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

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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.