Disease mechanisms of cone opsin mutants and treatment strategies

摘要 L视锥和M视锥约占视锥总种群的95%，主要集中在黄斑，它们 负责我们的日光、中央高分辨率和色觉。L视蛋白和M视蛋白的突变 基因与多种视觉缺陷有关，包括红绿视觉缺陷、蓝色视锥 单色性(BCM)，X连锁视锥细胞营养不良/功能障碍，高度近视视锥细胞功能异常。 目前对视锥细胞视蛋白突变致病机制的研究大多在体外进行， 因此，这些突变对锥体结构及其生理后果的影响并不是很好。 明白了。最近的研究表明，视紫红质二聚体在信号转导和 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 常染色体显性遗传性视网膜色素变性。相对于视紫红质，视锥细胞视蛋白在外周组织的研究 节段膜一直滞后，主要是因为锥体没有杆状细胞丰富，因此阻碍了 详细的结构分析。我们的目标是阐明视锥细胞视功能的分子机制 体内突变以开发有效的治疗方法，并了解锥体的组织 视锥细胞视蛋白二聚体破坏相关的外节膜视蛋白和病理生理学。 我们先前的研究表明，腺病毒介导的人L视蛋白和M-视蛋白的表达促进 经治疗的M-opsin基因敲除(Opn1mw-/-)中视锥外段的再生和挽救M-视锥功能 老鼠，BCM的模型。我们工作中的一个关键观察结果是，外部需要锥形光学元件 裂片形成，但不是为了圆锥体的生存能力。这些结果导致我们建议使用Opn1mw-/-小鼠 作为一个体内模型来研究与视锥蛋白突变体相关的疾病机制 发展了AAV介导的视锥靶向方法(AIM 1)。我们用这种方法得到的初步结果 表明锥体视蛋白C203R突变导致了超过一半的BCM群体，显示出 显性-负性表型。我们已经产生了一种携带这种突变的敲入鼠系，并将测试 基因治疗选择(目标2)。这些策略的成功也可用于治疗其他圆锥视盘。 突变表现为显性-负性表型。我们还将采用AAV技术的组合， 生化方法和转基因小鼠定义锥体视蛋白二聚化和 描述与二聚化破坏相关的病理生理学(目标3)。 完成这些目标将为我们制定有效的战略来对待不同的 视锥视蛋白突变引起的视网膜疾病的类别。这项研究也将提高我们对 视锥蛋白在外段视盘膜的形成和维持中的作用。

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.