Defining mechanisms of aortic root aneurysm in Loeys-Dietz syndrome using patients’ induced pluripotent stem cells and genome editing

使用患者诱导多能干细胞和基因组编辑来定义 Loeys-Dietz 综合征主动脉根部动脉瘤的机制

• 批准号: 10609896
• 负责人: Bo Yang
• 金额: $ 69.36万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609896
• 关键词: Affect; Aneurysm; Aorta; Aortic Aneurysm; Arteries; Biomechanics; Bioreactors; Blood Vessels; Bromouridine sequencing; CRISPR/Cas technology; Cardiovascular system; Cause of Death; Cell Differentiation process; Cell Lineage; Cell physiology; Cells; Cessation of life; Code; Control Groups; Coronary Arteriosclerosis; Data; Disease; Dissection; Enrollment; Extracellular Matrix; Family; Foundations; GKLF protein; Gene Mutation; Genes; Goals; Heart; Human; Impairment; Implant; In Vitro; Individual; Inferior; Knock-in; Knock-out; Knowledge; Lipids; Loeys-Dietz Syndrome; Mechanics; Medical; Modeling; Molecular; Muscle Cells; Mutate; Mutation; Neural Crest; Oryctolagus cuniculus; Pathogenicity; Pathway interactions; Patients; Persons; Phenotype; Property; Proteins; Pulsatile Flow; Research; Rest; Ruptured Aortic Aneurysms; Signal Transduction; Smooth Muscle Myocytes; Susceptibility Gene; TGFBR2 gene; Technology; Therapeutic; Thoracic Aortic Aneurysm; Thoracic aorta; Tissue Engineering; Tissue Transplantation; Tissues; Transforming Growth Factor beta; Transforming Growth Factors; United States; ascending aorta; density; drug testing; embryonic stem cell; gene correction; genome editing; improved; in vivo; induced pluripotent stem cell; inhibitor; loss of function mutation; mouse model; mutant; mutation correction; novel; novel therapeutics; prevent; receptor; response; screening; stem cells

ABSTRACT Aortic aneurysm is the 13th leading cause of death in the United States, 25% of which are thoracic aortic aneurysms (TAA). Approximately 15,000 people die every year of rupture of aortic aneurysm. Thoracic aortic dissection (often resulting from TAA) is another devastating condition that causes 10,000 deaths each year. Mouse models have been used to study TAA and dissection (TAAD) for years. However, there has been no significant improvement in medical treatments to prevent or reverse human TAAD. Gene mutations in the pathway of transforming growth factor (TGF) -β predispose patients to TAAD. Just as early research on mutations of low density lipid receptors opened a door to the medical treatment of coronary artery disease, studying the mechanisms of genetic alterations of the TGF-β pathway that cause TAAD, namely TGFBR1 and SMAD3 mutations which result in Loeys-Dietz syndrome (LDS) type 1 and 3, could open a door for the medical treatment for all TAAD in general. It is very interesting that patients with TGFBR1 or SMAD3 mutations frequently develop aortic root aneurysms first, initially sparing the rest of the aorta. The aortic root is composed of smooth muscle cells (SMCs) from the second heart field through Cardiovascular progenitor cell (CPC) lineage. TGF-β is critical for SMC differentiation from the second heart field. The mutations of TGFBR1 or SMAD3 in LDS patients are loss-of-function mutations. Our preliminary data showed that human induced pluripotent stem cells (iPSCs) with SMAD3 knockout or pathogenic TGFBR1 knockin (KI) mutations had defective differentiation of SMCs through CPC lineage compared to isogenic normal control iPSCs. Therefore, we hypothesize that pathogenic mutations in TGFBR1 or SMAD3 will disrupt SMC differentiation and thus decrease the contractile activity of CPC-derived SMCs and disrupt the extracellular matrix, resulting in aortic aneurysm. We have enrolled families of LDS type 1 and 3 patients, and normal controls, and generated iPSCs from all subjects. We will create the pathogenic LDS knock-in mutations of TGFBR1 or SMAD3 in normal control iPSCs, and correct the gene mutations in LDS iPSCs using CRISPR/Cas9 genome editing technology. We will then compare the SMC differentiation and function through CPC lineage (CPC-SMCs) in KI mutation vs. normal control groups; LDS patients vs. mutation-corrected groups. Using CPC-SMCs, we will create a tissue engineered blood vessel (TEBV) in a bioreactor with pulsatile flow, and compare the biomechanics of the TEBV with or without TGFBR1 or SMAD3 mutations. Finally, we will transplant the TEBV into nude rabbits to generate an in vivo human aneurysm with TGFBR1 or SMAD3 LDS mutations in rabbits to determine the molecular mechanism of the aortic aneurysm formation due to TGFBR1 or SMAD3 mutations, enabling screening of potential medical treatments. Our proposed study will provide in-depth knowledge of aneurysm formation in LDS patients and provide the foundation to develop novel medical therapies for TAAD in general.

摘要 主动脉瘤是美国第13大致死原因，其中25%是胸主动脉 动脉瘤(TAA)。每年约有15,000人死于主动脉瘤破裂。胸主动脉 夹层(通常由TAA引起)是另一种破坏性的疾病，每年导致10,000人死亡。 小鼠模型多年来一直被用于研究TAA和夹层(TAAD)。然而，目前还没有 在预防或逆转人类TAAD的医疗治疗方面有重大改进。人类基因组中的基因突变 转化生长因子-β途径使患者易患急性呼吸窘迫综合征。就像早期对 低密度脂蛋白受体的突变为冠心病的医学治疗打开了一扇门， 研究转化生长因子-β途径的基因改变导致TAAD的机制 导致Loeys-Dietz综合征(LDS)1型和3型的SMAD3突变可能为医学研究打开一扇门 对所有TAAD的一般治疗。非常有趣的是，携带TGFBR1或SMAD3突变的患者 通常首先发展为主动脉根部动脉瘤，最初保留了主动脉的其余部分。腹主动脉根部由 血管前体细胞(CPC)在第二心区的血管内皮细胞 血统。转化生长因子-β对SMC与第二心野的分化起关键作用。TGFBR1或TGFBR1突变 LDS患者中的SMAD3是功能丧失突变。我们的初步数据显示，人类诱导 具有SMAD3基因敲除或致病性TGFBR1敲除(KI)突变的多潜能干细胞(IPSCs) 与同基因的正常对照IPSCs相比，通过CPC谱系分化的SMC存在缺陷。因此， 我们假设TGFBR1或SMAD3的致病突变会扰乱SMC的分化，从而 降低CPC来源的SMC的收缩活性并破坏细胞外基质，导致主动脉 动脉瘤。我们招募了LDS 1型和3型患者的家庭，以及正常对照，并产生了IPSCs 从所有的科目。我们将在正常人群中创造致病的LDS敲入突变的TGFBR1或SMAD3 控制IPSCs，并使用CRISPR/Cas9基因组编辑技术纠正LDS IPSCs中的基因突变。 然后，我们将比较Ki突变中通过CPC谱系(CPC-SMC)的SMC分化和功能 与正常对照组；LDS患者与突变纠正组。使用CPC-SMCS，我们将创建 组织工程血管(TEBV)在脉动流生物反应器中的生物力学性能比较 有或没有TGFBR1或SMAD3突变的TEBV。最后，我们将把TEBV移植到裸兔体内 建立具有TGFBR1或SMAD3 LDS突变的兔体内人动脉瘤模型，以确定 TGFBR1或SMAD3突变导致主动脉瘤形成的分子机制 筛选可能的医疗治疗。我们提议的研究将提供对动脉瘤的深入了解。 在LDS患者中形成，并为开发治疗TAAD的新药物奠定了基础。