Translational studies of GAA deficiency in bioengineered human muscle

糖原累积病II型（庞贝氏症）是一种致命的退行性疾病， 酸性-α葡糖苷酶（GAA）或酸性麦芽糖酶。这种疾病的特点是进行性肌病 由骨骼肌和心肌细胞中溶酶体糖原的积累引起。酶 使用重组人GAA的替代疗法（ERT）是唯一FDA批准的庞贝氏症治疗方法 尽管有益，但非常昂贵且效率低下，需要100倍的酶剂量 比用于其他溶酶体疾病的那些更大。此外，ERT纠正重要问题的能力 疾病的各个方面，包括自噬、糖原积累和运动能力低下， 值得怀疑因此，显然需要开发ERT的替代或辅助疗法， 尽管小鼠GAA敲除（GAA-KO）模型经常用于此目的，但 小鼠和人类的大小和生理学以及小鼠中不太严重的疾病表型限制了翻译 这些研究的效用。从患者肌肉活检中分离出的人类细胞提供了一种可供研究的替代系统 然而，在体外肌肉疾病中，不存在产生功能性收缩肌纤维的方法， 从人体肌肉细胞中分离出来。在这个项目中，我们第一次描述了收缩工程， - 由使用以下获得的原代肌原细胞制成的响应性人肌肉组织（“生物人工肌肉”）： 正常人和庞贝氏症患者的标准肌肉活检。我们建议利用这些3D 细胞培养物作为预测性体外筛选用于人类肌肉的候选药物和基因治疗剂 疾病通过结合两位主要研究者的生物工程和临床专业知识，我们将开展 一系列体外和体内转化研究，以筛选和验证替代和辅助药物 以及庞贝氏症的基因疗法特别是，我们将：1）优化健康和 庞贝氏症人类生物人工肌肉组织，并系统地表征其分子，代谢 2）机械地研究用于庞贝氏症的新型候选药物和AAV疗法 使用GAA-KO小鼠，和3）使用工程化的基因工程在体外筛选这些候选方法的功效。 人庞贝氏症肌肉，并进一步验证最有前途的疗法，在体内使用一种新的 庞贝氏症的人源化小鼠模型。在未来，在此建立的实验框架 该项目将使我们能够进行类似的转化研究，以帮助治疗其他骨骼和心脏疾病。 肌肉紊乱

Glycogen storage disease type II (Pompe disease) is a fatal degenerative disease caused by the deficiency of acid-alpha glucosidase (GAA) or acid maltase. This disease is characterized by progressive myopathy resulting from the accumulation of lysosomal glycogen in skeletal and cardiac muscle cells. Enzyme replacement therapy (ERT) with recombinant human GAA is the only FDA-approved treatment for Pompe disease, which despite being beneficial, is highly expensive and inefficient, requiring enzyme doses 100-fold greater than those used for other lysosomal disorders. Furthermore, the ability of ERT to correct important aspects of the disease including autophagy, glycogen accumulation, and low exercise capacity, remains questionable. Therefore, the need for the development of alternative or adjuvant therapies to ERT is obvious, and although the mouse GAA knockout (GAA-KO) model is often utilized for this purpose, the differences in size and physiology of mice and humans and less severe disease phenotype in mice limit the translational utility of these studies. Human cells isolated from patients' muscle biopsies offer an alternative system to study muscle disease in vitro, however, no methods exist to generate functional contractile muscle fibers starting from human muscle cells. In this project we for the first time describe engineering of contractile, electrically responsive human muscle tissues ("bioartificial muscle") made of primary myogenic cells obtained using standard muscle biopsies from normal individuals and Pompe disease patients. We propose to utilize these 3D cell cultures as a predictive in vitro screen for candidate drug and gene therapeutics for human muscle disease. By combining bioengineering and clinical expertise of the two principal investigators, we will carry out a set of translational in vitro and in vivo studies in order to screen and validate alternative and adjuvant drug and gene therapies for Pompe disease. In particular, we will: 1) Optimize functional properties of healthy and Pompe disease human bioartifical muscle tissues and systematically characterize their molecular, metabolic and functional properties, 2) Mechanistically study novel candidate drug and AAV therapies for Pompe disease using GAA-KO mice, and 3) Screen the efficacy of these candidate approaches in vitro using engineered human Pompe disease muscle and further validate the most promising therapies in vivo using a novel humanized mouse model of Pompe disease. In the future, the experimental framework established in this project will allow us to undertake similar translational studies to aid treatment of other skeletal and cardiac muscle disorders.