Preclinical Development of Antisense Oligonucleotide Therapy for Spinocerebellar Ataxia Type 3

脊髓小脑性共济失调3型（SCA 3），也称为马查多-约瑟夫病（MJD），是世界上最常见的显性遗传性共济失调，由ATXN 3基因中编码多聚谷氨酰胺的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无情的渐进和致命的疾病。由于突变蛋白的表达是疾病发病机制中的早期和必要步骤，因此减少疾病基因本身表达的策略在潜在疗法中名列前茅。我们以前的治疗研究表明，需要广泛的中枢神经系统基因沉默试剂的交付。化学修饰的ASO可以广泛地递送至CNS，并且已知在体内高度稳定。采用ASO的先前研究已经记录了在神经退行性疾病（包括SMA、HD和ALS）的小鼠和非人灵长类动物模型中良好耐受的长期敲低。此外，脊髓性肌萎缩症（SMA）的阿索疗法最近在多次非常成功的人体临床试验后获得FDA和EMA批准。我们最近在SCA 3小鼠模型中建立了阿索疗法的概念验证，得出的结论是，降低患者非必需ATXN 3疾病蛋白水平的策略可能耐受良好。该U 01发现提案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先导阿索化合物。目标1将通过体外筛选啮齿动物和非人灵长类动物的炎症和脱靶效应以及体内耐受性来确认4-6导联ATXN 3 ASO的活力，这将支持IND使能研究。目的2将评估前导ATXN 3 ASO在表达全长人突变ATXN 3转录物的SCA 3小鼠模型中抑制突变ATXN 3表达并改善行为缺陷、改变疾病病理学和分子特征的体内功效。结果将最终选择一个单一的铅化合物为未来的IND使能研究。与这些研究同时，治疗成功的必要下一步是SCA 3疾病生物标志物的优化，可以直接评估治疗靶点参与和治疗反应。脑脊液（CSF）是寻找治疗生物标志物的理想生物样本，因为它易于获得，并且可以在疾病进展和治疗试验中重复采样。免疫测定技术的显著进步现在使得使用超灵敏单分子计数（SMC）免疫测定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免疫测定以检测来自SCA 3患者CSF样品的ATXN 3蛋白水平。开发这种新型ATXN 3 SMC免疫测定将能够检测SCA 3患者中阿索治疗期间的体内治疗靶点接合和疗效。

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.