Control of Breathing & Glycogen Storage Disease

描述（由申请人提供）：糖原储存病II型（GSDII）由酸a-葡萄糖苷酶（GAA）缺乏引起，GAA是一种降解糖原的溶酶体酶。GSDII导致婴儿心肺衰竭，青少年和成人出现进行性呼吸衰竭。虽然呼吸衰竭归因于肌肉病理，但尸检病例报告显示颈脊髓糖原积聚并提示运动神经元病理。我们观察到，在小鼠GSDII模型（GAA-/-“敲除”小鼠）中，输出膈放电、平均吸气气流和分钟通气量与代谢率（VE/VCO2）之比被减弱。此外，骨骼肌收缩力和GAA表达正常，但中枢神经系统（CNS）没有GAA的选择性敲除小鼠，也表现出明显的膈输出和通气减少。因此，中枢神经系统GAA缺乏与呼吸运动输出受损有关，GSDII的呼吸功能不全可能反映神经和肌肉病理。初步数据显示，GAA-/-小鼠颈脊髓糖原积累广泛，特别是在逆行识别的膈运动神经元内，也涉及神经机制。确定呼吸功能不全的机制很重要，因为静脉注射酶替代（目前的临床GSDII治疗）并不针对中枢神经系统，因为GAA不能穿过血脑屏障。重组腺相关病毒（rAAV）基因治疗是同时靶向肌肉和中枢神经系统的一种很有前途的方法。我们的初步数据表明，rAAV可以有效地转染膈运动神经元，并且可以通过椎管内或胸腔内注射给药。此外，胸腔内注射包装有GAA基因的rAAV （rAAV-GAA疗法）一个月后，GAA-/-小鼠的通气能力显著增强。这些实验代表了呼吸生理学（Fuller）、基因治疗（Byrne）和神经解剖学（Reier）等专业实验室之间的独特合作。我们提出验证三个假设：1)选择性和完全GAA-/-敲除小鼠膈肌和通气的神经驱动减弱；2)这些小鼠的呼吸缺陷与膈运动神经元的糖原积累并行发生，3)在选择性和GAA-/-敲除小鼠中，脊髓内和胸腔内给药rAAV-GAA分别可以改善脊髓糖原积累和增强呼吸运动输出。

DESCRIPTION (provided by applicant): Glycogen storage disease type II (GSDII) results from deficiency of acid a-glucosidase (GAA), a lysosomal enzyme that degrades glycogen. GSDII causes cardiorespiratory failure in infants, and progressive respiratory failure in juveniles and adults. Although respiratory failure has been attributed to muscle pathology, autopsy case reports show cervical spinal glycogen accumulation and suggest motoneuron pathology. We have observed that efferent phrenic discharge, mean inspiratory airflow, and the ratio of minute ventilation to metabolic rate (VE/VCO2) are blunted in a murine GSDII model, the GAA-/- "knockout" mouse. In addition, a selective knockout mouse with normal skeletal muscle contractility and GAA expression, but no GAA in the central nervous system (CNS), also exhibits marked reductions in phrenic output and ventilation. Accordingly, CNS GAA deficiency is associated with impaired respiratory motor output, and respiratory insufficiency in GSDII may reflect both a neural and a muscular pathology. A neural mechanism is also implicated by preliminary data showing extensive cervical spinal glycogen accumulation in GAA-/- mice, particularly within retrogradely identified phrenic motoneurons. Determining the mechanisms underlying respiratory insufficiency is important because i.v. enzyme replacement (the current clinical GSDII therapy) does not target the CNS as GAA cannot cross the blood brain barrier. A promising method for targeting both muscle and the CNS is recombinant adeno-associated virus (rAAV) gene therapy. Our preliminary data indicate that rAAV effectively transfects phrenic motoneurons, and can be delivered by intraspinal or intrathoracic injection. Further, ventilation is significantly enhanced in GAA-/- mice one month following intrathoracic injection of rAAV packaged with the GAA gene (rAAV-GAA therapy). These experiments represent a unique collaboration between laboratories specializing in respiratory physiology (Fuller), gene therapy (Byrne), and neuroanatomy (Reier). We propose to test three hypotheses: 1) neural drive to the diaphragm and ventilation are attenuated in both the selective and full GAA-/- knockout mice; 2) respiratory deficits in these mice occur in parallel with glycogen accumulation in phrenic motoneurons, and 3) intraspinal and intrathoracic rAAV-GAA delivery can ameliorate spinal glycogen accumulation and enhance respiratory motor output in the selective and GAA-/- knockout mice, respectively.