Gene Therapy Design Principles for Duchenne Muscular Dystrophy

项目摘要/摘要 这项研究的长期目标是开发治疗杜氏肌营养不良症(DMD)的基因疗法。 心肌病。DMD心肌病，以室腔扩大和心肌变薄为特征 室壁，最终导致心力衰竭。DMD心肌细胞的致病特征包括收缩 功能障碍、钙处理不良、活性氧升高、端粒缩短和细胞早熟 死亡。当心脏中大量细胞死亡时，就会形成疤痕组织，增加心脏的硬度。 虽然有可用于缓解扩张型心肌病症状的治疗方法，但目前还没有 预防或推迟该病发病的治疗方法。适用于基因治疗的较小版本的抗肌营养不良蛋白 已经显示出治疗DMD相关的严重骨骼肌萎缩的希望；然而，令人惊讶的是，几乎没有 已知它们在治疗心力衰竭方面的效果。这项研究计划将利用生物工程水凝胶 可调节的僵硬，带有dystrophin突变的人诱导多能干细胞(IPSCs)，以及生化 确定全长dystrophin是否可以拯救DMD心肌细胞使其免于病理性死亡的技术。 在K01颁奖期间，明日香博士将在海伦·布劳博士的指导下接受培训，海伦·布劳博士是一名 DMD。通过设计模仿僵硬的、患病的心脏组织的水凝胶，江口博士将能够测量 DMD IPSCs分化为心肌细胞的收缩参数。Aim 1将测试全长 Dystrophin可将DMD心肌细胞从收缩缺陷、钙调节异常和早产中拯救出来 细胞死亡。目标2将确定分裂载体或脂质纳米颗粒方法是否可以传递全长抗肌营养不良蛋白 到心肌细胞。目标3将测试这种传递全长抗肌营养不良蛋白的基因治疗策略是否可以延迟 DMD心肌病在小鼠模型中的发病。针对根本原因的基因治疗方法 疾病，即缺乏营养不良蛋白，对于延长DMD患者的寿命和提高他们的生活质量至关重要。 职业发展计划的目的是使江口博士能够成功地过渡到职业生涯 独立调查员。她的科学咨询委员会由贝丝·普鲁特博士组成，他是一名生物工程师， 在牵引力显微镜方面的专业知识，心血管疾病建模专家Joseph Wu博士和Dr。 儿科心脏病专家丹尼尔·伯恩斯坦说。这些合作者将共同帮助江口博士在 生物工程、细胞生物学和生物化学的接口，以在 心血管研究。

Project Summary/Abstract The long-term objective of this study is to develop gene therapies that treat Duchenne muscular dystrophy (DMD) cardiomyopathy. DMD cardiomyopathy, characterized by ventricular chamber enlargement and thinning of the ventricular wall, ultimately leads to heart failure. Pathogenic features of DMD cardiomyocytes include contractile dysfunction, poor calcium handling, elevated reactive oxygen species, telomere shortening, and premature cell death. When a large number of cells die in the heart, scar tissue forms, increasing the stiffness of the heart. Although there are treatments available to alleviate symptoms of dilated cardiomyopathy, there are currently no therapies to prevent or delay the onset of this disease. Smaller versions of dystrophin amenable to gene therapy have shown promise to treat DMD-associated severe skeletal muscle wasting; however, surprisingly little is known about their effects in treating heart failure. This research plan will leverage bioengineered hydrogels of tunable stiffness, human induced pluripotent stem cells (iPSCs) with dystrophin mutations, and biochemical techniques to determine if full-length dystrophin can rescue DMD cardiomyocytes from their pathogenic demise. During the K01 award period, Dr. Asuka Eguchi will train under the mentorship of Dr. Helen Blau, an expert on DMD. By engineering hydrogels that mimic stiff, diseased heart tissue, Dr. Eguchi will be able to measure parameters of contraction in cardiomyocytes differentiated from DMD iPSCs. Aim 1 will test if full-length dystrophin can rescue DMD cardiomyocytes from contractile deficits, aberrant calcium handling, and premature cell death. Aim 2 will determine if split vector or lipid nanoparticle approaches can deliver full-length dystrophin to cardiomyocytes. Aim 3 will test whether this gene therapy strategy to deliver full-length dystrophin can delay the onset of DMD cardiomyopathy in a mouse model. Gene therapy approaches targeting the root cause of disease, the lack of dystrophin, is critical for extending lifespan and improving the quality of life of DMD patients. The career development plan is designed to enable Dr. Eguchi to successfully transition to a career as independent investigator. Her scientific advisory committee consist of Dr. Beth Pruitt, a bioengineer with expertise in traction force microscopy, Dr. Joseph Wu, an expert on cardiovascular disease modeling, and Dr. Daniel Bernstein, a pediatric cardiologist. Collectively, these collaborators will help Dr. Eguchi develop skills at the interface of bioengineering, cell biology, and biochemistry to launch an independent research program in cardiovascular research.