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

项目摘要 Hutchinson-Gilford Progeria综合征(HGPS)是一种罕见的加速衰老的疾病，在 十几岁的病人。它是由LMNA基因的一个等位基因的从头突变引起的。LMNA是 通常交替拼接以产生负责稳定核膜的板层A和C 结构。HGPS突变导致被称为孕激素的前层蛋白A的截短、法尼化版本 聚集在核膜上，导致核膜形状异常。原发病理学 HGP的主要原因是动脉粥样硬化--动脉内斑块的堆积--导致中风和死亡。鉴于此， 病理学方面，本项目旨在研究HGPS对动脉血管内皮细胞的影响，以及 基因组编辑技术纠正其血管病理的潜力。人们对此知之甚少。 动脉腔内的内皮细胞(ECs)上的HGPS。ECS对施加在其上的剪应力很敏感 它们的基因表达在不同水平的剪应力下发生改变。另外 内皮功能障碍是动脉粥样硬化的早期事件。研究分子的变化 在HGPS的背景下发生在ECs中的这些信息对于全面了解疾病机制是重要的。结果, 本项目的目标1检测HGPS内皮细胞在层流剪应力作用下的基因表达。ECS 来自HGPS和健康供者的IPSCs将暴露在生理相关的剪应力下，使用 平行板流室24小时。将进行rna-seq以评估基因表达的差异。 在静态和流动状态下内皮细胞之间，以及在流动状态下HGPS和健康内皮细胞之间。这些 实验将深入了解HGPS内皮细胞的分子功能障碍，从而导致 动脉硬化。由于HGPS是一种单基因疾病，基因编辑是一种看似合理的 治疗。Lamin A蛋白对小鼠来说是不必要的，CRISPR/Cas9编辑LMNA以防止LMNA 最近有研究表明，在保留层粘连蛋白C的同时，A/孕激素转录降低了孕激素水平，并且略有下降。 延长HGPS小鼠模型的寿命。然而，这些模型并不能完全概括人类疾病。 关于血管病理的表型。因此，重要的是要说明是否完全击倒 拉明A在人性化的系统中是可以容忍的。因此，在目标2中，CRISPR/Cas9基因编辑对 用于治疗HGPS的人体细胞将进行测试。将构建组织工程化血管 使用iPS来源的平滑肌细胞和内皮细胞，以及CRISPR/Cas9机械将被添加到灌流中 媒体。本项目的目标3将阐明HGPS治疗后内皮细胞基因功能的变化。RNA- 针对Lamin A转录的CRISPR/Cas9处理的HGPS内皮细胞将进行SEQ，然后 暴露于流动中，如目标1所示。这将使我们深入了解HGPS治疗如何纠正EC对流动的反应。 总体而言，该项目将为HGPS中内皮细胞在动脉粥样硬化发展中的作用提供新的信息 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.