Spinal and brainstem respiratory neurons in Pompe disease

庞贝病中的脊髓和脑干呼吸神经元

• 批准号: 8426726
• 负责人: BARRY J BYRNE
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8426726
• 关键词: A Mouse; Acids; Alpha-glucosidase; Applications Grants; Automobile Driving; Blood - brain barrier anatomy; Brain Stem; Breathing; Cell Nucleus; Cervical spinal cord structure; Clinical; Clinical Trials; Communities; Complement; Data; Dependovirus; Development; Disease; Environmental air flow; Enzymes; Functional disorder; Gene Deletion; Gene Expression; Gene Transfer; Genes; Genetic; Genetic Recombination; Glycogen; Glycogen storage disease type II; Goals; Histopathology; Horns; Hypercapnic respiratory failure; Impairment; Infusion procedures; Injection of therapeutic agent; Knock-out; Left; Mediating; Motor; Motor Neurons; Motor output; Mus; Muscle; Muscle function; Mutation; Myocardium; Neuraxis; Neuromuscular Diseases; Neurons; Neurophysiology - biologic function; Output; Pathology; Patients; Pattern; Proteins; Recombinants; Relative (related person); Respiratory Diaphragm; Respiratory Insufficiency; Respiratory Muscles; Role; Scientist; Site; Skeletal Muscle; Specialist; Spinal; Spinal Cord; System; Testing; Therapeutic; Thinking; Time; Variant; Viral; Work; adeno-associated viral vector; clinically relevant; enzyme deficiency; functional disability; glucosidase; knockout gene; motor control; neuropathology; recombinase; research study; respiratory; retrograde transport; vector

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). PUBLIC HEALTH RELEVANCE: Pompe disease is a lysosomal storage disorder associated with systemic deficiency of an enzyme (acid alpha glucosidase) which is required to degrade glycogen. We hypothesize that part of the reason that breathing problems are common in Pompe disease is that brainstem neurons and spinal cord motoneurons fail to adequately control the respiratory muscles. To test this hypothesis, we propose to use genetic approaches to "knockout" the acid alpha glucosidase gene in spinal and medullary respiratory neurons of mice while leaving skeletal and cardiac muscle gene expression unaltered.

描述（由申请人提供）：庞贝疾病是一种由酸A-糖苷酶（GAA）的​​突变引起的神经肌肉疾病 - 一种降解溶酶体糖原所需的酶。衰减不足是所有形式的庞贝疾病的标志性特征，历史上一直归因于呼吸道肌肉病理学。 R21赠款应用中提出的实验将提供有关庞贝疾病呼吸不足的临床问题的基本机械信息。最重要的是，我们提出直接检验中枢神经系统功能障碍是导致庞贝疾病呼吸不足的主要因素。有证据支持这一假设，但缺乏明确的证据。为了实现这一目标，我们建议使用“特定位点”的CRE-LOX重组方法在小鼠的脊柱和髓质呼吸神经元中敲除GAA基因，同时留下骨骼和心脏肌肉基因表达未改变。如果确认该假设将告知临床界，请告知Pompe患者呼吸不足的根本原因。更重要的是，确认我们的假设将有必要从目前对纯肌肉定向疗法的重点转变为影响肌肉和神经功能的方法。因此，拟议的研究的总体目标是确定当呼吸神经元中的GAA基因表达“敲除”时，呼吸控制系统是否变得功能失调。我们还建议比较和对比脊柱运动神经元与髓质呼吸道控制神经元在庞贝病中呼吸运动障碍方面的作用。 GAA基因转移到Pompe患者的隔膜的初步临床试验正在进行中（ClinicalTrials.gov：NCT00976352）。这项工作将通过研究运动神经元与髓质神经元对呼吸不足的重要性来补充正在进行的试验。这很重要，因为可以通过注射后注射后逆行病毒转运来转导Phrenic的运动神经元，而髓质神经元则不会。因此，临床试验不太可能导致髓质呼吸神经元转导。在开发该应用时，我们获得了具有“ floxed” GAA基因的小鼠集落。我们建议使用驱动C​​RE重组酶表达的AAV载体的立体定位和/或逆行递送，以选择性地敲除脊髓呼吸道（PHRENIC）运动神经元（AIM 1）和脑干呼吸控制神经元中的GAA基因（AIM 2）。这项工作是呼吸控制科学家（Fuller），AAV专家和与Pompe患者（Byrne）（Byrne）合作的临床医生与具有立体定位交付专业知识（Mandel）专业知识的AAV专家之间的合作努力。 公共卫生相关性：庞贝疾病是一种溶酶体储存障碍，与降解糖原降解所需的酶（酸α葡萄糖酶）的全身性缺乏相关。我们假设呼吸问题在庞贝病中常见的部分原因是脑干神经元和脊髓运动神经元无法充分控制呼吸肌肉。为了检验这一假设，我们建议使用遗传学方法在小鼠的脊柱和髓质呼吸神经元中“敲除”酸α葡萄糖苷酶基因，同时留下未改变骨骼和心肌基因表达。