Antisense Oligonucleotides for treating Spinocerebellar Ataxia Type 2

神经退行性疾病是一个不断增加的社会和经济负担，世卫组织估计，到2040年，这些疾病将取代癌症，成为第二大死亡原因。在神经退行性疾病的研究中，已经发现了大量的通路，但它们与各自的人类疾病的直接和主要相关性一直很难证明，而且针对通路的定位仍然困难。这项拟议的工作将确定一种治疗脊髓小脑性共济失调2型(SCA2)的方法，这是一种遗传性神经退行性疾病，影响小脑浦肯野细胞(PC)和小脑、皮质下灰质和脊髓的其他神经元。SCA2的原因是ATXN2基因的CAG功能增强，导致ataxin-2的聚谷氨酰胺(PolyQ)结构域扩大。我们的目标是鉴定能够降低ATXN2表达的高效反义寡核苷酸(ASO)。我们的理论基础是基于对模式生物和人类的观察，结果表明，更高剂量的突变等位基因/蛋白会恶化疾病严重程度，即使在小鼠出现症状后，啮齿动物中突变PolyQ蛋白表达的下调也会逆转临床表现。此外，在小鼠中完全敲除ATXN2不会导致神经退化或过早死亡。这项研究的优点得到了正在进行的临床试验的积极结果的支持，这些试验测试了ASO治疗肌萎缩侧索硬化症和强直性肌营养不良1型疾病的疗效，以及SPINRAZA™(Nusinesen)治疗脊髓性肌萎缩症的成功，这是FDA批准的第一种治疗神经退行性疾病的ASO药物。这项研究的可行性是基于我们的积极概念验证数据，该数据表明，我们的主导ATXN2ASO在症状出现后延迟了进行性啮齿动物SCA2的运动、分子和神经生理表型。他们提出了五个具体目标：1)体外密集筛选靶向于整个ATXN2前体mRNA的ASO，包括SCA2患者细胞；2)在体内筛选以确定降低小鼠小脑ATXN2水平的先导药物；3)对啮齿动物和其他物种进行安全性毒性测试；4)测试ASO先导化合物用于延迟已建立的SCA2小鼠运动、分子和神经生理学表型的作用；以及5)GMP生产单一最有效的ASO候选药物，并与FDA成员举行预研新药(IND)会议。这项拟议的工作将为神经退行性疾病的治疗开辟新的天地，因为它证明了用反义寡核苷酸靶向显性作用突变的多聚Q基因的可行性。

Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease has been difficult to prove and targeting of pathways has remained difficult. The proposed work will identify a treatment for spinocerebellar ataxia type 2 (SCA2), a hereditary neurodegenerative disease affecting cerebellar Purkinje cells (PCs) and other neurons in the cerebellum, the subcortical grey matter and spinal cord. The cause of SCA2 is a gain-of-function CAG expansion in the ATXN2 gene resulting in an expanded polyglutamine (polyQ) domain in ataxin-2. Our objective is identification of highly-potent antisense oligonucleotides (ASOs) that lower ATXN2 expression. Our rationale is based on observations in model organisms and humans indicating that higher dosages of the mutant allele/protein worsen disease severity and that down-regulation of mutant polyQ protein expression in rodents reverses clinical manifestations even after mice have become symptomatic. Additionally, complete knock-out of ATXN2 in mice does not cause neurodegeneration or premature death. Merit for this study is supported by positive results in ongoing clinical trials to test ASOs for treating amyotrophic lateral sclerosis (ALS) and myotonic dystrophy type 1 (DM1), as well as the success of SPINRAZA™ (Nusinersen) for spinal muscular atrophy (SMA), the first ever FDA approved ASO drug for a neurodegenerative disorder. The feasibility of this study is based on our positive proof-of-concept data demonstrating that our lead ATXN2 ASO delays progressive rodent SCA2 motor, molecular, and neurophysiological phenotypes after symptom onset. Five specific aims are proposed: 1) an intensive in vitro screen of ASOs targeting throughout the ATXN2 pre-mRNA including cultured SCA2 patient cells, 2) in vivo screens to identify leads lowering cerebellar ATXN2 in mice, 3) safety toxicity testing in rodents and other species, 4) testing the ASO leads for delaying established SCA2 mouse motor, molecular, and neurophysiological phenotypes, and 5) GMP manufacturing of the single most potent ASO candidate and pre-investigative new drug (IND) meetings with members of the FDA. The proposed work will break new ground for treatment of neurodegenerative diseases by demonstrating feasibility of targeting dominant-acting mutated polyQ genes with antisense oligonucleotides.