In Vitro Human Tissue-Engineered Blood Vessel Disease Model of Progeria

摘要 Hutchinson-Gilford Progeria综合征(HGPS)是一种罕见的加速衰老的常染色体显性遗传病。 出现血管僵硬、血管钙化和纤维性动脉粥样硬化斑块形成的患者 导致血管闭塞，导致7至20岁的儿童因心脏病发作或中风而死亡。 这种疾病是由一个点突变(C.1824C&gt；T)引起的，它产生交替的剪接和法尼化 积聚在细胞核中的蛋白质孕激素。孕激素改变基因表达，导致 氧化应激、细胞凋亡和线粒体功能改变。HGPS动脉病变的病理分析 患者表现为内侧血管平滑肌细胞(SMCs)和内侧血管SMCs中的孕激素丢失， 外膜成纤维细胞和内皮细胞。虽然已经开发了几种潜在的治疗方法， 进展受到少数几个可以登记参加临床试验的HGPS个人的限制。在第一次获奖期间 期间，我们利用ECs和SMC建立了一种小动脉规模的组织工程血管(TEBV)模型 来源于从HGPS患者身上获得的诱导多能干细胞(IPSCs)。HGPS TEBV 显示疾病中观察到的病理，包括孕激素表达、SMC丢失和钙化。 HGPS内皮细胞表现出流动介导的基因表达减少，是促炎的，并且减少了 防止TEBV血管扩张的NOS3基因表达。HGPS TEBV在响应中显示更好的功能 对法尼基转移酶抑制剂Lonafarnib加或不加雷帕霉素类似物Everolimus。在这 竞争更新，我们将与刘大卫博士和曹侃博士合作评估假设 (1)腺苷碱基编辑(ABES)，精确的基因组编辑工具，可以直接纠正最常见的 HGPS基因突变，消除HGPS血管IPSC来源的内皮细胞(VECs)中孕激素积累 SMC(ViSMC)，恢复单个细胞和TEBV的正常功能；以及(2)功能和遗传 在ABE处理的HGPS小鼠模型中观察到ABE处理的TEBV的变化。我们会 检查HGPS viECs和viSMC的碱基编辑在多大程度上恢复功能和基因表达 经过生物力学刺激后。我们将评估用编辑过的细胞制作的TEBV的血管活性、僵硬、 细胞密度、内皮细胞功能、孕激素表达和炎症以确定是否维持正常功能 5周。为了模拟活体条件，我们将向HGPS TEBV灌注腺病毒载体，其中包含 引导RNA和ABES。我们将建立剂量，转导百分比，并测量病毒对 TEBV以确定实现HGPS血管病理有效纠正所需的条件。使用 小鼠G608 HGPS模型，我们将用TEBV研究中确定的条件进行处理，并比较细胞密度， 僵硬和EC炎症。将使用单细胞RNA-Seq来评估编辑对 ABE处理后小鼠血管中的血管细胞和TEBV。这项研究的结果将提供 促进ABES进入HGPS临床试验和使用ABES纠正遗传病的重要信息。

ABSTRACT Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare autosomal dominant disease of accelerated aging. Patients present with vascular stiffening, vascular calcification, and fibrous atherosclerotic plaque formation causing vessel occlusion, which causes death between 7 and 20 years of age due to heart attack or stroke. The disease arises from a point mutation (c.1824C>T) that produces the alternately spliced and farnesylated protein progerin that accumulates in the cell nucleus. Progerin alters gene expression, causing increased oxidative stress, apoptosis, and altered mitochondrial function. Pathological analysis of arteries of HGPS patients shows loss of medial vascular smooth muscle cells (SMCs) and progerin in the medial vascular SMCs, adventitial fibroblasts, and endothelial cells (ECs). While several potential therapeutics have been developed, progress is limited by the few HGPS individuals available to enroll in clinical trials. During the first award period, we developed an arteriole-scale tissue engineered blood vessel (TEBV) model using ECs and SMCs derived from induced pluripotent stem cells (iPSCs) obtained from individuals with HGPS. HGPS TEBVs exhibit the pathology observed in the disease including progerin expression, loss of SMCs, and calcification. HGPS ECs exhibit reduced expression of flow-mediated genes, are pro-inflammatory, and have reduced NOS3 gene expression that prevents TEBV vasodilation. HGPS TEBVs show improved function in response to the farnesyltransferase inhibitor Lonafarnib with or without the rapamycin analogue, Everolimus. In this competing renewal, in collaboration with Dr. David Liu and Dr. Kan Cao we will evaluate the hypotheses that (1) adenosine base editors (ABEs), precision genome editing tools that can directly correct the most common genetic mutation in HGPS, eliminate progerin accumulation in HGPS vascular iPSC-derived ECs (viECs) and SMCs (viSMCs), restoring normal function of individual cells and TEBVs; and (2) functional and genetic changes observed in ABE-treated TEBVs are observed in an HGPS mouse model treated with ABEs. We will examine the extent to which base editing of HGPS viECs and viSMCs restores function and gene expression after biomechanical stimulation. We will evaluate TEBVs made with edited cells for vasoactivity, stiffness, cellularity, EC function, progerin expression, and inflammation to establish if normal function is maintained over 5 weeks. To simulate in vivo conditions, we will perfuse HGPS TEBVs with adenovirus vectors containing guide RNAs and ABEs. We will establish dosage, percent transduction, and measure virus penetration into TEBVs to determine conditions needed to achieve effective correction of vascular pathology in HGPS. Using a mouse G608 HGPS model, we will treat with conditions identified in TEBV studies and compare cellularity, stiffness, and EC inflammation. Single cell RNA-Seq will be used to evaluate the impact of editing on the vascular cells in the mouse vessels and TEBVs after ABE treatment. Results of this study will provide important information to advance ABEs to clinical trials for HGPS and use of ABEs to correct genetic diseases.