Control of Breathing and Pompe Disease

项目概要/摘要： 庞贝氏症是由酸性α-葡萄糖苷酶（GAA）基因突变引起的，GAA是一种必需的酶， 降解溶酶体糖原。早发性疾病发生在缺乏功能性GAA的情况下，这导致 生命早期的心肺功能衰竭。迟发性疾病与GAA活性降低和渐进性 进展为呼吸衰竭。我们前两个资助周期的研究表明， Gaa-/-小鼠和庞贝氏症患者的呼吸衰竭。这是相关的，因为护理标准-静脉内 使用重组GAA的酶疗法不能到达中枢神经系统（CNS）， 进展为呼吸衰竭。我们的总体假设是，腺相关病毒（AAV）治疗是 能够在整个CNS、骨骼肌和心肌中恢复终生GAA表达，从而 保护心肺功能，延长生命。目的1关注用于早发性 需要早期治疗的疾病，可以预防呼吸和心脏衰竭。更好地 为了研究这个问题，我们建立了一个Gaa null（Gaa-/-）大鼠模型，该模型重现了早发性表型， 心肺病理学和早期死亡率。初步数据表明，新生儿AAV-GAA治疗 （结蛋白启动子，AAV 9血清型）不引起可检测的免疫应答，减轻心脏和呼吸系统疾病， 病理学和预防过早死亡。因此，我们假设在年轻人中单次静脉注射AAV-GAA剂量， 大鼠可以驱动持久和广泛的GAA表达并延长庞贝氏症大鼠的寿命。目的2 治疗晚发性庞贝氏症，其中呼吸衰竭是主要死亡原因。基于 根据我们前两个资助周期的数据，我们假设成年Pompe大鼠中的神经定向AAV-GAA治疗 足以防止呼吸功能下降并延长寿命。通过用肌肉包装AAV-GAA （肌酸激酶8）、神经（突触蛋白）或组织特异性（肌肉和神经、结蛋白）启动子，并递送 通过鞘内、静脉内或两者，我们可以驱动GAA表达的方式， 神经校正是必要的，足以防止衰退。将使用上述Gaa零大鼠 测试在缺乏内源性GAA活性的情况下神经与肌肉校正的概念验证。我们将 我还使用了另一种新的Pompe大鼠模型，其中CRISPR/cas9被用于插入最常见的 人类基因突变导致晚发型庞贝氏症（IVS 1）到大鼠基因组。这很重要 因为IVS 1突变导致GAA活性降低但并非缺失，并且与延迟的GAA相关。 进展为呼吸衰竭。这项工作是有意义的，因为目前的治疗策略， 庞贝氏症仅延迟疾病进展并最终导致呼吸衰竭。这里提出的战略 也将有助于推进神经退行性疾病的基因治疗的更广泛目标， 常染色体隐性遗传病

Project Summary / Abstract: Pompe disease results from mutations in the gene for acid α-glucosidase (GAA) – an enzyme necessary to degrade lysosomal glycogen. Early-onset disease occurs in the absence of functional GAA which leads to cardiorespiratory failure early in life. Late-onset disease is associated with reduced GAA activity and gradual progression to respiratory failure. Work from our first two grant cycles indicates neural involvement in respiratory failure in Gaa-/- mice and Pompe patients. This is relevant since the standard of care – intravenous enzyme therapy using recombinant GAA - does not reach the central nervous system (CNS) and patients still progress to respiratory failure. Our overarching hypothesis is that adeno-associated virus (AAV) therapy is capable of restoring life-long GAA expression throughout the CNS, skeletal and cardiac muscle, thereby preserving cardiorespiratory function and prolonging life. Aim 1 focuses on AAV therapy for early-onset disease which requires early life treatments that can prevent both respiratory and cardiac failure. To better study this problem, we created a Gaa null (Gaa-/-) rat model which recapitulates the early onset phenotype with cardiorespiratory pathology and early mortality. Preliminary data indicate that neonatal AAV-GAA therapy (desmin promoter, AAV9 serotype) evokes no detectable immune response, mitigates cardiac and respiratory pathology and prevents early death. Thus, we hypothesize that a single intravenous AAV-GAA dose in young rats can drive persistent and widespread GAA expression and extend the Pompe rat lifespan. Aim 2 addresses late onset Pompe disease in which respiratory failure is the primary cause of mortality. Based on data from our first two grant cycles we hypothesize that neural directed AAV-GAA therapy in adult Pompe rats is sufficient to prevent respiratory decline and extend the lifespan. By packaging AAV-GAA with muscle (creatine kinase 8), neural (synapsin) or tissue specific (muscle and neural, desmin) promoters, and delivering the vector intrathecally, intravenously, or both, we can drive GAA expression in a manner that will determine if neural correction is necessary and sufficient to prevent decline. The aforementioned Gaa null rat will be used to test proof-of-concept for neural vs. muscle correction in the absence of endogenous GAA activity. We will also use another new Pompe rat model in which CRISPR/cas9 has been used to insert the most common human gene mutation causing late-onset Pompe disease (IVS1) into the rat genome. This is important because the IVS1 mutation leads to low but not absent GAA activity and is associated with delayed progression to respiratory failure. The proposed work is significant because current therapeutic strategies in Pompe disease only delay disease progression with eventual respiratory failure. The strategies proposed here will also contribute to the broader goal of advancing gene therapy for neurodegenerative conditions and autosomal recessive diseases.