iPSC lines for modeling age-related macular degeneration

描述（由申请人提供）：本研究的目标是创建从独特来源-成人视网膜色素上皮（RPE）-重新编程的iPSC集合，并使用其创建干性年龄相关性黄斑变性（AMD）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，影响超过1100万患者，黄斑（视网膜中心）视力丧失，需要高敏锐度视力（阅读、书写、识别面部）。AMD没有改变疾病的疗法。干细胞衍生的RPE眼内移植方法正在临床试验中，但这可能不是许多老年患者的选择。更好的 对疾病过程的了解可能会为这种高度流行和破坏性的疾病带来新的治疗选择。 干性AMD通过没有黄斑的鼠物种或通过不表达人RPE的关键表型特征的转化细胞系如ARPE-19不完全建模。人iPSC可以产生具有天然RPE的显著特性的细胞。我们提出iPSC衍生的RPE可以形成有价值的干性AMD模型的基础。几项GWAS研究已经证明了AMD的基因连锁，使得筛选这些SNP的iPSC系以获得已知遗传风险的概况成为可能。此外，许多环境因素已被证明会增加干性AMD的风险，包括广泛认可的因素，慢性氧化应激。黄斑RPE暴露于异常水平的氧化应激，这是由于聚焦光和含有非常高水平的氧化产物的视锥光感受器外节的昼夜吞噬作用。这种压力导致RPE细胞内的显著变化，导致吞噬和代谢效率低下，并且在某些个体中，导致物质积聚成沉积在RPE下的大赘生物，引起炎症反应和进一步损伤-AMD的标志是这些“玻璃疣”沉积物。因此，数十年的慢性应激导致RPE的改变，为从这种疾病靶组织本身产生iPSC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患者的眼睛中产生RPE细胞，并从这些细胞中产生iPSC细胞系，这些细胞系将被记录为AMD相关的SNP。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细胞。这一目标将与NEI的RPE表征专家谢尔顿米勒合作进行。在目标3中，我们将确定这些不同的细胞系如何在我们为原发性人类RPE开发的新型干性AMD模型中响应慢性氧化应激，该模型测量几种玻璃疣蛋白的产生。因此，我们将为干性AMD创建一个有价值的基于iPSC-RPE的模型。通过比较来自几个个体的年轻、正常老年和AMD RPE-iPSC-RPE的反应，我们将揭示与正常老化和黄斑疾病相关的变化。这些CNS衍生的iPSC可能对建模其他CNS衰老疾病有价值。 公共卫生相关性：黄斑变性是老年人失明的主要原因，仅在美国就有超过1100万患者，并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品。我们建议使用新的人类诱导多能干细胞（iPSC）技术来创建年轻，老年和AMD人类iPSC系的集合，并使用这些来开发AMD的新模型，以加速治疗进展。这种“盘中疾病”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衰老而衰退的分子途径，以及AMD（一种流行的、破坏性的年龄相关神经退行性疾病）的基础。

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.