细胞和更少的视锥细胞之下并支撑它们。RPE酶 限制导致与年龄相关的脂褐素在吞噬溶酶体中积累。脂褐素具有蛋白质、脂质和 在JMD/dAMD中观察到的RPE内和RPE下沉积物（视网膜色素瘤、玻璃疣）是由碳水化合物组分引起的，并且有助于JMD/dAMD中观察到的RPE内和RPE下沉积物（视网膜色素瘤、玻璃疣）。 脂褐素在蓝光激发下具有明亮的自发荧光和光反应性， 存在有毒的双类视色素（TBR），如吡啶鎓盐（A2 E）和视黄醇二聚体（RetDi）。这些 衍生自共价反应，在PR外段，两个分子的视网膜醛，导致从视觉 色素漂白和再生，与脂质和蛋白质。正常情况下，TBR色素在RPE细胞中积聚 年龄（> 30岁），多年积累。任何压力源（例如，黄斑变性过程）， 促进加速的TBRs积累将对RPE细胞产生毒性凋亡作用，失去覆盖的 PR和细胞死亡（例如，dAMD/JMD）。TBR是经验证的治疗分子靶标。每日成交额为 视杆细胞中大量的视杆细胞色素对视网膜色素上皮细胞是一个巨大的负担（大多数是代谢上的 活跃的细胞），并有助于细胞内和视网膜下碎片的积累; 50% 正常人视杆细胞视紫红质（RHO）的减少没有退化作用，人们仍然能看到星星。90%的 视杆细胞质量是RHO。RHO是一个有效的目标。我们的假设是，dAMD/JMD可以是 通过减少RHO以及PR和RPE中碎片和TBR的相关时间依赖性积累进行治疗 细胞基本原理或策略是RHO和相关TBRs的稳态降低将减缓年龄- 副产物的相关毒性积累，降低代谢负担，并维持活性RPE细胞更长的寿命。 这将保护覆盖的视杆细胞和视锥细胞，维持中央视锥细胞视觉，并减缓或停止视锥细胞的出现。 地图状萎缩长期目标是开发一种安全有效的dAMD/JMD基因治疗方法。 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）RHO表达以缓解日常视网膜外应激（TBRs， 代谢应激）。在dAMD/JMD（ABCR-/-//RDH 8-/-; VL 4 mut、SOD 2ko），它们都具有外视网膜PR/RPE变性。预期的结果是， RHO减少将挽救外部视网膜变性，保留明视敏感度（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性损失（<-0.3log 功能具体目标是：目标1。优化新型先导候选抗RHO hhRzs和shRNA 并为这些试剂开发稳定、无毒的细胞表达构建体。目标2. I期模型 视网膜下或玻璃体内rAAV后候选治疗剂（hhRz，shRNA）的毒性研究 在针对正常RHO人源化的小鼠中递送。目标3。II期模型研究，用于挽救视网膜病变 视网膜下或玻璃体内rAAV递送抗RHO治疗候选物后的视网膜变性 dAMD/JMD的多种小鼠模型，具有不同的应激，测量为内在速率的变化 在视网膜光损伤范例中，视网膜变性或保护感光细胞损失。 dAMD在我们的老年退伍军人群体中非常普遍，是一种视觉破坏性疾病。目前， 维生素是唯一的（有限的）疗法。在小鼠模型中的成功拯救为临床前- 在非人灵长类动物dAMD模型中进行研究性新药（IND）测试，作为迈向临床的一步 在VA医疗保健系统中进行的新型dAMD基因治疗试验。

In common dry age-related macular degeneration (dAMD) and orphan juvenile macular degeneration (JMD) (e.g., Stargardt) cellular and biochemical debris accumulates within and beneath the retinal pigment epithelium (RPE). These diseases reflect 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 and JMD starts, a single RPE cell underlies and supports about 30-35 rods and fewer cones. RPE enzymatic limitations cause age-related lipofuscin to accumulate in phagolysosomes. Lipofuscin has protein, lipid and carbohydrate components and contributes to intra- and sub-RPE deposits (flecks, drusen) seen in JMD/dAMD. Lipofuscin has a brilliant autofluorescence and photoreactivity 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 retinaldehydes, resulting from visual pigment bleaching and regeneration, with lipids and proteins. TBR pigments accumulate in RPE cells normally with age (> 30 years) integrated over many years. Any stressors (e.g., macular degenerative processes) that promote accelerated TBRs buildup will exert toxic apoptotic effects on RPE cells, loss of support of overlying PRs and cell death (e.g., dAMD/JMD). TBRs are validated molecular targets for therapy. Daily turnover of massive amounts of rod visual pigment in the parafovea is a huge burden for the RPE (most metabolically active cells in human body) and contributes to accumulation of intracellular and subretinal debris; 50% reduction of normal human rod rhodopsin (RHO) has no degenerative effect and people still see stars. 90% of rod photoreceptor mass is RHO. RHO is a validatable target. Our hypothesis is that dAMD/JMD can be treated by reducing RHO and related time-dependent accumulation of debris and TBRs in PR and RPE cells. The rationale or strategy is that steady-state reductions in RHO and related TBRs would slow age- related toxic buildup of byproducts, decrease metabolic burden, and maintain viable RPE cells longer into life. This would preserve overlying rods and cones, maintain central cone vision, and slow or halt emergence of 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) RHO expression to relieve daily outer retinal stresses (TBRs, metabolic stresses). This novel strategy is tested in multiple mouse models of dAMD/JMD (ABCR-/-//RDH8-/-; ELOVL4mut, SOD2ko), which all have outer retinal PR/RPE degenerations. The expected outcome is that RHO reduction will rescue outer retinal degenerations, with preserved photopic sensitivity (cones use a Müller cell retinoid visual cycle) at the expense of slight scotopic sensitivity loss (< -0.3 log) but preserved scotopic function. Specific Aims are: Aim 1. Optimize the novel lead candidate anti-RHO hhRzs, and shRNAs and develop stable, non-toxic cellular expression constructs for these agents. Aim 2. Phase I model study for toxicity of candidate therapeutic agents (hhRz, shRNA) after subretinal or intravitreal rAAV delivery in a mouse humanized for normal RHO. Aim 3. Phase II model study for rescue of retinal degeneration after subretinal or intravitreal rAAV delivery of anti-RHO therapeutic candidates in multiple mouse models of dAMD/JMD, with diverse stresses, measured as changes in the intrinsic rate of retinal degeneration or protection against photoreceptor loss in a retinal light damage paradigm. dAMD is highly prevalent in our aging Veteran population and is a visually devastating disease. Currently, vitamins are the only (limited) therapy. Successful rescue in mouse models sets the stage for preclinical- Investigational New Drug (IND) testing in nonhuman primate dAMD models, as a step toward clinical trials of a novel dAMD gene therapy in the VA Healthcare System.