3D Bioprinted Human Model of Duchenne Muscular Dystrophy (DMD) Cardiomyopathy to Study Disease Progression with Imposed Force and Precise Gene Editing

杜氏肌营养不良症（DMD）心肌病是普遍存在的，致命的，并导致突变的基因， dystrophin基因肌营养不良蛋白是心脏机械传导（MT）的重要组成部分， 肌膜上的机械应力在DMD中，肌营养不良蛋白的缺乏导致心肌细胞， 容易受到收缩引起的损伤，从而加速疾病的进展。我们理解 DMD心肌病的早期进展是有限的，这是由于模型不能完全重现人类 疾病，从而限制了有效疗法的发展。鉴于肌营养不良蛋白在连接神经元中的重要作用， 由于MT是细胞外基质（ECM）的收缩装置，因此它对增强DMD心肌病至关重要 模型，包括细胞-ECM接合与施加的生理力，以重现细胞-ECM接合时的早期变化。 测试新疗法所需的器官水平。人类腔室肌肉泵（hChaMP）可以做到这一点。 hChaMP是通过3D打印人类诱导多能干细胞和生物墨水产生的， 可以被加压以施加渐进应变。长期目标是确定DMD的机制， 心肌病的进展是开发新的疾病特异性疗法来预防心肌病。整体 目的是评估在缺乏抗肌萎缩蛋白的DMD中MT和ECM动力学改变的作用 心肌病疾病进展和测试DMD hChaMP中的肌营养不良蛋白基因编辑， 加载中核心假设是肌营养不良蛋白的缺失导致对机械应力的脆弱性增加 导致早期改变的心脏MT和ECM动力学，其促进疾病进展， 肌营养不良蛋白替代将通过恢复MT和ECM稳态来限制收缩诱导的损伤， 挽救DMD心肌病我们的中心假设将在两个特定的目标进行测试：1）评估 使用hChaMP模型系统，改变MT对DMD心肌病疾病进展的影响 进行性容积负荷; 2）确定DMD精确基因对肌营养不良蛋白恢复的影响 编辑心脏重塑机制，决定DMD心肌病的疾病进展。在目标1中，我们 将产生DMD hChaMP以评估增加的体积压力对人的生理影响 DMD表型早期和晚期。在目标2中，我们将引入DMD精确基因编辑来恢复肌营养不良蛋白， 早期和晚期的DMD hChaMPs与体积负载，并评估生理和转录的变化。 在成功完成拟议的研究，我们的研究的预期成果是 早期DMD心肌病负荷进展的特征及其潜在的分子机制 机制等拟议的研究是创新的，因为它结合了使能技术来开发3D 人类DMD心肌病的临床前模型，其模拟疾病进展并通过精确的 基因编辑这些发现将对人类健康产生重大影响， 疾病进展和治疗和预防DMD心肌病的坚实基础。

Duchenne muscular dystrophy (DMD) cardiomyopathy is ubiquitous, deadly, and results from mutations in the dystrophin gene. Dystrophin is an essential component of cardiac mechanotransduction (MT) and distributes mechanical stress across the sarcolemma. In DMD, the absence of dystrophin results in cardiomyocytes that are vulnerable to contraction-induced damage, which accelerates disease progression. Our understanding of the early progression of DMD cardiomyopathy is limited due to models that do not fully recapitulate human disease, thus limiting development of effective therapies. Given the essential role of dystrophin in connecting the contractile apparatus to the extracellular matrix (ECM) for MT, it is critical to augment DMD cardiomyopathy models to include cell-ECM engagement with applied physiological force to recapitulate early changes at the organ level necessary to test novel therapies. The human chambered muscle pump (hChaMP) can do just that. The hChaMP is generated by 3D printing human induced pluripotent stem cells and bio-ink to yield a pump that can be pressurized to impose progressive strain. The long-term goal is to determine mechanisms by which DMD cardiomyopathy progresses to develop novel disease specific therapies to prevent cardiomyopathy. The overall objective is to assess the role of altered MT and ECM dynamics in the absence of dystrophin on DMD cardiomyopathy disease progression and to test dystrophin gene editing in a DMD hChaMP with volumetric loading. The central hypothesis is that the loss of dystrophin leads to increased vulnerability to mechanical stress resulting in early altered cardiac MT and ECM dynamics that promote disease progression and that early dystrophin replacement will limit contraction-induced injury by restoring MT and ECM homeostasis and thereby rescue DMD cardiomyopathy. Our central hypothesis will be tested in two specific aims: 1) To evaluate the impact of altered MT on DMD cardiomyopathy disease progression using the hChaMP model system with progressive volumetric loading; 2) To determine the impact of dystrophin restoration with DMD precise gene editing on cardiac remodeling mechanisms dictating disease progression in DMD cardiomyopathy. In aim 1, we will generate a DMD hChaMPs to assess the physiologic impact of increased volumetric pressure on the human DMD phenotype early and later. In aim 2, we will introduce DMD precise gene editing to restore dystrophin both early and late in DMD hChaMPs with volumetric loading and assess the physiologic and transcriptional changes. At the successful completion of the proposed research, the expected outcomes of our study is the characterization of early DMD cardiomyopathy progression with loading and the underlying molecular mechanisms. The proposed research is innovative as it combines enabling technologies to develop a 3D preclinical model of human DMD cardiomyopathy that mimics disease progression and correction with precise gene editing. These findings will have a significant impact on human health by increasing our understanding of disease progression and a strong basis for treating and preventing DMD cardiomyopathy.