Novel Adjunctive Therapies for Pompe Disease

项目摘要 候选人和环境：候选人的短期和长期目标是获得技能，知识， 成为一名成功的神经肌肉疾病基因治疗独立研究者所必需的经验 呼吸道疾病（NMD），如庞贝氏症。杜克大学是 候选人实现这些目标与世界著名的庞贝氏症临床和研究中心，沿着 与肌肉学和基因治疗专家，谁经常合作，谁主持每月的研究研讨会。 研究：庞贝氏症是一种致命的糖原累积病，由编码酸的基因突变引起。 α-葡萄糖苷酶（GAA），负责水解溶酶体糖原。GAA缺乏导致 肌肉（心肌、骨骼肌、平滑肌）和运动神经元溶酶体中的糖原积累。唯一的 FDA批准的治疗是重组人GAA（rhGAA）的酶替代疗法（ERT）。ERT 有效治疗心肌糖原积聚，但不能完全纠正骨骼肌病理 因为自噬被破坏了此外，ERT不能穿过血脑屏障治疗运动神经元， 因此，呼吸衰竭持续存在。因此，为了预防庞贝氏症患者的呼吸窘迫，需要 治疗，可以清除糖原和修复自噬在呼吸肌和运动神经元。的 总体目标是确定新的连续治疗，以改善呼吸肌和运动神经元 庞贝氏症的病理学我们建议注射三种可以穿过血液的自噬激活剂- 脑屏障并评估其对庞贝氏症小鼠（Gaa-/-）运动单位的治疗影响。急性间歇性 缺氧（AIH）、雷帕霉素和重组腺相关病毒（rAAV）基因治疗是潜在的治疗方法 治疗庞贝氏症，将在三个目标进行评估。每种治疗方法都有独特的， 互补的能力来解决自噬，庞贝氏症细胞治疗的关键组成部分， 呼吸肌和运动神经元。(1)缺氧是自噬体起始复合物的激活剂。 此外，AIH诱导神经再生障碍中呼吸运动神经元的神经可塑性。（二） 雷帕霉素目前作为免疫抑制剂施用于庞贝氏症患者，然而，雷帕霉素也具有免疫抑制作用。 对主代谢调节因子mTORC 1产生直接的负面影响，从而激活自噬。(3)rAAV-GAA 提供缺乏的酶，从而减少糖原和改善溶酶体健康，这是 恢复正常的自噬在本提案的最终目标中，我们将联合收割机rAAV-GAA治疗与AIH相结合， 雷帕霉素以确定累积效应。分析的关键方法包括神经生理学，以评估神经 呼吸神经输出，全身体积描记法评估呼吸，分子蛋白质组学分析 用于自噬途径蛋白，以及免疫组织化学以鉴定肌肉和CNS的细胞结构。 这些新的干预措施已经用于临床试验，并有可能迅速转化为临床 庞贝氏症患者的护理患有呼吸窘迫。

PROJECT SUMMARY Candidate & Environment: The short and long-term goals of the candidate are to gain skills, knowledge, and experience necessary to become a successful independent investigator in gene therapy for neuromuscular diseases (NMDs) with respiratory pathology, such as Pompe disease. Duke University is the optimal location for the candidate to achieve these goals with a world-renowned Pompe disease clinical and research center, along with myology and gene therapy experts, who frequently collaborate and who host monthly research seminars. Research: Pompe disease is a fatal glycogen storage disease caused by mutations in the gene encoding acid α-glucosidase (GAA), which is responsible for hydrolyzing lysosomal glycogen. GAA deficiency results in glycogen accumulation in the lysosomes of muscles (cardiac, skeletal, smooth) and motor neurons. The only FDA approved treatment is enzyme replacement therapy (ERT) of recombinant human GAA (rhGAA). ERT effectively treats cardiac muscle glycogen accumulation but cannot completely correct skeletal muscle pathology due to disrupted autophagy. In addition, ERT cannot cross the blood-brain barrier to treat motor neurons, and therefore, respiratory failure persists. Thus, to prevent respiratory distress in Pompe patients, there is a need for therapies that can clear glycogen and repair autophagy in the respiratory muscles and motor neurons. The overall goal is to identify novel adjunctive therapies to improve respiratory muscle and motor neuron pathology in Pompe disease. We propose to administer three autophagy activators which can cross the blood- brain barrier and assess their therapeutic impact on the motor unit of Pompe mice (Gaa-/-). Acute intermittent hypoxia (AIH), rapamycin, and recombinant adeno-associated virus (rAAV) gene therapy are potential therapies to treat Pompe disease, which will be evaluated across three aims. Each therapeutic has a unique and complementary ability to address autophagy, a critical component of Pompe disease cellular therapy, in key respiratory muscles and motor neurons. (1) Hypoxia is an activator of the autophagosome initiation complex. Additionally, AIH induces neuroplasticity in respiratory motor neurons in neurogenerative disorders. (2) Rapamycin is currently administered to Pompe patients as an immune suppressor, however, rapamycin also has a direct negative impact on master metabolic regulator, mTORC1, thus activating autophagy. (3) rAAV-GAA provides the deficient enzyme, thereby reducing glycogen and improving lysosome health which is a key to restoring normal autophagy. In the final aim of this proposal, we will combine rAAV-GAA therapy with AIH and rapamycin to determine the cumulative effect. Key methods of analysis include neurophysiology to assess neural output of respiratory nerves, whole body plethysmography to assess respiration, molecular proteomics analysis for autophagy pathway proteins, and immunohistochemistry to identify cellular architecture of muscle and CNS. These novel interventions are already used in clinical trials and have the potential to quickly translate to clinical care in Pompe patients suffering from respiratory distress.