Antisense oligonucleotide treatment for Pompe disease

项目摘要： Pompe病是一种破坏性的肌肉疾病，由溶酶体酶酸性α-缺乏引起。 葡萄糖苷酶(GAA)，导致糖原在溶酶体和细胞质中积聚。护理的标准是 重组人(Rh)GAA的酶替代疗法。庞培病患者通常会有 由于该药物在清除肌肉糖原储存方面的疗效有限，结果很差。底物还原 治疗(SRT)对其他溶酶体储存性疾病有效，如高谢病和法布里病。我们建议 通过下调糖原合成酶(GSY1)，我们将减少肌肉中糖原的合成，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/减少庞培病肌肉疾病的进展。主要差距：尽管进行了ERT，但患者 继续在溶酶体中积累糖原，导致肌肉纤维化和脂肪替代。有效治疗 因为庞培病的进行性肌肉无力因此是一个巨大的未得到满足的需求。初步结果： 反义寡核苷酸(ASO)技术已经成为一种强大的直接治疗替代疗法 治疗遗传性疾病的传统小分子方法或基因替换策略 比如脊髓性肌萎缩症。ASO是一种短的、合成的单链DNA序列，旨在 RNA由具有良好特性的Watson-Crick碱基配对，并一旦与靶RNA结合，即可调控RNA 通过使用RnaseH1的降级机制发挥作用。Ionis制药公司设计和筛选ASOS 体外靶向小鼠GYS1，以确定最有效的ASO。屏幕上的主演ASO是 在剂量反应研究中得到了验证，并对排名前10位的ASO进行了筛选，以便在野生型小鼠身上进行测试。三 GYS1反义寡核苷酸表现出最好的耐受性和有效性，导致GYS1 mRNA的下调 大约50%的控制权。GYS1Asos 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GYS1 ASOS 有效地将GYS1降低了约85%，并在清算方面改进了约50% 肌肉糖原水平。因此，我们认为早期减少糖原底物或联合治疗 ERT可能是庞贝病治疗成功的关键。假设：我们提出GYS1 ASOS Pompe小鼠的治疗将减少糖原的产生并防止肌肉无力，如果开始作为单一治疗 早期或联合ERT，以获得最大的治疗效益。具体目标：目标1：确定 抑制ASO介导的糖原合成酶(GYS1)抑制糖原蓄积的最有效途径 在庞贝病的小鼠模型中。目的2：使用GYS1矫正肌肉糖原和虚弱 反义寡核苷酸联合酶替代疗法(ERT)在Pompe小鼠中的应用成功 这项翻译研究将为庞贝病的新的有效治疗方法铺平道路。

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.