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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的哈钦森-吉尔福德早衰综合症 (HGPS) 是一种罕见的常染色体显性疾病，加速衰老。患者出现血管僵硬、血管钙化和纤维动脉粥样硬化斑块形成导致血管闭塞，导致 7 至 20 岁之间因心脏病发作或中风而死亡。该疾病源于点突变 (c.1824C>T)，该突变产生交替剪接和法呢基化蛋白质早老素在细胞核中积累。早老蛋白改变基因表达，导致增加氧化应激、细胞凋亡和线粒体功能改变。 HGPS动脉病理分析患者表现出内侧血管平滑肌细胞（SMC）和内侧血管 SMC 中早老素的丢失，外膜成纤维细胞和内皮细胞（EC）。虽然已经开发出几种潜在的治疗方法，由于能够参加临床试验的 HGPS 个人数量很少，进展受到限制。第一次获奖期间在此期间，我们利用 EC 和 SMC 开发了小动脉规模的组织工程血管 (TEBV) 模型源自具有 HGPS 的个体的诱导多能干细胞 (iPSC)。 HGPS TEBV 显示疾病中观察到的病理学，包括早老素表达、SMC 损失和钙化。 HGPS ECs 表现出流介导基因的表达减少，具有促炎性，并且减少了 NOS3 基因表达可防止 TEBV 血管舒张。 HGPS TEBV 的响应功能得到改善法尼基转移酶抑制剂 Lonafarnib（含或不含雷帕霉素类似物依维莫司）。在这个竞争性更新，与 David Liu 博士和 Kan Cao 博士合作，我们将评估以下假设：（1）腺苷碱基编辑器（ABE），精准基因组编辑工具，可直接纠正最常见的碱基编辑器 HGPS 中的基因突变，消除 HGPS 血管 iPSC 衍生的 EC（viEC）中早老素的积累，以及 SMC（viSMC），恢复单个细胞和TEBV的正常功能； (2) 功能和遗传在经 ABE 处理的 TEBV 中观察到的变化在经 ABE 处理的 HGPS 小鼠模型中观察到。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生物力学刺激后。我们将评估由编辑细胞制成的 TEBV 的血管活性、硬度、细胞结构、EC 功能、早老蛋白表达和炎症，以确定是否能维持正常功能 5周。为了模拟体内条件，我们将用含有以下成分的腺病毒载体灌注 HGPS TEBV：引导 RNA 和 ABE。我们将确定剂量、转导百分比并测量病毒渗透到 TEBV 确定在 HGPS 中实现血管病理学有效校正所需的条件。使用小鼠 G608 HGPS 模型，我们将使用 TEBV 研究中确定的条件进行治疗并比较细胞结构，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将 ABE 推进 HGPS 临床试验以及使用 ABE 纠正遗传疾病的重要信息。