Preclinical Development of Antisense Oligonucleotide Therapy for Spinocerebellar Ataxia Type 3

脊髓小脑性共济失调3型（SCA3），也称为Machado-Joseph病（MJD），是世界上最常见的显性遗传性共济失调，由ATXN3基因中编码多谷氨酰胺的CAG重复扩增引起。目前还没有有效的治疗方法来治疗这种不断发展和致命的疾病。由于突变蛋白的表达是疾病发病的早期和必要步骤，因此减少疾病基因本身表达的策略在潜在治疗方法列表中名列前茅。我们之前的治疗研究表明，基因沉默试剂需要广泛的中枢神经系统递送。化学修饰的ASOs可以广泛地传递到中枢神经系统，并且在体内具有高度稳定性。先前使用ASOs的研究已经证明，在小鼠和非人灵长类动物模型中，包括SMA、HD和ALS在内的神经退行性疾病具有良好的耐受性和长期的抑制作用。此外，经过多次非常成功的人体临床试验，ASO治疗脊髓性肌萎缩症（SMA）最近获得了FDA和ema的批准。我们最近在SCA3小鼠模型中建立了ASO治疗的概念验证，结论是降低患者非必需ATXN3疾病蛋白水平的策略可能具有良好的耐受性。该U01发现提案从概念验证研究扩展到描述用于ind研究的最终人类候选先导ASO化合物。Aim 1将通过体外筛选炎症和脱靶效应以及啮齿动物和非人类灵长类动物的体内耐受性来证实4-6先导ATXN3 ASOs的可行性，这将支持ind研究。目的2将在表达全长突变ATXN3转录物的SCA3小鼠模型中评估ATXN3 ASOs在抑制突变ATXN3表达、改善行为缺陷、改变疾病病理和分子特征方面的体内疗效。这些结果将最终为未来的ind研究选择一种先导化合物。在这些研究的同时，治疗成功的一个必要的下一步是优化SCA3疾病生物标志物，这些生物标志物可以直接评估治疗靶点的参与和治疗反应。脑脊液（CSF）是寻找治疗性生物标志物的理想生物样本，因为它易于获取，并且可以在疾病进展和治疗试验期间反复取样。免疫分析技术的重大进步现在使得使用超灵敏单分子计数（SMC）免疫分析检测系统来量化低丰度蛋白质成为可能。在Aim 3中，我们将优化SMC免疫分析法来检测SCA3患者CSF样本中的ATXN3蛋白水平。开发这种新的ATXN3 SMC免疫分析法将能够检测SCA3患者ASO治疗期间的体内治疗靶点参与和疗效。

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD), is the most common dominantly inherited ataxia in the world and caused by an expansion of a polyglutamine-coding CAG repeat in the ATXN3 gene. There currently is no effective treatment for this relentlessly progressive and fatal disease. Because expression of the mutant protein is an early and necessary step in disease pathogenesis, strategies to reduce expression of the disease gene itself are high on the list of potential therapies. Our previous therapeutic studies suggested the need for broad CNS delivery of gene silencing reagents. Chemically modified ASOs can be delivered broadly to the CNS and are known to be highly stable in vivo. Prior studies employing ASOs have documented well-tolerated, long-term knockdown in mouse and non-human primate models of neurodegenerative diseases including SMA, HD and ALS. Moreover, ASO therapy in spinal muscular atrophy (SMA) was recently FDA- and EMA-approved following multiple highly successful human clinical trials. We recently established ASO therapy proof-of-concept in a SCA3 mouse model, concluding that the strategy to reduce levels of the nonessential ATXN3 disease protein in patients would likely be well tolerated. This U01 discovery proposal extends from the proof-of-concept studies to characterize a final human candidate lead ASO compound for IND-enabling studies. Aim 1 will confirm the viability of 4-6 lead ATXN3 ASOs through in vitro screens for inflammatory and off-target effects and in vivo tolerability in rodent and non-human primates that will support IND-enabling studies. Aim 2 will assess the in vivo efficacy of the lead ATXN3 ASOs to suppress mutant ATXN3 expression and ameliorate behavioral deficits, alter disease pathology and molecular signatures in a SCA3 mouse model expressing the full-length human mutant ATXN3 transcript. The results will culminate in the selection of a single lead compound for future IND-enabling studies. Concurrent with these studies, a necessary next step for therapeutic success is the optimization of SCA3 disease biomarkers that may directly assess therapeutic target engagement and treatment response. Cerebrospinal fluid (CSF) is an ideal biological sample in which to look for therapeutic biomarkers as it is readily accessible and can be sampled repeatedly throughout disease progression and therapeutic trials. Significant advances in immunoassay technology now make it possible to quantify low abundance proteins using an ultrasensitive single molecule counting (SMC) immunoassay detection system. In Aim 3, we will optimize a SMC immunoassay to detect ATXN3 protein levels from SCA3 patient CSF samples. Developing this novel ATXN3 SMC immunoassay will enable detection of in vivo therapeutic target engagement and efficacy during ASO treatment in SCA3 patients.