Control of Breathing & Glycogen Storage Disease

描述(申请人提供)：糖原储存疾病II型(GSDII)由酸性α-葡萄糖苷酶(GAA)缺乏引起，GAA是一种降解糖原的溶酶体酶。GSDII会导致婴儿的心肺衰竭，以及青少年和成年人的进行性呼吸衰竭。虽然呼吸衰竭被归因于肌肉病理，但尸检病例报告显示颈椎糖原积聚，并提示运动神经元病理。我们观察到，在GSDII小鼠模型--GAA-/-“基因敲除”小鼠中，传出的膈神经放电、平均吸气气流和每分钟通气量与代谢率的比率(VE/VCO2)被钝化。此外，选择性基因敲除小鼠骨骼肌收缩能力和GAA表达正常，但中枢神经系统(CNS)没有GAA，也显示出明显的膈输出和换气减少。因此，中枢神经系统GAA缺乏与呼吸运动输出受损有关，而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，在GAA-/-小鼠中，尤其是在逆行识别的膈运动神经元中，颈椎糖原广泛积累，这也暗示了一种神经机制。确定呼吸功能不全的机制很重要，因为静脉注射。酶替代疗法(目前的临床GSDII疗法)不针对中枢神经系统，因为GAA不能通过血脑屏障。重组腺相关病毒(RAAV)基因治疗是一种很有前途的同时针对肌肉和中枢神经系统的方法。我们的初步数据表明，rAAV可以有效地转染膈运动神经元，并可以通过椎管内或胸腔内注射来传递。此外，在胸腔内注射包装有GAA基因的rAAV(rAAV-GAA疗法)一个月后，GAA-/-小鼠的通气性显著增强。这些实验代表了专门从事呼吸生理学(Fuller)、基因疗法(Byrne)和神经解剖学(Reier)的实验室之间的独特合作。我们建议检验三个假设：1)选择性和完全GAA/-基因敲除小鼠对横隔膜的神经驱动和通风都减弱；2)这些小鼠的呼吸缺陷与膈运动神经元中的糖原积聚平行发生；3)在选择性和GAA/-基因敲除小鼠中，椎管内和胸腔内注射rAAV-GAA可以分别改善脊髓糖原积聚和提高呼吸运动输出。

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.