Disease mechanisms of cone opsin mutants and treatment strategies

视锥细胞突变体的致病机制及治疗策略

• 批准号: 10228662
• 负责人: Wen-Tao Deng
• 金额: $ 36.45万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10228662
• 关键词: Affect; Biochemical; Biogenesis; CRISPR/Cas technology; Categories; Cell Death; Color; Color Visions; Complementary DNA; Cone; Cone dystrophy; Defect; Dimerization; Disease; Dominant-Negative Mutation; Dorsal; Endoplasmic Reticulum; Foundations; Functional disorder; Future; Genes; Goals; Human; In Vitro; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Lead; Light; Link; Maintenance; Mediating; Membrane; Messenger RNA; Modeling; Molecular; Mus; Mutation; Myopia; Natural regeneration; Opsin; Outcome; Pathogenicity; Patients; Phenotype; Photoreceptors; Phototransduction; Physiological; Play; Point Mutation; Population; Property; Proteins; Research; Resistance; Resolution; Retina; Retinal Cone; Retinal Diseases; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod; Role; Signal Transduction; Small Interfering RNA; Solid; Structure; Supplementation; Technology; Testing; Therapeutic; Transgenic Mice; Vertebrate Photoreceptors; Vision Disorders; Visual; Work; adeno-associated viral vector; blue cone monochromacy; dimer; effective therapy; functional loss; gene replacement therapy; gene therapy; improved; in vivo; in vivo Model; innovation; macula; mutant; rational design; retinal rods; spatial vision; success; treatment strategy

ABSTRACT L- and M- cones constitute about 95% of the total cone population, primarily concentrated in the macula, they are responsible for our daylight, central high resolution, and color vision. Mutations in the L-opsin and M-opsin genes are associated with a variety of visual defects including red-green color vision deficiency, blue cone monochromacy (BCM), X-linked cone dystrophy/dysfunction, and high myopia with abnormal cone function. Currently studies on disease mechanisms of cone opsin mutations have been mostly carried out in vitro, therefore the impact of these mutations on cone structure and their physiological consequences are not well understood. Recent studies suggest that rhodopsin dimerization plays a central role in signal transduction and that defects in dimerization are one molecular mechanism associated with some forms of rhodopsin-related autosomal dominant retinitis pigmentosa. Relative to rhodopsin, studies of cone opsin organization in outer segment membranes have been lagging primarily because cones are less abundant than rods thus hampering a detailed structural analysis. Our goals are to elucidate the molecular mechanisms underlying cone opsin mutations in vivo to develop effective treatment approaches, and to understand the organization of cone opsins in outer segment membranes and pathophysiology associated with cone opsin dimerization disruption. Our prior studies have demonstrated that AAV-mediated expression of human L-opsin and M-opsin promotes regrowth of cone outer segments and rescues M-cone function in the treated M-opsin knockout (Opn1mw-/- ) mouse, a model for BCM. One critical observation from our work is that cone opsins are required for outer segment formation, but not for cone viability. These results lead us to propose the use of the Opn1mw-/- mice as an in vivo model to investigate disease mechanisms associated with cone opsin mutants via our well- developed AAV-mediated cone targeting approach (Aim 1). Our preliminary results using this approach indicate that the cone opsin C203R mutation, responsible for more than half of the BCM population, displays a dominant-negative phenotype. We have generated a knock-in mouse line carrying this mutation and will test gene therapy options (Aim 2). The success of these strategies can be employed to treat other cone opsin mutations displaying dominant-negative phenotypes. We will also employ a combination of AAV technology, biochemical approaches, and transgenic mice to define domains involved in cone opsin dimerization and characterize the pathophysiology associated with dimerization disruption (Aim 3). Completing these goals will provide us a solid foundation for developing effective strategies to treat different categories of retinal disease caused by cone opsin mutations. This study will also improve our knowledge of the roles cone opsins play in outer segment disc membrane formation and maintenance.

摘要 L-和M-视锥细胞约占视锥细胞总数的95%，主要集中在黄斑区， 负责我们的日光，中央高分辨率和彩色视觉。L-视蛋白和M-视蛋白的突变 基因与多种视觉缺陷相关，包括红绿色觉缺陷、蓝锥 单色性（单眼）、X连锁视锥细胞营养不良/功能障碍和具有异常视锥细胞功能的高度近视。 目前关于视锥蛋白突变的疾病机制的研究大多在体外进行， 因此，这些突变对锥体结构的影响及其生理后果尚不清楚。 明白最近的研究表明，视紫红质二聚化在信号转导中起着核心作用， 二聚化缺陷是与某些形式的视紫红质相关的分子机制之一， 常染色体显性视网膜色素变性相对于视紫红质，外膜视锥蛋白结构的研究 节膜已经落后，主要是因为锥比杆少，从而阻碍 详细的结构分析。我们的目标是阐明视锥细胞视蛋白的分子机制 体内突变，以开发有效的治疗方法，并了解锥细胞的组织 外节膜中的视蛋白和与视锥视蛋白二聚化破坏相关的病理生理学。 我们先前的研究已经证明，AAV介导的人L-视蛋白和M-视蛋白的表达促进了人视蛋白的表达。 视锥细胞外节的再生长，并在经治疗的M视蛋白敲除（Opn 1 mw-/-）中挽救M视锥细胞功能 小鼠，一种动物模型。我们工作中的一个关键观察是，外视锥细胞需要视蛋白。 节的形成，但不是锥的活力。这些结果使我们提出使用Opn 1 mw-/-小鼠 作为体内模型，通过我们的良好- 开发了AAV介导的视锥靶向方法（Aim 1）。我们使用这种方法的初步结果 表明，锥视蛋白C203 R突变，负责超过一半的人群，显示 显性阴性表型。我们已经产生了一个携带这种突变的敲入小鼠品系， 基因治疗选择（目标2）。这些策略的成功可用于治疗其他视锥细胞视蛋白 显示显性阴性表型的突变。我们还将采用AAV技术， 生物化学方法和转基因小鼠来定义参与视锥视蛋白二聚化的结构域， 表征与二聚化破坏相关的病理生理学（目的3）。 完成这些目标将为我们制定有效的战略， 视锥细胞视蛋白突变引起的视网膜疾病的分类。这项研究还将提高我们对以下方面的认识： 视锥细胞视蛋白在外节盘膜形成和维持中的作用。