iPSC lines for modeling age-related macular degeneration

描述(申请人提供)：这项研究的目标是创建一组从独特来源-成人视网膜色素上皮(RPE)-重新编程的IPSCs，并使用它来创建干性老年性黄斑变性(AMD)的模型，AMD是最常见的衰老神经退行性疾病之一。AMD是50岁以上患者失明的主要原因，影响着1100多万名患者，黄斑是视网膜的中心，需要高视力的视力(阅读、书写、识别人脸)。AMD目前还没有改变疾病的治疗方法。干细胞来源的RPE眼内移植方法正在进行临床试验，但对于许多老年患者来说，这可能不是一种选择。更好的 对疾病过程的了解可能会为这种高度流行和毁灭性的疾病带来新的治疗选择。干性AMD由没有斑点的小鼠物种或不表达人类RPE关键表型特征的转化细胞系(如ARPE-19)模拟得不完美。人的IPSCs可以产生具有天然RPE显著特性的细胞。我们认为IPSC来源的RPE可以构成干性AMD模型的基础。几项GWAS研究已经证明了AMD的基因联系，使筛选这些SNP的IPSC株成为可能，以获得已知遗传风险的概况。此外，许多环境因素已被证明增加了干性AMD的风险，包括公认的因素，慢性氧化应激。由于聚光和视锥感光细胞外节的吞噬作用，黄斑RPE暴露在极高水平的氧化应激下，而视锥感光细胞外节含有非常高水平的氧化产物。这种压力会导致RPE细胞内的显著变化，导致吞噬和新陈代谢效率低下，在某些人中，会导致物质积聚成大量多余的物质，沉积在RPE下，导致炎症反应和进一步的损害-AMD的标志是这些“玻璃样变”沉积。因此，数十年的慢性应激导致RPE的变化，为从这种疾病的靶组织本身生产IPSCs提供了强有力的理由。在目标1中，我们将从胎儿、正常老年人和AMD患者的眼睛中培养RPE细胞，并从这些细胞中产生将被记录为AMD相关SNP的IPSC株。在目标2中，我们将这些细胞分化为RPE，并通过全面的表型和功能分析来表征这些细胞。这一目标将与NEI的谢尔登·米勒合作实现，谢尔顿·米勒是RPE表征方面的专家。在目标3中，我们将在我们为初级人类RPE开发的干性AMD新模型中，确定这些不同的品系如何应对慢性氧化应激，该模型测量了几种玻璃体蛋白的产生。因此，我们将为干性AMD创建一个有价值的IPSC-RPE模型。通过比较年轻人、正常老年人和几个个体的AMD RPE-IPSC-RPE的反应，我们将揭示与正常衰老和黄斑病相关的变化。这些中枢神经系统来源的IPSCs可能对其他中枢神经系统衰老疾病的建模有价值。 公共卫生相关性：老年性黄斑变性是老年人失明的主要原因，仅在美国就有1100多万患者，而且没有有效的治疗方法。动物模型没有正确的视网膜解剖来模拟这种疾病，因此正在积极寻找替代方案。我们建议使用新的人类诱导多能干细胞(IPSC)技术来创建一组年轻、老年和AMD人类IPSC系，并利用这些细胞系开发一种新的AMD模型，以加速治疗进展。这种“盘子里的疾病”模型在整个过程中使用了相关的人类视网膜细胞，因此可以帮助揭示随着年龄的增长而下降的分子途径，以及AMD的基础，AMD是一种普遍的、毁灭性的与年龄相关的神经退行性疾病。

DESCRIPTION (provided by applicant): The goal of this research is to create a collection of iPSCs reprogrammed from a unique source - the adult human retinal pigment epithelium (RPE) - and use this to create a model of dry age-related macular degeneration (AMD), one of the most prevalent neurodegenerative diseases of aging. AMD is the leading cause of blindness in patients over age 50 and affects over 11 million patients, with vision loss in the macula, the center of the retina, needed for high acuity vision (reading, writing, recognizing faces). There ar no disease-altering therapies for AMD. A stem cell-derived RPE intraocular transplantation approach is in clinical trials, but this might not be an option for many elderly patients. A better understanding of the disease process could lead to new therapeutic options for this highly prevalent and devastating condition. Dry AMD is imperfectly modeled by murine species, which do not have a macula, or by transformed cell lines such as ARPE-19 that do not express key phenotypic features of human RPE. Human iPSCs can produce cells with the salient properties of native RPE. We propose that iPSC-derived RPE can form the basis of a valuable model of dry AMD. Several GWAS studies have demonstrated gene linkages for AMD, making it possible to screen iPSC lines for these SNPs to obtain a profile of known genetic risk. In addition a number of environmental factors have been shown to increase the risk of dry AMD, including the widely acknowledged factor, chronic oxidative stress. The macula RPE is exposed to an extraordinary level of oxidative stress due to focused light and to the diurnal phagocytosis of cone photoreceptor outer segments which contain very high levels of oxidized products. This stress causes significant changes within the RPE cells, resulting in inefficient phagocytosis and metabolism, and in some individuals, to the build-up of material into large excrescences that are deposited under the RPE causing inflammatory reactions and further damage - the hallmark of AMD is these 'drusen' deposits. Consequently, decades of chronic stress lead to alterations in the RPE, providing a strong rationale for producing iPSCs from this disease target tissue itself. In aim 1 we will produce RPE cells from fetal, normal aged and AMD patient eyes, and from these generate iPSC lines that will be documented for AMD-related SNPs. In aim 2 we will differentiate these cells into RPE and characterize the cells with a comprehensive phenotypic and functional analysis. This aim will be carried out in collaboration with Sheldon Miller at NEI, an expert in RPE characterization. In aim 3, we will determine how these different lines respond to chronic oxidative stress in a novel model for dry AMD we developed for primary human RPE that measures production of several drusen proteins. Thus we will create a valuable iPSC-RPE based model for dry AMD. By comparing the responses of young, normal aged and AMD RPE-iPSC-RPE from several individuals, we will reveal changes associated with normal aging and macula disease. These CNS-derived iPSCs could be valuable for modeling other CNS aging diseases. PUBLIC HEALTH RELEVANCE: Age-related macular degeneration is the leading cause of blindness in the elderly with over 11 million sufferers in the US alone and no effective treatments. Animal models do not have the correct retinal anatomy to model this disease, so alternatives are actively being sought. We propose to use the new human induced pluripotent stem cell (iPSC) technology to create a collection of young, old and AMD human iPSC lines, and use these to develop a novel model for AMD to accelerate progress towards therapeutics. This 'disease in a dish' model uses the relevant human retinal cells throughout the process, and therefore can help reveal the molecular pathways that decline with aging and that underlie AMD, a prevalent, devastating age-related neurodegenerative disease.