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

 描述（由申请人提供）： 视网膜相关性黄斑变性（AMD）是视网膜损伤的主要原因，是西方国家失明的主要原因，顾名思义，是一种衰老疾病。与其他与年龄相关的疾病一样，AMD的研究特别具有挑战性，因为它需要数十年的时间才能发展，因此任何研究模型都必须概括老年生物体的状况。 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。这些研究确定了三个单核苷酸多态性（SNP）是AMD的强风险因素。1一个SNP位于CFH基因的402 H等位基因中，另外两个SNP紧密连锁，位于相邻的HTRA 1和ARMS 2基因中。这些SNP赋予人类遗传学GWAS研究历史上最重要的遗传风险因素。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，超氧化物歧化酶（SOD）机制在受影响的细胞中受到干扰，随着时间的推移，这会引入反应性氧化物质（ROS）介导的细胞损伤，最终表现为AMD。如果活性氧代谢确实在AMD中被破坏，那么我们可能最终开始解决疾病的原因。 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，我们已经找到了一种方法，将患者的干细胞分化为视网膜细胞。此外，我们开发了一种方案，概括了这些患者干细胞衍生的视网膜细胞的衰老。最后，通过基因靶向技术，我们可以操纵干细胞基因组，靶向疾病相关的SNP，以确定每个SNP的个体贡献。通过应用这些强大的方法，我们相信我们可以最终确定AMD的根本原因，并因此开始开发新的疗法。 我们的目标将在两个具体目标中实现：目标1A。使用CRISPR/Cas9系统将HTRA 1和ARMS 2等位基因从患者来源的干细胞中的低风险转化为高风险。目标1B。确定人类HTRA 1和ARMS 2等位基因对AMD发病机制的个体贡献。测试Aim 1A中从低风险AMD等位基因到高风险AMD等位基因的CRISPR转换是否影响细胞中的ROS水平。目标2.在人-小鼠嵌合体中测试患者干细胞衍生的RPE的功能，体内测定。

 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.