Translational studies of GAA deficiency in bioengineered human muscle

描述(申请人提供)：糖原储存疾病II型(Pompe病)是一种致命的退行性疾病，由酸性-α葡萄糖苷酶(GAA)或酸性麦芽糖酶缺乏引起。这种疾病的特征是由于骨骼和心肌细胞中溶酶体糖原的积聚而引起的进行性肌病。重组人GAA的酶替代疗法(ERT)是FDA批准的唯一治疗Pompe病的方法，尽管有益，但非常昂贵且效率低下，需要的酶剂量是其他溶酶体疾病的100倍。此外，ERT纠正疾病的重要方面的能力仍然值得怀疑，这些方面包括自噬、糖原积累和运动能力低下。因此，开发ERT的替代或辅助治疗方法的必要性是显而易见的，尽管经常利用小鼠GAA基因敲除(GAA-KO)模型来实现这一目的，但小鼠和人类的大小和生理差异以及小鼠不太严重的疾病表型限制了这些研究的翻译效用。人类细胞 从患者的肌肉活组织中分离出来，为在体外研究肌肉疾病提供了一种替代系统，然而，还没有从人类肌肉细胞产生功能性收缩肌肉纤维的方法。在这个项目中，我们首次描述了利用从正常人和庞培病患者的标准肌肉活检获得的原代肌源性细胞制成的收缩、电响应的人类肌肉组织(生物人工肌肉)的工程。我们建议利用这些3D细胞培养作为候选药物和基因治疗人类肌肉疾病的体外筛选。通过结合两位首席研究人员的生物工程和临床专业知识，我们将开展一系列体内外转化性研究，以筛选和验证庞培病的替代和辅助药物和基因疗法。具体地说，我们将：1)优化健康和Pompe病人类生物人造肌肉组织的功能特性，并系统地表征它们的分子、代谢和功能特性；2)利用GAA-KO小鼠以机械方式研究治疗Pompe病的新型候选药物和AAV疗法；3)使用改造的人Pompe病肌肉体外筛选这些候选方法的有效性，并使用新型人源化Pompe病小鼠模型进一步验证最有希望的治疗方法。在未来，这个项目中建立的实验框架将允许我们进行类似的翻译研究，以帮助治疗其他骨骼肌和心肌疾病。

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.