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.

年龄相关性黄斑变性 (AMD) 影响着近 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，最初会导致中心视力丧失，并可能导致 几年内剩余视力迅速退化。虽然这是一种普遍存在的疾病，给双方造成了损失 影响个人和整个社会，目前治疗方法很少，也没有治愈方法。为 AMD缺乏治疗方法，导致发病机制的潜在遗传因素仍未解决。 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，但没有一个基因被确定为疾病的危险因素。 已被证明是致病因素。表观遗传学是 AMD 发病机制中尚未得到充分探索的领域。在 我们与 Margaret DeAngelis 博士合作，对 140 个干细胞的 RPE 进行了 DNA 甲基化研究 AMD 供体，识别了 400 多个差异甲基化区域。将此数据与 RNA-Seq 数据相结合 iPSC-RPE 在氧化应激下，我们已经鉴定出 81 个基因被差异甲基化和 差异表达。在此列表的顶部是胸腺嘧啶 DNA 糖基化酶，这是一种执行最后一项任务的酶。 DNA去甲基化步骤。我们假设 TDG 抑制会导致自然环境的扰动 细胞利用甲基化/去甲基化循环来调节基因表达以响应环境 刺激，例如氧化应激。在目标 1 中，我们将使用基于 CRISPR/Cas9 的基因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。一个 AMD 缺乏治疗方法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。再生过程中需要克服的一个障碍 眼科药物的难点在于生产与天然组织高度相似的视网膜组织。视网膜 色素上皮 (RPE) 是眼睛后部的色素单层，具有最强的再生能力 医学潜力，因为它可以很容易地从患者特异性 iPSC 中衍生出来。多项动物和人类研究 然而，使用这些 iPSC-RPE 细胞无法产生持久的结果，因为细胞要么死亡，要么产生免疫 反应，或者没有产生功能改善，可能是因为这些 iPSC-RPE 只是类似 RPE，并不精确 原生 RPE 的复制品。我们的小组和其他小组在转录景观方面表现出显着差异 iPSC-RPE，相对于天然 RPE。许多定义 RPE 的蛋白质编码基因都会表达，但是 与天然 RPE 相比，iPSC-RPE 中的水平显着降低。有充分记录表明 将体细胞重编程为 iPSC 会消除大部分表观遗传记忆，但仍有一些保留。这 记忆在分化过程中传递给目标细胞类型，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确定。 定义的。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数据来检验这一假设 死后血液和天然 RPE。血液将被重新编程为 iPSC，随后将被 与 RPE 不同。从这些样本中，我们将表征表观基因组以识别表观遗传标记 从血液（亲代细胞）到 iPSC 到 iPSC-RPE（表观遗传记忆）中保留的信息，并将其与 来自同一个体的天然 RPE 表观基因组。这两个目标的实现将带来更好的结果 对 AMD 病理学和 iPSC-RPE 生物学的了解将对治疗策略产生直接影响 对于退伍军人来说。