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

早衰症体外人体组织工程血管疾病模型

• 批准号: 10445145
• 负责人: George A Truskey
• 金额: $ 70.21万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445145
• 关键词: 20 year old; Adenosine; Adenovirus Vector; Aging; Alternative Splicing; Apoptosis; Architecture; Arterial Fatty Streak; Arteries; Award; Biological Assay; Biomechanics; Blood Vessels; CCI-779; Cause of Death; Cell Nucleus; Cell physiology; Cells; Cellularity; Child; Chromatin Structure; Clinical Trials; Collaborations; DNA; DNA Sequence Alteration; Development; Disease; Disease model; Endothelial Cells; Enrollment; Epigenetic Process; Exhibits; Exons; Exposure to; FDA approved; Farnesyl Transferase Inhibitor; Fibroblasts; Fibrosis; First Independent Research Support and Transition Awards; Gene Expression; Genes; Genetic Diseases; Guide RNA; Human; Human Engineering; In Vitro; Individual; Inflammation; Inflammatory; Lead; Lentivirus; Lipids; Lonafarnib; Measures; Medial; Mediating; Mitochondria; Mitosis; Modeling; Mus; Mutation; Myocardial Infarction; NOS3 gene; Nuclear; Oxidative Stress; Pathologic; Pathology; Patients; Penetration; Perfusion; Periodicity; Point Mutation; Pre-Clinical Model; Process; Progeria; Proteins; RNA; RNA Splicing; Rare Diseases; Recovery; SDZ RAD; Site; Smooth Muscle Myocytes; Stimulus; Stretching; Stroke; Structure; Syndrome; System; Testing; Therapeutic; Time; Tissue Engineering; Translations; Variant; Vascular Smooth Muscle; Vascular calcification; Vasodilation; Virus; arteriole; base editing; base editor; calcification; cell growth; cellular engineering; disease phenotype; dosage; effectiveness testing; functional restoration; genome editing; histone methylation; human tissue; improved functioning; in vivo; induced pluripotent stem cell; mouse model; novel; novel therapeutics; prevent; rare genetic disorder; response; shear stress; single-cell RNA sequencing; tool; transcriptome sequencing

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.

抽象的 Hutchinson-Gilford Progeria综合征（HGP）是一种罕见的加速衰老的常染色体显性疾病。 患者存在血管僵硬，血管钙化和纤维性动脉粥样硬化斑块的形成 引起血管阻塞，这会导致由于心脏病发作或中风而导致7至20岁之间的死亡。 该疾病是由点突变（C.1824C> t）产生的 积累在细胞核中的蛋白质。后代蛋白改变基因表达，导致增加 氧化应激，凋亡和线粒体功能改变。 HGP动脉的病理分析 患者显示内侧血管平滑肌细胞（SMC）和内侧血管SMC中的雌激素的损失， 外在成纤维细胞和内皮细胞（EC）。尽管已经开发了几种潜在的治疗剂 进展受到可用于参加临床试验的少数HGP个人的限制。在第一个奖项中 时期，我们使用ECS和SMC开发了一个动脉尺度组织工程血管（TEBV）模型 源自从HGP的个体获得的诱导多能干细胞（IPSC）。 hgps tebvs 展示了该疾病中观察到的病理学，包括孕激素表达，SMC的丧失和钙化。 HGPS ECS表现出降低流量介导的基因的表达，促炎，并且已经降低 NOS3基因表达可防止TEBV血管舒张。 HGPS TEBV显示出改进的响应功能 带有或不带有雷帕霉素类似物的everolimus，到Farnesylsylansferase抑制剂Lonafarnib。在这个 竞争更新，与David Liu博士和Kan Cao博士合作，我们将评估假设 （1）腺苷基础编辑器（ABES），精确的基因组编辑工具，可以直接纠正最常见的 HGP中的遗传突变，消除了HGP的血管IPSC衍生EC（VIEC）和 SMC（VISMC），恢复单个细胞和TEBV的正常功能； （2）功能和遗传 在用ABS处理的HGP小鼠模型中观察到在ABE处理的TEBV中观察到的变化。我们将 检查HGPS VIEC和VISMC的基础编辑在多大程度上恢复功能和基因表达 生物力学刺激后。我们将评估用编辑细胞制成的TEBV，以进行血管活性，刚度， 细胞性，EC功能，孕激素表达和炎症，以确定是否保持正常功能超过 5周。为了模拟体内条件，我们将用含有腺病毒载体的hgps tebvs灌注HGPS TEBV 引导RNA和ABES。我们将建立剂量，转导百分比并测量病毒渗透到 TEBV确定有效纠正HGP中血管病理所需的条件。使用 小鼠G608 HGPS模型，我们将使用TEBV研究中确定的疾病进行治疗，并比较细胞性， 僵硬和EC炎症。单细胞RNA-Seq将用于评估编辑对 ABE处理后，小鼠血管和TEBV中的血管细胞。这项研究的结果将提供 重要的信息，以促进HGP的临床试验和使用AB来纠正遗传疾病。