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

老年性黄斑变性(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，最初会导致中央视力丧失，并可能导致 剩余视力在几年内迅速退化。虽然这是一种流行的疾病，但对 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。贡献于 缺乏对AMD的治疗，是导致AMD发病的潜在遗传因素仍未解决的事实。 虽然34个基因中或大约34个基因的变异已被确定为疾病的风险因素，但没有一个基因被确定为疾病的危险因素 被证明是致病因素。表观遗传学是AMD发病机制中尚未充分探讨的领域。在……里面 与玛格丽特·迪安吉利斯博士合作，我们对140个干RPE进行了DNA甲基化研究 AMD捐赠者，确定了400多个差异甲基化区域。将该数据与来自 IPSC-RPE在氧化应激下，我们已经确定了81个基因同时存在差异甲基化和 差异表达。排在榜首的是胸腺嘧啶DNA糖基酶，这是一种执行最后一种 DNA去甲基化步骤。我们假设TDG的抑制会导致自然的 细胞用来调节基因表达以响应环境的甲基化/去甲基化循环 刺激，如氧化应激。在目标1中，我们将使用基于CRISPR/Cas9的击倒 TDG在正常和氧化应激条件下诱导的多能干细胞来源的RPE(IPSC-RPE)。一个 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，这是一种再生医学。再生能源需要克服的一个障碍 眼科药物的难点是制造出与天然组织高度相似的视网膜组织。视网膜 色素上皮(RPE)是位于眼球后部的色素单层，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定的IPSCs中衍生出来。多种动物和人体研究 然而，使用这些IPSC-RPE细胞不能产生持久的结果，因为细胞要么死亡，要么产生免疫 响应，或者没有产生功能改进，可能是因为这些IPSC-RPE只是类似RPE的，并不确切 本机RPE的复制副本。我们组和其他组在转录格局上表现出显著的差异 IPSC-RPE相对于本地RPE。许多定义RPE的蛋白质编码基因都得到了表达，但 与本地RPE相比，IPSC-RPE中的水平显著降低。已经有很好的证据表明 将体细胞组织重新编程为iPSCs可以移除大部分表观遗传记忆，但也有一些保留下来。这 记忆在分化过程中传递给目标细胞类型，并可以促进 去分化为原始细胞类型。然而，这些细胞的确切表观遗传学图景尚不清楚。 已定义。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来检验这一假设 死后的血液和天然的RPE。血液将被重新编程为IPSC，随后将被 分化为RPE。从这些样本中，我们将对表观基因组进行表征，以确定表观遗传标记 从血液(亲本细胞)到iPSCs再到iPSC-RPE(表观遗传记忆)的保留，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更好的 对AMD病理和IPSC-RPE生物学的了解将直接影响治疗策略 为老兵准备的。

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.