A 3D IN VITRO DISEASE MODEL OF ATRIAL CONDUCTION

项目总结 在美国，近十分之一的65岁以上的成年人患有心房颤动(AF)，导致大约 每年的医疗成本为60亿美元。由于高龄是发生房颤的主要危险因素， 随着人口老龄化，预计总发病率在未来几十年内将稳步上升。当前 治疗干预效果非常差和/或不良副作用在很大程度上是由于我们的 不能针对疾病的根本原因，并为心房和患者提供特异性。中环 该方案的目的是建立和验证一个健壮的三维异常人体微生理学模型 使用患者的诱导多能干细胞进行心房传导。该模型将模拟重要的 房颤的要素，如传导速度，并开发使用腺病毒的新治疗策略 基因干扰传递(CRISPRi)将改变的基因调控通路作为房颤的来源。我们 将通过完成以下具体目标来实现这一目标：1)确定转录组的特征， 表观基因组和成年人心房肌细胞(正常和房颤)的电生理学；2)在 利用人iPS细胞来源的心房肌细胞(iPS-1)建立人心房传导的体外疾病模型 ACM)和心房调节基因表达；3)设计和测试腺病毒基因递送策略 特异性靶向心房(不是心室或结节)心肌细胞；4)展示心房特异性腺病毒 体外人心房组织中CRISPRi的传递(SA3)和PITX2的基因干扰；5)产生iPS- 一组正常人和患者的ACM和相应的体外心房传导模型 使用一组现有的药物和药物，在体外模型中表征药物疗效和基因传递 我们构建的针对PITX2(SA3)CRISPR基因干扰的心房特异性腺病毒载体。完成特定的 AIMS将提供一个人体心房传导的模型，可以作为理解药物的广泛平台 对房颤等疾病的有效性和安全性。

PROJECT SUMMARY Nearly 1 in 10 adults over the age of 65 in the U.S. suffer from atrial fibrillation (AF) leading to approximately $6 billion annually in healthcare costs. Because advanced age is a primary risk factor for developing AF, the overall incidence is expected to rise steadily over the coming decades as our population ages. Current therapeutic interventions have remarkably poor efficacy and/or untoward side effects due in large part to our inability to target the root cause of the disease and provide specificity for the atria and the patient. The central objective of this proposal is to create and validate a robust 3D microphysiological model of abnormal human atrial conduction using induced pluripotent stem cells from the patient. The model will simulate important elements of AF, such as conduction velocity, and develop novel therapeutic strategies that employ adenoviral delivery of gene interference (CRISPRi) that target altered gene regulatory pathways as the source of AF. We will accomplish this objective by completing the following specific aims: 1) characterize the transcriptome, epigenome, and electrophysiology of adult human atrial cardiomyocytes (normal and AF); 2) create a 3D in vitro disease model of human atrial conduction leveraging human iPS cell-derived atrial cardiomyocytes (iPS- aCM) and atrial regulatory gene expression; 3) design and test an adenoviral gene delivery strategy to specifically target atrial (not ventricular or nodal) cardiomyocytes; 4) demonstrate atrial specific adenoviral delivery (SA3) of CRISPRi and gene interference of PITX2 in ex vivo human atrial tissue; and 5) create iPS- aCM and the corresponding in vitro model of atrial conduction from a cohort of normal subjects and patients with AF; characterize drug efficacy and gene delivery in the in vitro models using a panel of existing drugs and our atrial specific adenoviral construct for CRISPR gene interference of PITX2 (SA3). Completing the specific aims will provide a model of human atrial conduction that can be used as a broad platform to understand drug efficacy and safety for diseases such as AF.!