Progeria and Vasculature: Investigating Genetic Effects and the Potential of Genome Editing for Treatment

项目摘要 Hutchinson-Gilford早衰综合征（HGPS）是一种罕见的加速衰老疾病， 患者在十几岁时就开始了它是由LMNA基因的一个等位基因中的从头点突变引起的。LMNA是 通常交替剪接产生核纤层蛋白A和C，它们负责稳定核膜 结构HGPS突变导致前核纤层蛋白A的截短的法尼基化形式，称为早老蛋白， 积累在核膜上并导致异常的核膜形状。主要病理 动脉粥样硬化是一种斑块在动脉内的积聚，导致中风和死亡。鉴于这种 病理学，该项目旨在研究HGPS对动脉内皮细胞的影响， 基因组编辑技术纠正其血管病理的潜力。人们对以下因素的影响知之甚少： HGPS作用于动脉管腔内皮细胞（EC）。内皮细胞对切应力敏感 它们的基因表达在不同水平的剪切应力下会发生改变。另外 内皮功能障碍是一般动脉粥样硬化中值得注意的早期事件。检测分子变化 在HGPS的背景下，在EC中发生的变化对于充分理解疾病机制很重要。因此，在本发明中， 本研究的目的一是检测HGPS内皮细胞在层流剪切应力作用下的基因表达。ECS 来自HGPS和健康供体的iPSC将暴露于生理学相关的剪切应力， 平行板流动室中24小时。将进行RNA-seq以评估基因表达的差异 在静态和流动条件下的EC之间，以及在流动条件下的HGPS和健康EC之间。这些 实验将深入了解HGPS ECs的分子功能障碍，导致HGPS ECs的发展。 动脉粥样硬化由于HGPS是一种单基因疾病，基因编辑呈现为一种合理的过程， 治疗核纤层蛋白A蛋白在小鼠中表达，CRISPR/Cas9编辑LMNA以防止核纤层蛋白 A/早老蛋白转录，同时保留核纤层蛋白C，最近已显示减少早老蛋白和轻微 增加HGPS小鼠模型寿命。然而，这些模型并不能完全概括人类疾病 关于血管病理学的表型。因此，重要的是要说明是否完全敲除 核纤层蛋白A在人源化系统中是可耐受的。因此，在目标2中，CRISPR/Cas9基因编辑对 将测试用于治疗HGPS的人类细胞。组织工程血管将被构建 使用iPS衍生的平滑肌细胞和EC，将CRISPR/Cas9机器添加到灌注中， 媒体本项目的目的3将阐明HGPS处理后EC中基因功能的变化。RNA- seq将在用靶向核纤层蛋白A转录的CRISPR/Cas9处理的HGPS EC上进行，然后 如目标1中那样暴露于流动。这将使我们深入了解HGPS治疗如何纠正EC对血流的反应。 总的来说，该项目将提供关于内皮细胞在HGPS动脉粥样硬化发展中的作用的新信息 并研究一种可以恢复血管功能和延长患者生命的治疗方法。

Project Summary Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, accelerated-aging disease that leads to death in patients by their early teens. It is caused by a de novo point mutation in one allele of the LMNA gene. LMNA is typically alternately spliced to produce Lamins A and C which are responsible for stable nuclear envelope structure. The HGPS mutation results in a truncated, farnesylated version of pre-lamin A called progerin which accumulates on the nuclear membrane and leads to abnormal nuclear envelope shape. The primary pathology of HGPS is atherosclerosis – a buildup of plaque within arteries – leading to stroke and death. Given this pathology, this project aims to investigate the effects of HGPS on the endothelium that lines arteries, and the potential of genome-editing technology to correct its vascular pathology. Little is known about the effects of HGPS on endothelial cells (ECs) that line the arterial lumen. ECs are sensitive to shear stress exerted on them by the flow of blood, and their gene expression is altered under different levels of shear stress. Additionally endothelial dysfunction is a notable early event in general atherosclerosis. Examining the molecular changes that occur in ECs in the context of HGPS is important to fully understand the disease mechanism. As a result, Aim 1 of this project examines gene expression of HGPS ECs upon exposure to laminar shear stress. ECs derived from iPSCs of HGPS and healthy donors will be exposed to physiologically relevant shear stress using parallel-plate flow chambers for 24 hrs. RNA-seq will be performed to assess differences in gene expression between ECs in static and flow conditions, and between HGPS and healthy ECs under flow. These experiments will give insight into molecular dysfunction of HGPS ECs, leading to the development of atherosclerosis. Because HGPS is a single gene disorder, gene editing presents as a plausible course of treatment. The Lamin A protein is dispensable in mice, and CRISPR/Cas9 editing of LMNA to prevent Lamin A/progerin transcription while preserving Lamin C has recently been shown to decrease progerin and slightly increase life span in mouse models of HGPS. However, these models do not fully recapitulate human disease phenotypes with regards to vascular pathology. It is important therefore to illustrate whether a full knockout of Lamin A is tolerable in a humanized system. Therefore in Aim 2, the efficacy of CRISPR/Cas9 gene editing on human cells for the treatment of HGPS will be tested. Tissue-engineered blood vessels will be constructed using iPS-derived smooth muscle cells and ECs, and CRISPR/Cas9 machinery will be added to the perfusion media. Aim 3 of this project will then elucidate changes in gene function after HGPS treatment in ECs. RNA- seq will be performed on HGPS ECs that were treated with CRISPR/Cas9 targeting Lamin A transcription, then exposed to flow as in Aim 1. This will give insight into how HGPS treatment corrects EC responses to flow. Overall this project will provide new information about the role of ECs in atherosclerosis development in HGPS and investigate a treatment that may restore vascular function and prolong patient life.