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: 10622613
• 负责人: George A Truskey
• 金额: $ 67.25万
• 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/10622613
• 关键词: 20 year old; Acceleration; 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; Lentivirus; Lipids; Lonafarnib; Measures; Medial; Mediating; Mitochondria; Mitosis; Modeling; Mus; Muscle function; Mutation; Myocardial Infarction; NOS3 gene; Nuclear; Oxidative Stress; Pathologic; Pathology; Patients; Penetration; Perfusion; Periodicity; Point Mutation; Pre-Clinical Model; Process; Progeria; Proliferating; Protein Farnesylation; RNA; RNA Splicing; Rare Diseases; Recovery; SDZ RAD; Site; Smooth Muscle Myocytes; Stimulus; Stretching; Stroke; Structure; Syndrome; System; Testing; Therapeutic; Tissue Engineering; Translations; Variant; Vascular Smooth Muscle; Vascular calcification; Vasodilation; Virus; arteriole; autosome; base editing; base editor; calcification; cell growth; cellular engineering; disease phenotype; dosage; effectiveness testing; functional improvement; functional restoration; genome editing; histone methylation; human tissue; improved; 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.

摘要 Hutchinson-Gilford早衰综合征（HGPS）是一种罕见的加速衰老的常染色体显性遗传病。 患者表现为血管硬化、血管钙化和纤维性动脉粥样硬化斑块形成 导致血管闭塞，导致7至20岁之间的人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>T）引起的，该点突变产生了交替剪接和法尼基化的 在细胞核中积累的蛋白质早老蛋白。早老蛋白改变基因表达， 氧化应激、凋亡和线粒体功能改变。高脂血症动脉病理分析 患者显示中膜血管平滑肌细胞（SMC）和中膜血管SMC中的早老蛋白丢失， 外膜成纤维细胞和内皮细胞（EC）。虽然已经开发了几种潜在的治疗方法， 进展受限于可用于临床试验的少数HGPS个体。第一次颁奖时 本研究利用内皮细胞和平滑肌细胞建立了小动脉规模的组织工程血管模型 来源于从患有HGPS的个体获得的诱导多能干细胞（iPSC）。HGPS TEBV 表现出在疾病中观察到的病理学，包括早老蛋白表达、SMC损失和钙化。 HGPS EC表现出降低的流动介导的基因表达，是促炎性的，并且具有降低的 NOS 3基因表达可阻止TEBV血管舒张。HGPS TEBV显示出响应功能的改善 法尼基转移酶抑制剂洛那法尼与或不与雷帕霉素类似物依维莫司。在这 竞争性更新，与大卫刘博士和曹侃博士合作，我们将评估假设， (1)腺苷碱基编辑器（ABE），精确的基因组编辑工具，可以直接纠正最常见的 HGPS中的遗传突变，消除HGPS血管iPSC衍生的EC（viEC）中的早老蛋白积累， SMC（viSMC），恢复单个细胞和TEBV的正常功能;以及（2）功能和遗传 在用ABE处理的HGPS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的碱基编辑恢复功能和基因表达的程度 在生物力学刺激之后。我们将评估用编辑过的细胞制成的TEBV的血管活性，硬度， 细胞结构、EC功能、早老蛋白表达和炎症，以确定正常功能是否维持超过 五周。为了模拟体内条件，我们将用腺病毒载体灌注HGPS TEBV，所述腺病毒载体含有 指导RNA和ABE。我们将建立剂量，百分比转导，并测量病毒渗透到 TEBV，以确定在HGPS中实现血管病理学有效矫正所需的条件。使用 小鼠G608 HGPS模型，我们将用TEBV研究中鉴定的条件处理并比较细胞结构， 僵硬和EC炎症。单细胞RNA-Seq将用于评估编辑对细胞的影响。 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，以推进ABE的临床试验HGPS和使用ABE纠正遗传疾病。