Complement in AMD: Mechanisms and Therapeutic Intervention

描述（由申请人提供）：本项目的长期目标是阐明补体在年龄相关性黄斑变性（AMD）发病机制中的作用，并确定预防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，但我们对潜在分子过程的了解和治疗选择的可用性仍然很有限。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，几项基因分型研究已经确定C3、因子B和因子H的基因多态性是这种疾病发展的重要风险因素。然而，这些遗传发现的功能意义仍然未知，需要转化为疾病模型。为了从蛋白质水平阐明补体在AMD中的作用，我们提出了两个具体的目标。在目的1中，我们通过在哺乳动物细胞中表达补体蛋白及其AMD相关的同种异型物并通过从AMD患者和健康个体的血浆中分离它们来进行补体蛋白及其AMD相关的同种异型物的全面深入分析。将通过质谱法测定序列修饰，并将在一组成熟的生物物理和生物化学测定中系统地检测所有单个蛋白质及其各种组合的直接结合和功能活性。最后，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将通过将我们的发现与可用的晶体结构、基于溶液的结构分析和计算模型相关联来分析。AMD靶向药物的开发和测试通常受到疾病相关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，我们将利用黄斑变性的临床相关猴模型来测试补体抑制剂对疾病进展的影响。将玻璃体内注射肽抑制剂坎普他汀，并评价其对玻璃疣形成的影响。此外，将探索作为玻璃体内凝胶形成的结果的眼睛中的持续药物释放。系统评估AMD相关补体修饰的功能后果，并在疾病相关动物模型中测试补体抑制，将有助于我们了解AMD，并有助于开发更有效的治疗方法。 公共卫生相关性：本研究的目的是描述补体激活和抑制对年龄相关性黄斑变性发病机制的贡献。这将通过系统评价疾病相关多态性对补体成分的结构、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模型中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.