Spinal and brainstem respiratory neurons in Pompe disease

描述（由申请方提供）：庞贝氏症是一种神经肌肉疾病，由酸性α-葡糖苷酶（GAA）基因突变引起，GAA是降解溶酶体糖原所必需的酶。换气不足是所有形式的庞贝氏症的标志性特征，其历史上被归因于呼吸肌病理。在这项R21资助申请中提出的实验将提供有关庞贝氏症呼吸功能不全临床问题的基本机制信息。最重要的是，我们提出了一个直接测试的假设，中枢神经系统功能障碍是一个主要的贡献者呼吸功能不全的庞贝氏症。支持这一假设的证据越来越多，但缺乏确凿的证据。为了实现这一目标，我们建议使用“位点特异性”Cre-Lox重组方法敲除小鼠脊髓和延髓呼吸神经元中的GAA基因，同时保持骨骼肌和心肌基因表达不变。如果这一假设得到证实，它将告知临床社区有关庞贝氏症患者呼吸功能不全的根本原因。更重要的是，要证实我们的假设，就必须从目前强调的单纯肌肉定向治疗转向同时影响肌肉和神经功能的方法。因此，所提出的研究的首要目标是确定当呼吸神经元中的GAA基因表达被“敲除”时，呼吸控制系统是否变得功能障碍。我们还建议比较和对比脊髓运动神经元与延髓呼吸控制神经元在庞贝氏症呼吸运动输出受损方面的作用。将GAA基因转移到庞贝氏症患者膈肌的初步临床试验正在进行中（ClinicalTrials.gov：NCT 00976352）。这项工作将通过检查运动神经元与延髓神经元对呼吸功能不全的重要性来补充正在进行的试验。这一点很重要，因为膈运动神经元可以通过膈注射后的逆行病毒转运进行转导，而髓神经元则不会。因此，临床试验不太可能导致延髓呼吸神经元的转导。在开发该应用程序时，我们获得了具有“floxed”GAA基因的小鼠群体。我们建议使用立体定位和/或逆行递送驱动Cre重组酶表达的AAV载体来选择性地敲除脊髓呼吸（膈）运动神经元（Aim 1）和脑干呼吸控制神经元（Aim 2）中的GAA基因。这项工作是呼吸控制科学家（富勒），AAV专家和临床医生与庞贝氏症患者（伯恩），和AAV专家与立体定位交付（曼德尔）的专业知识之间的合作努力。

DESCRIPTION (provided by applicant): Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid a- glucosidase (GAA) - an enzyme necessary to degrade lysosomal glycogen. Hypoventilation is a hallmark feature of all forms of Pompe disease that has historically been attributed to respiratory muscle pathology. The experiments proposed in this R21 grant application will provide fundamental, mechanistic information about the clinical problem of respiratory insufficiency in Pompe disease. Most importantly, we propose a direct test of the hypothesis that central nervous system dysfunction is a primary contributor to respiratory insufficiency in Pompe disease. Evidence is mounting in support of this hypothesis, but definitive proof is lacking. To accomplish this goal we propose to use a "site-specific" Cre-Lox recombination approach to knockout the GAA gene in spinal and medullary respiratory neurons of mice while leaving skeletal and cardiac muscle gene expression unaltered. If the hypothesis is confirmed it will inform the clinical community about the underlying causes of respiratory insufficiency in Pompe patients. More importantly, confirmation of our hypothesis would necessitate a shift from the current emphasis on purely muscle directed therapies towards approaches which would impact on both muscle and neural function. Thus, overarching goal of the proposed studies is to determine if the respiratory control system becomes dysfunctional when GAA gene expression is "knocked out" in respiratory neurons. We also propose to compare and contrast the role of spinal motoneurons vs. medullary respiratory control neurons with regard to impaired respiratory motor output in Pompe disease. An initial clinical trial of GAA gene transfer to the diaphragm of Pompe patients is underway (ClinicalTrials.gov: NCT00976352). This work will complement the ongoing trial by examining the importance of motoneurons vs. medullary neurons to respiratory insufficiency. This is important since phrenic motoneurons can be transduced via retrograde viral transport post-diaphragm injection whereas medullary neurons will not. Thus, the clinical trial is not likely to result in transduction of medullary respiratory neurons. In developing this application we obtained a mouse colony with a "floxed" GAA gene. We propose to use stereotaxic and/or retrograde delivery of AAV vectors driving Cre recombinase expression to selectively knockout the GAA gene in spinal respiratory (phrenic) motoneurons (Aim 1) and brainstem respiratory control neurons (Aim 2). This work is a collaborative effort between a respiratory control scientist (Fuller), an AAV specialist and clinician working with Pompe patients (Byrne), and an AAV specialist with expertise in stereotaxic delivery (Mandel).