Antisense oligonucleotide treatment for Pompe disease

项目摘要： 庞贝氏症是一种破坏性的肌病，由于缺乏溶酶体酶酸性α- 葡萄糖苷酶（GAA），导致糖原在溶酶体和细胞质中的积累。护理标准是 重组人（rh）GAA的酶替代疗法（ERT）。庞贝氏症患者通常有 由于药物在清除肌糖原储备方面的疗效有限，结果不佳。底物减少 SRT疗法对其他溶酶体贮积病如戈谢病和法布里病有效。我们提出 通过敲低糖原合成酶（GSY 1），我们将减少肌肉中的糖原合成，从而减少 需要被酶清除的底物的量。我们的长期目标是开发一种有效的治疗方法 以阻止/减少庞贝氏症中肌肉疾病的进展。主要差距：尽管有ERT， 继续在溶酶体中积累糖原，导致肌肉纤维化和脂肪替代。有效疗法 因此，庞贝氏症的进行性肌无力是一个巨大的未满足的需求。初步结果： 反义寡核苷酸（ASO）技术已成为一种强大的直接治疗替代品， 用于治疗遗传性疾病的常规小分子方法或基因置换策略 例如脊髓性肌萎缩症。ASO是短的合成单链DNA序列，旨在靶向 RNA通过良好表征的沃森-克里克碱基配对，一旦与靶RNA结合，可以调节RNA 通过使用RnaseH1的降解机制发挥作用。Ionis Pharmaceuticals设计并筛选了ASO 体外靶向小鼠GYS 1以鉴定最有效的ASO。屏幕上的主要ASO是 在剂量反应研究中验证，并筛选前10个ASO用于在野生型小鼠中测试。三 GYS 1 ASO显示出最佳的耐受性和功效特征，导致GYS 1 mRNA通过免疫抑制被敲低。 控制的50%左右。GYS 1 ASO 25 mg/kg/周对肌肉影响的临床前研究 1月龄和3月龄Pompe小鼠的糖原、组织学和肌肉功能表明，GYS 1 ASO 有效地敲除GYS 1约85%，清除率提高约50 肌糖原水平因此，我们认为，早期减少糖原底物或联合治疗， 可能是成功治疗庞贝氏症的关键。假设：我们提出GYS 1 ASO 在庞贝氏症小鼠中，如果开始作为单一疗法， 早期或与ERT联合使用，以获得最大的治疗获益。具体目标：目标1：确定 最有效的ASO介导的糖原合酶（GYS 1）敲低，以防止糖原积累 在庞贝氏症的小鼠模型中。目的2：使用GYS 1纠正肌糖原和肌无力 反义寡核苷酸与酶替代疗法（ERT）的组合在庞贝氏症小鼠中的应用。成功 这项转化研究将为庞贝氏症的新的有效治疗铺平道路。

Project Abstract: Pompe disease is a devastating myopathy resulting from the deficiency of the lysosomal enzyme acid alpha- glucosidase (GAA),resulting in accumulation of glycogen in lysosomes and cytoplasm. The standard of care is enzyme replacement therapy (ERT) with recombinant human (rh) GAA. Patients with Pompe disease often have poor outcomes due to limited efficacy of the drug in clearing muscle glycogen stores. Substrate Reduction Therapy (SRT) is effective in other lysosomal storage diseases such as Gaucher and Fabry disease. We propose that by knocking down glycogen synthase (GSY1), we will reduce glycogen synthesis in muscle, thus reducing the amount of substrate requiring to be cleared by enzyme. Our long-term goal is to develop a potent therapy to stop/reduce the progression of the muscle disease in Pompe disease. Major gaps: Despite ERT, patients continue to accumulate glycogen in lysosomes leading to muscle fibrosis and fatty replacement. Effective therapy for the progressive muscle weakness of Pompe disease is thus a huge unmet need. Preliminary results: Antisense Oligonucleotides (ASO) technology has emerged as a powerful direct therapeutic alternative to conventional small molecule approaches or gene replacement strategies for the treatment of genetic disorders such as spinal muscular atrophy. ASOs are short, synthetic single-stranded DNA sequences designed to target RNA by well-characterized Watson-Crick base pairing, and once bound to the target RNA, can modulate RNA function through a degradative mechanism using RnaseH1. Ionis Pharmaceuticals designed and screened ASOs targeting mouse GYS1 in vitro to identify the most efficacious ASO. The lead ASOs from the screen were validated in a dose response study and the top 10 ASOs were screened for testing in wild type mice. Three GYS1 ASOs showed the best tolerability and efficacy profile leading to knock down of GYS1 mRNA by approximately 50% of control. Preclinical studies of the effects of the GYS1 ASOs 25 mg/kg/weekly on muscle glycogen, histology, and muscle function in 1 month and 3-month-old Pompe mice indicated that GYS1 ASOs were effective in knocking down GYS1 by approximately 85%, with approximately 50% improvement in clearing muscle glycogen levels. We therefore believe that early reduction of glycogen substrate or a combination therapy with ERT may be the key to successful treatment in Pompe disease. Hypothesis: We propose that GYS1 ASOs in Pompe mice will reduce glycogen production and prevent muscle weakness, either as monotherapy if started early, or in combination with ERT, to obtain maximum therapeutic benefit. Specific Aims: Aim 1: Identification of the most effective ASO-mediated glycogen synthase (GYS1) knock down to prevent glycogen accumulation in a mouse model of Pompe disease. Aim 2: Correction of muscle glycogen and weakness using GYS1 antisense oligonucleotides in combination with Enzyme Replacement Therapy (ERT) in Pompe mice. Success with this translational study will pave the way for novel effective treatments for Pompe disease.