Translational studies of GAA deficiency in bioengineered human muscle

描述（由申请方提供）：II型糖原累积病（庞贝氏症）是一种由酸性α-葡萄糖苷酶（GAA）或酸性麦芽糖酶缺乏引起的致死性退行性疾病。这种疾病的特征是由骨骼肌和心肌细胞中溶酶体糖原的积累引起的进行性肌病。使用重组人GAA的酶替代疗法（ERT）是唯一FDA批准的用于庞贝氏症的治疗，尽管是有益的，但其非常昂贵且效率低，需要比用于其他溶酶体疾病的酶剂量高100倍的酶剂量。此外，ERT纠正疾病的重要方面，包括自噬、糖原积累和运动能力低下的能力仍然值得怀疑。因此，开发ERT的替代或辅助疗法的需求是显而易见的，尽管小鼠GAA敲除（GAA-KO）模型经常用于此目的，但小鼠和人的大小和生理学差异以及小鼠中较不严重的疾病表型限制了这些研究的转化效用。人类细胞 从患者肌肉活检中分离的肌纤维提供了一种体外研究肌肉疾病的替代系统，然而，不存在从人肌肉细胞开始产生功能性收缩肌纤维的方法。在该项目中，我们首次描述了由使用来自正常个体和庞贝氏症患者的标准肌肉活检获得的原代肌源性细胞制成的可收缩、电响应的人类肌肉组织（"生物人工肌肉"）的工程化。我们建议利用这些3D细胞培养物作为预测性的体外筛选候选药物和基因治疗人类肌肉疾病。通过结合两位主要研究者的生物工程和临床专业知识，我们将进行一系列体外和体内转化研究，以筛选和验证庞贝氏症的替代和辅助药物和基因疗法。我们尤其会：1）优化健康和庞贝氏症人类生物人工肌肉组织的功能特性，并系统地表征其分子、代谢和功能特性，2）使用GAA-KO小鼠机械地研究用于庞贝氏症的新候选药物和AAV疗法，和3）使用工程化的人庞贝氏症肌肉在体外筛选这些候选方法的功效，并使用免疫组织化学方法在体内进一步验证最有希望的疗法。庞贝氏症的新型人源化小鼠模型。在未来，该项目建立的实验框架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肌疾病。

DESCRIPTION (provided by applicant): 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 candidat 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 itro 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.