Complement in AMD: Mechanisms and Therapeutic Intervention

描述(申请人提供)：该项目的长期目标是阐明补体在老年性黄斑变性(AMD)发病机制中的作用，并确定可防止疾病进展的补体抑制剂。虽然AMD已经成为老年人失明的主要原因，但我们对潜在的分子过程和治疗方案的可用性的了解在很大程度上仍然有限。越来越多的证据表明补体在AMD的进展中起着至关重要的作用，一些基因分型研究发现C3、B因子和H因子基因的多态性是AMD发生的重要危险因素。然而，这些基因发现的功能意义仍然未知，需要转化为疾病模型。为了在蛋白质水平上阐明补体在AMD中的作用，我们提出了两个特定的目标。在目标1中，我们通过在哺乳动物细胞中表达补体蛋白及其与AMD相关的同种异型，并从AMD患者和健康人的血浆中分离它们，对补体蛋白及其相关同种异型进行了全面深入的分析。序列修改将通过质谱学确定，所有单个蛋白质及其各种组合的直接结合和功能活性将在一系列成熟的生物物理和生化分析中进行系统测试。最后，通过将我们的发现与现有的晶体结构、基于溶液的结构分析和计算模型相关联，分析这些蛋白质修饰对所涉及的补体成分的结构和接触界面的影响。AMD靶向药物的开发和测试经常受到与疾病相关的动物模型的限制和不利的药代动力学特征的阻碍。在目标2中，我们将利用临床相关的黄斑变性猴子模型来测试补体抑制剂对疾病进展的影响。多肽抑制剂Compstatin将被注射到玻璃体内，并将评估其对玻璃体形成的影响。此外，还将探索由于玻璃体内凝胶形成而导致的药物在眼内的持续释放。系统地评估AMD相关补体修饰的功能后果，并在与疾病相关的动物模型中测试补体抑制将有助于我们理解AMD，并有助于开发更有效的治疗方法。 公共卫生相关性：我们研究的目的是描述补体激活和抑制在老年性黄斑变性发病机制中的作用。这将通过系统地评估与疾病相关的多态对补体成分的结构、结合和功能的影响，以及通过评估补体抑制剂对黄斑变性猴子模型疾病进展的影响来实现。因此，这些研究可能会提高我们描述和治疗这种疾病的能力。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the role of complement in the pathogenesis of age-related macular degeneration (AMD), and to identify complement inhibitors that prevent disease progression. Though AMD has emerged as the predominant cause of blindness in elderly people, our knowledge of the underlying molecular processes and the availability of treatment options remain largely limited. Accumulating evidence suggests a crucial role for complement in the progression of AMD, and several genotyping studies have identified polymorphisms in the genes for C3, factor B, and factor H as important risk factors for development of this disease. However, the functional significance of these genetic findings remains unknown and need to be translated into a disease model. To elucidate the functions of complement in AMD on the protein level, we propose two specific aims. In Aim 1 we perform a comprehensive in-depth analysis of complement proteins and their AMD- associated alloforms, by expressing them in mammalian cells and by isolating them from the plasma of AMD patients and healthy individuals. Sequence modifications will be determined by mass spectrometry, and the direct binding and functional activities of all individual proteins and their various combinations will be systematically tested in a panel of well-established biophysical and biochemical assays. Finally, the effect of these protein modifications on the structure and contact interface of the involved complement components will be analyzed by correlating our findings with available crystal structures, solution-based structural analysis, and computational models. The development and testing of AMD-targeting drugs is often hampered by restricted access to disease-relevant animal models and unfavorable pharmacokinetic profiles. In Aim 2, we will utilize a clinically relevant monkey model of macular degeneration for testing the effect of complement inhibitors on disease progression. The peptidic inhibitor compstatin will be injected intravitreally and its effect on drusen formation will be evaluated. Furthermore, a sustained drug release in the eye as a result of intravitreal gel formation will be explored. The systematic assessment of the functional consequences for AMD-associated complement modifications and the testing of complement inhibition in a disease-relevant animal model will contribute to our understanding of AMD, as well as contribute to the development of more effective treatments. PUBLIC HEALTH RELEVANCE: The purpose of our study is to describe the contributions of complement activation and inhibition to the pathogenesis of age-related macular degeneration. This will be accomplished by systematically evaluating the effect of disease-related polymorphisms on the structure, binding, and function of complement components, and by assessing the impact of complement inhibitors on disease progression in a monkey model of macular degeneration. Thus, these studies will likely improve our capacity for describing and treating the disease.