Control of Breathing and Pompe Disease

描述(申请人提供)：庞培病是一种神经肌肉疾病，由酸性基因突变引起。 α-葡萄糖苷酶--一种降解溶酶体糖原所必需的酶。换气不足是庞培病的一个显著特征，从这笔赠款的第一个周期开始的工作表明，神经病理学导致庞贝呼吸问题。这一点很重要，因为目前治疗庞培病的方法--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)。这一新的应用旨在优化以腺相关病毒(AAV)为基础的治疗庞培病中枢神经系统的方法。目标1将使用AAV9的逆行运输将被用于确定选择性地针对整个运动单位(肌肉和运动神经元)的基因疗法是否可以纠正特定的运动系统。具体地说，目标1将检验这样的假设，即将编码GAA基因的AAV9载体(AAV9-GAA)注射到Pompe(GAA-/-)小鼠的舌内将导致肌肉和运动神经元中GAA的表达，并将恢复舌体运动功能。由于Pompe病的舌下运动系统受损，导致言语、吞咽和呼吸障碍，对静脉注射ERT无效，因此受到重视。目的2将通过测试脑池内和静脉注射AAV9-GAA将在GAA-/-小鼠体内传递脊髓和脑干神经元，并将恢复舌肌和横隔膜运动功能的假说，专注于广泛的中枢神经系统转导。目的2强调舌和横隔膜，因为呼吸障碍、呼吸机依赖和舌运动问题是庞贝病的主要问题。目的3是基于提高GAA清除神经糖原积聚的能力。我们最近评估了一种改良形式的重组GAA，其中人GAA与胰岛素样生长因子II受体(IGF-IIR)的配体融合。得到的融合蛋白对糖原具有完全的催化活性，并在我们的小鼠模型中显示出增强的减少糖原积聚的能力。对GAA转基因的另一个修改将使我们能够评估GAA的一个高度保守的区域，该区域促进对酶的最催化活性的70 kDa成熟形式的加工。为了达到最终目的，我们建议将这些增强形式的GAA的基因包装到AAV9中，以检验修改后的GAA蛋白更有效地靶向运动神经元的假设。对于所有这三个目标，将使用一系列全面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)和呼吸控制科学家(D.D.Fuller)共同努力的结果。我们认为这项工作意义重大，因为庞培病治疗的现状是肌肉导向的ERT。两周一次的ERT治疗所需的巨大努力、每年50万美元的费用、潜在的免疫反应以及ERT有限的成功证明了改进方法的必要性。这项工作的总体创新之处在于，我们正在开发基于AAV9的治疗庞贝病呼吸功能不全的新疗法。

DESCRIPTION (provided by applicant): Pompe disease is a neuromuscular disorder resulting from mutations in the gene for acid α-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.