Development of a SYF2 antisense oligonucleotide treatment for ALS and FTD

开发SYF 2反义寡核苷酸治疗ALS和FTD 项目摘要 肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）是由许多疾病引起的复杂疾病， 不同的遗传病因。尽管靶向特定因果突变（例如C9 ORF 72反义寡核苷酸）的治疗策略是有效的，但是， 尽管这些方法（例如，寡核苷酸（ASO））可以证明对个体形式的ALS或FTD有效，但这些方法不能解决 绝大多数病例的遗传病因不明。此外，考虑到大量不同的基因， 可能导致ALS和FTD，并且每种遗传形式都相对罕见，这种策略可能很难 适用于所有情况。因此，迫切需要新的治疗策略来挽救多种形式的ALS。 和FTD，特别是那些遗传病因不明的患者。 ALS和FTD的一个标志性病理学特征是TAR DNA结合蛋白43（TDP-43）从 在脑和脊髓中的神经元的细胞核中，它聚集成不溶性包涵体， >95%的ALS病例和~45%的FTD病例是死后发生的。虽然研究表明这些神经元TDP-43聚集体 驱动神经退行性变58，TDP-43从细胞核中的减少也改变了超过 1，500 RNA 9，包括STMN 2等疾病标志。因此，TDP-43从细胞核的丢失及其 细胞质中的聚集导致神经变性，开发解决这两个问题的治疗方法至关重要。 这一具有挑战性的病理学。 我们发现，抑制剪接体相关因子SYF 2的编码基因可抑制TDP-43的聚集 和错误定位，改善TDP-43活性，并挽救C9 ORF 72和散发性ALS存活。此外，Syf 2 抑制可改善TDP-43小鼠的神经变性、神经肌肉接头损失和运动功能障碍。小鼠 具有一个Syf 2功能丧失拷贝的人是健康的，携带SYF 2功能丧失等位基因的人不受 儿科疾病或已知在成年期受疾病影响。因此，剪接体相关因子的抑制 如SYF 2是ALS和FTD的一个有前途和广泛有效的遗传靶点。 反义寡核苷酸（Antisense oligonucleotides，ASO）是一种很有吸引力的中枢神经系统（central nervous system，CNS）基因靶点研究方法 像SYF 2一样，因为它们可以直接注射到脊髓中，在整个脊髓中实现持续的靶点接合， CNS，并且不太可能引起外周毒性。利用我们专有的阿索设计软件， 计算机模拟可能的SYF 2阿索序列，并优先考虑数百个预测具有增强稳定性的导联， 这有助于提高可制造性，并降低免疫原性和脱靶效应。该项目的目标是直接进入第二阶段， 从这些线索中确定真正开发候选SYF 2阿索，以促进研究性新产品 药物（IND）毒性研究。该项目的广泛目标是建立Syf 2的治疗指数 在TDP-43小鼠中的抑制，评估SYF 2在大组ALS中的效力、功效和耐受性 患者来源的神经元和细菌人工染色体人SYF 2转基因小鼠，最后评估线索 在啮齿类动物的探索性毒性研究中。

Development of a SYF2 antisense oligonucleotide treatment for ALS and FTD Project Summary Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are complex diseases that result from many diverse genetic etiologies. Although therapeutic strategies that target specific causal mutations (e.g. C9ORF72 antisense oligonucleotides (ASOs)) may prove effective against individual forms of ALS or FTD, these approaches cannot address the vast majority of cases that have unknown genetic etiology. Moreover, given the large number of different genes that likely contribute to ALS and FTD and the fact that each genetic form is relatively rare, this strategy may be difficult to implement for all cases. Thus, there is a pressing need for new therapeutic strategies that rescue multiple forms of ALS and FTD, particularly those with unknown genetic etiologies. A hallmark pathological feature of ALS and FTD is the depletion of TAR DNA-binding protein 43 (TDP-43) from the nucleus of neurons in the brain and spinal cord to the cytoplasm where it aggregates into insoluble inclusion bodies in >95% of ALS cases and ~45% of FTD cases post mortem. While studies suggest that these neuronal TDP-43 aggregates drive neurodegeneration58, reduction in TDP-43 from the nucleus also alters the splicing or expression levels of more than 1,500 RNAs9, including disease hallmarks such as STMN2. Thus, both the loss of TDP-43 from the nucleus and its aggregation in the cytoplasm contribute to neurodegeneration, and it is critical to develop treatments that address both aspects of this challenging pathology. We found that suppressing the gene encoding the spliceosome-associated factor SYF2 alleviates TDP-43 aggregation and mislocalization, improves TDP-43 activity, and rescues C9ORF72 and sporadic ALS survival. Moreover, Syf2 suppression ameliorates neurodegeneration, neuromuscular junction loss, and motor dysfunction in TDP-43 mice. Mice with one loss-of-function copy of Syf2 are healthy and humans carrying a loss-of-function SYF2 allele are not affected by pediatric diseases or known to be affected by disease in adulthood. Thus, suppression of spliceosome-associated factors such as SYF2 is a promising and broadly-effective genetic target for ALS and FTD. Antisense oligonucleotides (ASOs) are an attractive approach for genetic targets in the central nervous system (CNS) like SYF2 because they can be injected directly into the spinal cord, achieve sustained target engagement throughout the CNS, and are less likely to cause peripheral toxicity. Leveraging our proprietary ASO design software, we assessed all possible SYF2 ASO sequences in silico and prioritized several hundred leads predicted to have enhanced stability and manufacturability, and reduced immunogenicity and off-target effects. The objective of this Direct to Phase 2 project is to identify a bona fide development candidate SYF2 ASO from among these leads for advancement in investigational new drug (IND)-enabling toxicity studies. The broad aims of the project are to establish the therapeutic index of Syf2 suppression in TDP-43 mice, assess SYF2 leads for potency, efficacy and tolerability in a large panel of ALS patient-derived neurons and a bacterial artificial chromosome human SYF2 transgenic mouse, and lastly to assess the leads in an exploratory toxicity study in rodents.