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

摘要 主动脉瘤是美国第13大死亡原因，其中25%是胸主动脉瘤。 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）是另一种毁灭性的疾病，每年导致10，000人死亡。 小鼠模型已被用于研究TAA和夹层（TAAD）多年。然而，没有 预防或逆转人TAAD的医学治疗的显著改进。基因突变 转化生长因子（TGF）-β通路使患者易患TAAD。正如早期的研究一样， 低密度脂蛋白受体的突变为冠状动脉疾病的药物治疗打开了大门， 研究导致TAAD的TGF-β途径的遗传改变机制，即TGFBR 1和 SMAD 3突变导致Loeys-Dietz综合征（LDS）1型和3型，可能为医学治疗打开大门。 一般来说，所有TAAD的治疗。非常有趣的是，TGFBR 1或SMAD 3突变的患者 通常首先发展主动脉根部动脉瘤，最初保留主动脉的其余部分。主动脉根由 通过心血管祖细胞（CPC）从第二心脏领域的平滑肌细胞（SMC） 脉TGF-β对第二心脏区域的SMC分化至关重要。TGFBR 1或TGFBR 2的突变 LDS患者中的SMAD 3是功能缺失突变。我们的初步数据显示，人类诱导 具有SMAD 3敲除或致病性TGFBR 1敲入（KI）突变的多能干细胞（iPSC） 与同基因正常对照iPSC相比，SMC通过CPC谱系的分化缺陷。因此，我们认为， 我们假设TGFBR 1或SMAD 3的致病性突变会破坏SMC的分化， 降低CPC衍生的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iPSC 所有科目。我们将在正常人中产生TGFBR 1或SMAD 3的致病性LDS敲入突变， 控制iPSC，并使用CRISPR/Cas9基因组编辑技术纠正LDS iPSC中的基因突变。 然后我们将通过KI突变的CPC谱系（CPC-SMCs）比较SMC的分化和功能 vs.正常对照组; LDS患者与突变校正组。使用CPC-SMC，我们将创建 组织工程化血管（TEBV）的生物力学性能，并比较 具有或不具有TGFBR 1或SMAD 3突变的TEBV。最后，我们将TEBV移植到裸鼠体内 在兔中产生具有TGFBR 1或SMAD 3 LDS突变的体内人动脉瘤，以确定 TGFBR 1或SMAD 3突变导致的主动脉瘤形成的分子机制， 筛选潜在的医疗方法。我们提出的研究将提供动脉瘤的深入知识， 在LDS患者中的形成，并提供了基础，以开发新的药物治疗TAAD一般。

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.