Epigenetic-guided studies of AMD pathology and iPSC-RPE transplantation therapy

AMD 病理学和 iPSC-RPE 移植治疗的表观遗传学引导研究

• 批准号: 9892385
• 负责人: Michael Farkas
• 金额: --
• 依托单位: VA WESTERN NEW YORK HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-10-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9892385
• 关键词: Address; Affect; Age; Age related macular degeneration; Aging; American; Animals; Area; Automobile Driving; Autopsy; Back; Basic Science; Biology; Blindness; Blood; CRISPR/Cas technology; Caring; Cell Cycle; Cell Lineage; Cells; Choroidal Neovascularization; Clinical; Clinical Trials; Code; Collaborations; Complex; DNA; DNA Methylation; Data; Diet; Disease; Early treatment; Elderly; Environmental Risk Factor; Enzymes; Epigenetic Process; Etiology; Exhibits; Eye; Functional disorder; Gene Expression; General Population; Genes; Genetic; Genetic Transcription; Geography; Goals; Hour; Human; Immune response; In Vitro; Individual; Lead; Mediating; Memory; Methodology; Methods; Methylation; Nerve Degeneration; Neurodegenerative Disorders; Nonexudative age-related macular degeneration; Outcome; Oxidative Stress; Parents; Pathogenesis; Pathology; Patients; Phenotype; Pigments; Predisposition; Process; Proteins; Regenerative Medicine; Replacement Therapy; Repression; Research; Retina; Retinal Degeneration; Risk; Risk Factors; Role; Sampling; Smoking; Societies; Stimulus; Structure of retinal pigment epithelium; Testing; Therapeutic; Thymine DNA Glycosylase; Time; Tissues; Transplantation; Variant; Veterans; Vision; base; biological adaptation to stress; case control; cell type; clinically relevant; cost; demethylation; differential expression; disorder risk; effective therapy; epigenetic memory; epigenome; functional improvement; gene therapy; genome wide association study; genome-wide; geographic atrophy; induced pluripotent stem cell; knock-down; methylation pattern; monolayer; novel strategies; response; restoration; retinal progenitor cell; success; therapeutic target; transcriptome sequencing; treatment strategy; whole genome

Age-related macular degeneration (AMD) affects nearly 5% of aging veterans, and millions of civilian Americans. AMD is caused by genetic and environmental factors, which lead to central vision loss initially, and can lead to rapid degeneration of remaining vision in a few years. While this is a prevalent disease with costs to both the affected individuals and society as a whole, few treatments, and no cures, currently exist. Contributing to the lack of treatments for AMD, is the fact the underlying genetic factors resulting in pathogenesis remain unsolved. While variants in, or around, 34 genes have been identified as risk factors for disease, none have been demonstrated to be a causative agent. Epigenetics is an area yet to be fully explored in AMD pathogenesis. In collaboration with Dr. Margaret DeAngelis, we have performed DNA methylation studies on RPE from 140 dry AMD donors, identifying over 400 differentially methylated regions. Combining this data with RNA-Seq data from iPSC-RPE under oxidative stress, we have identified 81 genes to be both differentially methylated and differentially expressed. At the top of this list is Thymine DNA Glycosylase, an enzyme that performs the last step of DNA demethylation. We hypothesize that TDG repression results in perturbation of the natural methylation/demethylation cycle that the cell uses to regulate gene expression in response to environmental stimuli, such as oxidative stress. In Aim 1, we will test this hypothesis using CRISPR/Cas9-based knockdown of TDG in induced pluripotent stem cell-derived RPE (iPSC-RPE) under normal and oxidative stress conditions. A second contributing factor to the lack of treatments for AMD is the limited progress observed in clinical trials using cell replacement therapy, a form of regenerative medicine. One hurdle to overcome in regenerative medicine for the eye is the difficulty in producing retinal tissues with high similarity to native tissue. The retinal pigment epithelium (RPE) is a pigmented monolayer at the back of the eye, which has the most regenerative medicine potential, since it can be readily derived from patient-specific iPSCs. Multiple animal and human studies using these iPSC-RPE cells, however, fail to produce lasting results, as the cells either die, produce an immune response, or yield no functional improvement, likely because these iPSC-RPE are only RPE-like and not exact replicas of native RPE. Our group, and others, have shown significant differences in the transcriptional landscape of iPSC-RPE, relative to native RPE. Many of the protein-coding genes that define the RPE are expressed, but at significantly lower levels in iPSC-RPE when compared to native RPE. It has been well documented that reprogramming somatic tissue to iPSCs removes most of the epigenetic memory, but some remains. This memory is passed along during the differentiation process to the target cell type, and can promote dedifferentiation to the original cell type. The exact epigenetic landscape of these cells, however, has yet to be defined. We hypothesize that a retained epigenetic memory in iPSC-RPE is driven, in part, by continued expression of genes that define the parent cell lineage. In Aim 2, we will test this hypothesis by collecting human post-mortem blood and native RPE. The blood will be reprogrammed to iPSC, which will subsequently be differentiated to RPE. From these samples, we will characterize the epigenome to identify the epigenetic marks that are retained from blood (parent cell) to iPSCs to iPSC-RPE (the epigenetic memory) and compare this to the epigenome of native RPE from the same individual. The outcome of these two aims will lead to a better understanding of AMD pathology and iPSC-RPE biology that will have a direct impact on treatment strategies for veterans.

视网膜相关性黄斑变性（AMD）影响近5%的老年退伍军人和数百万美国平民。 AMD是由遗传和环境因素引起的，最初导致中心视力丧失，并可导致 在几年内迅速退化的剩余视力。虽然这是一种流行病， 对于受影响的个人和整个社会，目前几乎没有治疗方法，也没有治愈方法。有助于 缺乏AMD的治疗，是导致发病机制的潜在遗传因素仍未解决的事实。 虽然34个基因中或周围的变异已被确定为疾病的危险因素，但没有一个是 被证明是一种病原体。表观遗传学是AMD发病机制中尚未充分探索的领域。在 与Margaret DeAngelis博士合作，我们对140个干细胞的RPE进行了DNA甲基化研究。 AMD供体，鉴定超过400个差异甲基化区域。将这些数据与来自 在氧化应激下的iPSC-RPE中，我们已经鉴定了81个基因是差异甲基化的， 差异表达。在这个列表的顶部是胸腺嘧啶DNA糖基化酶，一种酶，执行最后一个 DNA去甲基化步骤。我们假设TDG抑制导致了对正常细胞的干扰， 甲基化/去甲基化周期，细胞用来调节基因表达，以响应环境 刺激，如氧化应激。在目标1中，我们将使用基于CRISPR/Cas9的敲除来测试这一假设。 正常和氧化应激条件下诱导多能干细胞衍生的RPE（iPSC-RPE）中的TDG。一 AMD缺乏治疗的第二个因素是临床试验中观察到的进展有限 使用细胞替代疗法，一种再生医学。再生能源领域需要克服的一个障碍 用于眼睛的药物的困难在于难以产生与天然组织高度相似的视网膜组织。视网膜 色素上皮（RPE）是位于眼睛后部的色素单层，其具有最多的再生能力。 这是因为它可以很容易地从患者特异性iPSC中获得。多项动物和人体研究 然而，使用这些iPSC-RPE细胞并不能产生持久的效果，因为细胞要么死亡，要么产生免疫反应， 这可能是因为这些iPSC-RPE只是RPE样的，而不是精确的 原生RPE的复制品。我们的研究小组和其他研究小组在转录水平上表现出了显著的差异 iPSC-RPE相对于天然RPE的。许多定义RPE的蛋白质编码基因表达，但 与天然RPE相比，iPSC-RPE中的水平显著较低。有充分的证据表明， 将体细胞组织重新编程为iPSC会消除大部分表观遗传记忆，但仍有一些保留下来。这 记忆在向靶细胞类型分化的过程中沿着传递，并且可以促进 去分化为原始细胞类型。然而，这些细胞的确切表观遗传景观尚未被发现。 定义了我们假设iPSC-RPE中保留的表观遗传记忆部分是由持续的 定义亲本细胞谱系的基因的表达。在目标2中，我们将通过收集人类样本来验证这一假设。 尸体血液和自体视网膜色素上皮血液将被重新编程为iPSC，随后将 分化为RPE。从这些样本中，我们将表征表观基因组，以确定表观遗传标记 从血液（亲本细胞）到iPSC再到iPSC-RPE（表观遗传记忆），并将其与 来自同一个体的天然RPE的表观基因组。这两个目标的结果将导致一个更好的 了解AMD病理学和iPSC-RPE生物学，这将直接影响治疗策略 为退伍军人。