Control of Breathing and Pompe Disease

描述（由申请人提供）：庞贝病是一种由酸基因突变引起的神经肌肉疾病。-葡萄糖苷酶(GAA) -降解溶酶体糖原所必需的酶。通气不足是庞培病的一个标志性特征，从该资助的第一个周期开始的工作表明，神经病理学有助于庞培呼吸问题。这一点很重要，因为目前Pompe病的治疗方法——使用重组GAA的静脉酶替代疗法（ERT）——并不针对中枢神经系统（CNS）。这项更新应用的目标是优化基于腺相关病毒（AAV）的治疗方法来治疗庞贝病的中枢神经系统。Aim 1将使用AAV9的逆行转运来确定选择性靶向整个运动单元（肌肉和运动神经元）的基因治疗是否可以纠正特定的运动系统。具体而言，Aim 1将验证将编码GAA基因的AAV9载体（AAV9-GAA）注射到Pompe （GAA -/-）小鼠的舌头中会引起GAA在肌肉和运动神经元中的表达，从而恢复舌头运动功能的假设。我们强调舌下运动系统，因为它在庞贝病中受损，影响言语、吞咽和呼吸，并且对静脉ERT没有反应。目的2将通过测试Gaa-/-小鼠内胆和静脉注射AAV9-GAA会传导脊髓和脑干神经元，并恢复舌和膈肌运动功能的假设，重点关注广泛的中枢神经系统转导。目的2强调舌头和横膈膜，因为呼吸受损、呼吸机依赖和舌头运动问题是庞贝病的主要问题。目的3是基于提高GAA清除神经元糖原积累的能力。我们最近评估了一种重组GAA的修饰形式，其中人GAA与胰岛素样生长因子II受体（IGF-IIR）的配体融合。合成的融合蛋白对糖原具有充分的催化活性，并且在我们的小鼠模型中显示出增强的减少糖原积累的能力。GAA转基因的另一个修饰将使我们能够评估GAA的高度保守区域，该区域促进加工到最具催化活性的70kDa成熟形式。为了最终目的，我们建议将这些增强形式的GAA基因包装到AAV9中，以测试修饰的GAA蛋白更有效地靶向运动神经元的假设。为了实现这三个目标，将使用一系列综合的结果测量来表征呼吸功能和AAV9转导。这项工作是临床医生和基因治疗研究员（B.J. Byrne）以及呼吸控制科学家（D.D. Fuller）的合作成果。我们认为这项工作意义重大，因为庞贝病治疗的现状是肌肉定向ERT。两周一次的ERT治疗需要大量的努力，每年50万美元的费用，潜在的免疫反应和ERT有限的成功需要改进的方法。这项工作的整体创新在于我们正在开发新的基于AAV9的庞贝病呼吸功能不全的治疗方法。

DESCRIPTION (provided by applicant): Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid ?-glucosidase (GAA) - an enzyme necessary to degrade lysosomal glycogen. Hypoventilation is a hallmark feature of Pompe disease and work from the first cycle of this grant demonstrated that neuropathology contributes to Pompe breathing problems. This is important since the current therapy for Pompe disease - intravenous enzyme replacement therapy (ERT) using recombinant GAA - does not target the central nervous system (CNS). This renewal application targets optimization of adeno-associated virus (AAV) based therapies to treat the CNS in Pompe disease. Aim 1 will use retrograde transport of AAV9 will be used to determine if gene therapy that selectively targets the entire motor unit (muscle and motoneuron) can correct a specific motor system. Specifically, Aim 1 will test the hypotheses that injection of AAV9 vector encoding the GAA gene (AAV9-GAA) into the tongue of Pompe (Gaa-/-) mice will cause GAA expression in muscle and motoneurons, and will restore tongue motor function. The hypoglossal motor system is being emphasized since it is impaired in Pompe disease with consequences to speech, swallow and breathing and does not respond to intravenous ERT. Aim 2 will focus on widespread CNS transduction by testing the hypotheses that intracisternal and intravenous AAV9-GAA delivery in Gaa-/- mice will transduce spinal cord and brainstem neurons, and will restore both tongue and diaphragm motor function. Aim 2 emphasizes the tongue and diaphragm since impaired breathing, ventilator-dependence and tongue motor problems are primary concerns in Pompe disease. Aim 3 is based on improving the ability of GAA to clear neuronal glycogen accumulation. We recently evaluated a modified form of recombinant GAA in which human GAA is fused to the ligand of the insulin-like growth factor II receptor (IGF-IIR). The resultant fusion protein has full catalytic activity for glycogenand shows an enhanced ability to reduce glycogen accumulation in our mouse model. Another modification of the GAA transgene will allow us to evaluate a highly conserved region of GAA which promotes processing to the most catalytically active 70kDa mature form of the enzyme. For the final aim we propose to package the gene for these enhanced forms of GAA into AAV9 to test the hypotheses that the modified GAA proteins more effectively target motoneurons. For all three aims a comprehensive series of outcome measures will be used to characterize respiratory function and AAV9 transduction. This work is a collaborative effort between a clinician and gene therapy researcher (B.J. Byrne) and a respiratory control scientist (D.D. Fuller). We believe this work is significant because the status quo in Pompe disease therapy is muscle-directed ERT. The substantial effort needed for bi-weekly ERT treatment, cost of >$500K per year, potential immune responses and limited success of ERT warrant an improved approach. The overall innovation of this work is that we are developing new AAV9 based therapies for respiratory insufficiency in Pompe disease.