Preclinical Development of Antisense Oligonucleotide Therapy for Spinocerebellar Ataxia Type 3

脊髓小脑型共济失调3型(SCA3)，又称马查多-约瑟夫病(MJD)，是世界上最常见的遗传性共济失调，由ATXN3基因编码多谷氨酰胺的CAG重复序列扩增引起。目前还没有有效的治疗方法来治疗这种持续不断和致命的疾病。由于突变蛋白的表达是疾病发病机制中早期和必要的步骤，因此减少疾病基因本身表达的策略是潜在治疗方法的重点。我们以前的治疗研究表明，需要广泛的中枢神经系统传递基因沉默试剂。化学修饰的ASO可以广泛地传递到中枢神经系统，并在体内高度稳定。之前使用ASOS的研究已经证明，在包括SMA、HD和ALS在内的神经退行性疾病的小鼠和非人类灵长类动物模型中，ASOS具有良好的耐受性和长期的击倒作用。此外，在多次非常成功的人类临床试验之后，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要ATXN3疾病蛋白水平的策略可能会被很好地耐受。这项U01发现建议从概念验证研究扩展到表征用于IND使能研究的最终人类候选ASO化合物。目的1将通过体外筛选炎症和非靶点效应以及在啮齿动物和非人类灵长类动物中的体内耐受性来确认4-6先导ATXN3 ASO的生存能力，这将支持IND使能研究。目的2评价先导ATXN3反义寡核苷酸在SCA3小鼠模型中抑制突变ATXN3表达、改善行为缺陷、改变疾病病理和分子特征的效果。结果将最终选定一种单一的先导化合物用于未来的IND研究。在这些研究的同时，治疗成功的下一个必要步骤是优化SCA3疾病生物标记物，这些标记物可以直接评估治疗靶点的参与和治疗反应。脑脊液(CSF)是寻找治疗生物标志物的理想生物样本，因为它很容易获得，并且可以在疾病进展和治疗试验中重复采样。免疫分析技术的重大进步现在使得使用超灵敏的单分子计数(SMC)免疫分析检测系统来定量低丰度蛋白质成为可能。在目标3中，我们将优化SMC免疫分析方法，以检测SCA3患者脑脊液样本中ATXN3蛋白水平。开发这一新的ATXN3 SMC免疫分析将使在SCA3患者ASO治疗期间能够检测体内治疗靶点的参与和疗效。

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.