A Ribozyme Rescue Strategy for Dry Age-Related Macular Degeneration

干性年龄相关性黄斑变性的核酶救援策略

• 批准号: 9892813
• 负责人: JOHN M. SULLIVAN
• 金额: --
• 依托单位: VA WESTERN NEW YORK HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9892813
• 关键词: 11 cis Retinal; Adolescent; Age; Aging; American; Amines; Apoproteins; Apoptosis; Apoptotic; Biochemical; Bruch' s basal membrane structure; Carbohydrates; Catalytic RNA; Cell Death; Cell physiology; Cells; Clinic; Clinical Trials; Color; Cone; Cultured Cells; Cyclic GMP; Darkness; Deposition; Development; Digestion; Disease; Drusen; Elderly; Elements; Energy Metabolism; Evolution; Flecks; Free Radicals; Gene therapy trial; Genetic; Geography; Health; Healthcare; Healthcare Systems; Human; Human body; In Vitro; Inherited; Investigational Drugs; Ions; Kinetics; Length; Life; Light; Lipids; Lipofuscin; Macular degeneration; Measures; Mediating; Messenger RNA; Metabolic; Metabolic stress; Modeling; Modernization; Muller' s cell; Mus; Natural regeneration; Nonexudative age-related macular degeneration; Opsin; Orphan; Outcome; Outcome Study; Pathogenesis; Performance; Phagocytosis; Phagolysosome; Phase; Phosphatidylethanolamine; Photoreceptors; Phototoxicity; Pigments; Population; Preclinical Testing; Prevalence; Primates; Process; Proteins; RNA Interference; Reaction; Recombinant adeno-associated virus (rAAV); Reducing Agents; Retina; Retinal Cone; Retinal Degeneration; Retinal Photoreceptors; Retinal Pigments; Retinaldehyde; Retinoids; Rhodopsin; Rod; Rod Outer Segments; Salts; Societies; Stress; Structure of retinal pigment epithelium; Study models; Supporting Cell; Surface; Testing; Therapeutic Agents; Time; Toxic effect; Vertebrate Photoreceptors; Veterans; Vision; Visual; Vitamins; age related; circadian; clinically relevant; density; dimer; disability; drug testing; experimental study; gene therapy; geographic atrophy; hammerhead ribozyme; innovation; intraepithelial; knock-down; lead candidate; macula; molecular targeted therapies; mouse model; nonhuman primate; novel; novel strategies; null mutation; pre-clinical; preservation; retinal rods; small hairpin RNA; stressor; success; therapeutic candidate; tool; visual cycle

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.

常见的干性老年性黄斑变性(DAMD)和少年性孤儿黄斑变性(JMD) (例如，Stargardt)细胞和生化碎片在视网膜色素上皮内和下方堆积 (RPE)。这些疾病反映了自然发生的视杆和视锥感光细胞(PR)外部的昼夜节律脱落。 节段末端，RPE细胞的吞噬和溶酶体消化。在人类的副乳房，在那里damd 而JMD开始时，单个RPE细胞支撑约30-35个视杆和更少的视锥。RPE酶 局限性会导致与年龄相关的脂褐素在吞噬酶体内积聚。脂褐素具有蛋白质、脂肪和 碳水化合物组成，并有助于在JMD/DAMD中看到的RPE内部和亚RPE沉积(斑点、玻璃样斑)。 脂褐素在蓝光激发下具有辉煌的自体荧光和光反应活性，这是由于 存在有毒的双维A酸(TBR)，如吡啶盐(A2E)和视黄醛二聚体(RetDi)。这些 来自PR外段的两个视黄醛分子的共价反应，由视觉产生 色素漂白和再生，含脂类和蛋白质。TbR色素在RPE细胞中正常积累 年龄(&gt；30岁)综合多年。任何应激源(例如，黄斑变性过程) 促进TBRs加速积聚会对RPE细胞产生毒性的凋亡效应，失去覆盖的支持 PRS和细胞死亡(例如，dAMD/JMD)。TBRs是有效的治疗分子靶点。每日成交量 视旁窝中大量的视杆状视觉色素对RPE(最新陈代谢)是一个巨大的负担 人体内的活跃细胞)，并导致细胞内和视网膜下碎片的积累；50% 正常人视紫红质(RHO)的减少没有退化作用，人们仍然可以看到星星。90%的 杆状感光器质量为Rho。Rho是一个可验证的目标。我们的假设是DAMD/JMD可能是 通过减少PR和RPE中的Rho和相关的随时间积累的碎屑和TBR进行处理 细胞。其理论基础或策略是，RHO和相关TBR的稳定减少将减缓年龄- 相关的有毒副产物积聚，减少代谢负担，并保持活着的RPE细胞更长的生命。 这将保留覆盖的视杆和视锥，保持中央视锥的视觉，并减缓或阻止 地理萎缩。长期目标是开发一种安全有效的DAMD/JMD基因治疗方法。 建议的实验目的是使用锤头状核酶(Hhrz)或rna干扰。 (ShRNA)作为基因工具敲除(KD)Rho表达，以缓解日常视网膜外应激(TBR， 代谢压力)。这一新策略在多个dAMD/JMD小鼠模型上进行了测试(ABCR-/-//RDH8-/-； ELOVL4mut，SOD2ko)，均有外周视网膜PR/RPE变性。预期的结果是 Rho减压术将挽救视网膜外部的退化，并保留明视敏感度(视锥细胞使用Müler 细胞视黄样视觉周期)，但暗视敏感度略有下降(-0.3log)，但暗视被保留 功能。具体目标是：目标1.优化新的领先候选抗Rho hhRzs和shRNAs 并为这些制剂开发稳定、无毒的细胞表达载体。目标2.第一阶段模型 视网膜下或玻璃体内rAAV后候选治疗药物(hhRz、shRNA)的毒性研究 在人性化的老鼠体内分娩，以获得正常的Rho。目的3.视网膜挽救的二期模型研究 抗Rho治疗候选药物视网膜下或玻璃体内注射后的变性 多个dAMD/JMD小鼠模型，具有不同的应激，测量内在率的变化 在视网膜光损伤范例中，视网膜退化或防止光感受器丢失的保护。 DAMD在我们老龄化的退伍军人中非常普遍，是一种视觉破坏性疾病。目前， 维生素是唯一(有限的)治疗方法。在小鼠模型上的成功救援为临床前- 新药(IND)在非人灵长类动物DAMD模型中的试验，作为迈向临床的一步 退伍军人管理局医疗系统中一种新的damd基因疗法的试验。