Evaluating GWAS AMD Candidate Loci by Gene Editing in Human iPS Cells

 描述(由申请人提供)： 老年性黄斑变性(AMD)是视网膜损伤的主要原因，在西方国家是导致失明的主要原因，顾名思义，它是一种衰老疾病。像其他与年龄相关的疾病一样，AMD的研究尤其具有挑战性，因为它需要几十年的时间才能形成，因此任何研究模型都必须概括一个较老的有机体的状况。全基因组关联研究(GWAS)和连锁分析为可能导致AMD的原因提供了第一条线索。这些研究发现了三个单核苷酸多态(SNP)，它们是AMD的强危险因素。1其中一个SNP位于CFH基因的402H等位基因，另外两个紧密连锁，位于邻近的HTRA1和ARMS2基因。这些SNPs赋予了人类遗传学研究历史上最重要的遗传风险因素。携带这些SNP纯合子的人患AMD的风险增加50倍。然而，这些突变如何导致视力恶化尚不清楚，因为AMD的潜在分子机制尚不清楚。然而，最近，我们的无偏蛋白质组分析表明，在受影响的细胞中，超氧化物歧化酶(SOD)机制受到干扰，随着时间的推移，这会引入反应性氧化物种(ROS)介导的细胞损伤，最终表现为AMD。如果AMD患者的ROS代谢确实受到干扰，那么我们可能最终开始着手解决该疾病的病因。我们相信，干细胞技术可以绕过寻找AMD病因和治疗方法所面临的障碍。为此，我们找到了一种将患者的干细胞分化为视网膜细胞的方法。此外，我们开发了一种方案，可以概括这些患者干细胞来源的视网膜细胞的衰老情况。最后，通过基因打靶技术，我们可以操纵干细胞基因组，打靶与疾病相关的SNPs，以确定每个SNPs的个体贡献。通过应用这些强大的方法，我们相信我们最终可以确定AMD的根本原因，从而开始开发新的治疗方法。我们的目标将通过两个具体目标来实现：目标1A。使用CRISPR/Cas9系统在患者来源的干细胞中将HTRA1和ARMS2等位基因从低风险转换为高风险。目标1B。确定人类HTRA1和ARMS2等位基因在AMD发病机制中的个体贡献。测试Aim 1A中低风险AMD等位基因到高危AMD等位基因的CRISPR转换是否会影响细胞内的ROS水平。目的2.在人-鼠嵌合体实验中测试患者干细胞来源的RPE的功能。

 DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is a major cause of retinal damage, the leading cause of blindness in Western countries and, as the name implies, a disease of aging. Like other age-related diseases, AMD is particularly challenging to study because it takes decades to develop and so any research model must recapitulate the conditions of an older organism. Genome-wide association studies (GWASs) and linkage analyses have provided the first clues to what might cause AMD. These studies identified three single nucleotide polymorphisms (SNPs) that are strong risk factors for AMD.1 One SNP lies in the 402H allele in the CFH gene and the two others are tightly linked and lie in the neighboring HTRA1 and ARMS2 genes. These SNPs confer the most significant genetic risk factors in the history of GWAS studies in human genetics. People homozygous for these SNPs have a 50-fold increased risk of AMD. How these mutations might cause sight to deteriorate is unclear, however, because the underlying molecular mechanisms of AMD are unknown. Recently, however, our unbiased proteome analysis suggested super oxide dismutase (SOD) mechanisms are perturbed in affected cells and that, over time, this introduces reactive oxidative species (ROS) mediated cellular insults that eventually manifest as AMD. If ROS metabolism is indeed disrupted in AMD, then we might finally begin to address the causes of the disease. We believe the hurdles faced in finding causes and treatments for AMD could be circumvented by stem cell technologies. To this end we have found a way to differentiate stem cells from patients into retinal cells. Moreover, we developed a protocol that recapitulates aging in these patient-stem-cell-derived retinal cells. Finally, through gene-targeting technology, we can manipulate the stem cell genome, targeting disease- associated SNPs, to determine the individual contributions of each. By applying these powerful methodologies, we believe we can finally identify the root causes of AMD and so begin to develop new therapies. Our goals will be accomplished in two specific aims: Aim 1A. Use the CRISPR/Cas9 system to convert HTRA1 and ARMS2 alleles from low-risk to high risk in patient-derived stem cells. Aim 1B. Determine the individual contribution of human HTRA1 and ARMS2 alleles to AMD pathogenesis. Test whether CRISPR conversion from low to high-risk AMD alleles in Aim 1A affects ROS levels in cells. Aim 2. Test the function of patient-stem-cell-derived RPE in a human-mouse chimera, in vivo assay.