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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的 L锥和M锥构成约95％的总锥形人群，主要集中在黄斑中，它们负责我们的日光，中央高分辨率和色觉。 L-粘蛋白和M-元素中的突变基因与各种视觉缺陷有关，包括红绿色色觉缺乏症，蓝色锥单色（BCM），X连锁的锥体营养不良/功能障碍和具有异常锥功能的高近视。目前，关于锥体蛋白酶突变疾病机制的研究主要是在体外进行的，因此，这些突变对锥体结构及其生理后果的影响不太好理解。最近的研究表明，视紫红蛋白二聚化在信号转导和二聚化缺陷是一种与某些形式的视紫红质相关的分子机制常染色体显性视网膜炎色素。相对于视紫红质，外部的锥蛋白OPSIN组织的研究片段膜一直滞后，主要是因为锥体比杆不那么丰富，从而阻碍了棒详细的结构分析。我们的目标是阐明锥蛋白的分子机制体内突变以开发有效的治疗方法，并了解锥体的组织外部段膜中的蛋白和与锥蛋白二聚化破坏相关的病理生理学。我们先前的研究表明，AAV介导的人L-粘蛋白和M-粘蛋白的表达促进锥外段的再生和拯救M-CONE功能在处理过的M-oppin敲除（OPN1MW - / - ）中鼠标，BCM的模型。我们工作中的一个关键观察是，外部需要锥蛋白段形成，但不是用于锥生存力。这些结果使我们提出了OPN1MW - / - 鼠标的使用作为一个体内模型，用于研究与锥蛋白的疾病机制通过我们的良好开发了AAV介导的锥体靶向方法（AIM 1）。我们使用这种方法的初步结果表明锥形Opsin C203R突变，负责超过一半的BCM人群主要阴性表型。我们已经产生了一条载有此突变的敲门鼠标线，并将测试基因治疗选择（AIM 2）。这些策略的成功可以用于治疗其他锥体Opsin 表现出显性阴性表型的突变。我们还将采用AAV技术的组合，生化方法和转基因小鼠，以定义锥体opsin二聚化和表征与二聚化破坏相关的病理生理学（AIM 3）。完成这些目标将为我们提供稳固的基础，以制定有效的策略来治疗不同锥蛋白突变引起的视网膜疾病类别。这项研究还将提高我们对角色锥蛋白在外部片段圆盘膜的形成和维护中发挥作用。