Expanding the Chemical Diversity of Therapeutic Oligonucleotides

项目总结/摘要 治疗性寡核苷酸化合物（例如，siRNA，反义）有望成为 治疗许多遗传学定义的神经退行性疾病，包括亨廷顿病（HD）和 肌萎缩侧索硬化症（ALS）。治疗性寡核苷酸通过靶向沉默疾病基因， 降解mRNA，从而阻止有毒基因产物的表达。序列特异性和长- 治疗性寡核苷酸的持久作用提供了强有力的治疗范例，只要它们可以 递送到相关的靶组织。在NINDS的资助下，我们已经鉴定了两类治疗性siRNA， 在中枢神经系统（CNS）中显示出稳健的分布和功效：二价siRNA和亲脂性siRNA 共轭这一发现导致了变革性的治疗候选人向正式的 临床调查然而，有效、均匀、长期、等位基因选择性、无毒的递送仍然是一个挑战。 在扩大寡核苷酸药物治疗神经退行性疾病方面存在重大障碍。 该提案旨在开发和表征能够实现均匀分布的创新化学品， 多靶点沉默和体内CNS中的核内基因调节。该提案描述了一类 多价siRNA化合物，其表现出完全的代谢稳定性、在脊髓中的稳健分布 和脑（啮齿动物、绵羊、NHP），当通过脑脊液（CSF）输注时，神经元有效摄取，以及 有效和持久的沉默无毒性至少六个月后，单次注射。而发展中 CNS活性的二价siRNA，我们开发了称为exNA的稳定骨架化学。在这里，我们建议 结构-功能研究，以研究化合价（以上两种）、接头和基于exNA的稳定性如何影响 治疗活性。值得注意的是，化合价的增加进一步降低了CSF的清除率，而exNA- 基于稳定化增强了效果的持续时间。此外，调节化学结构可以改变 siRNA从细胞质到细胞核的细胞内分布，以靶向细胞核定位的mRNA。 最后，使用正交化学来合成多靶向化合物对于治疗 病因复杂的疾病。 该提案的完成将：（i）优化多价，超稳定的配置，支持统一， 在中枢神经系统中有效、特异和持久的多靶点沉默（超过6个月）;以及 (ii)为HD和ALS的新型组合治疗建立发展途径。另外这款 一项提案寻求建立一个技术平台，直接针对任何基因或基因组合 在中枢神经系统的任何区域表达。因此，顺利完成这项工作将使 中枢神经系统基因功能的研究和基于阿糖胞苷的新型药物的开发 基因定义的神经退行性疾病的治疗方法。

Project Summary/Abstract Therapeutic oligonucleotide compounds (e.g., siRNA, antisense) hold promise as transformative drugs for the treatment of many genetically-defined neurodegenerative disorders, including Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). Therapeutic oligonucleotides silence disease genes by targeting and degrading mRNA, thus preventing the expression of toxic gene products. The sequence specificity and long- lasting effect of therapeutic oligonucleotides provide a powerful therapeutic paradigm, as long as they can be delivered to the relevant target tissue. Funded by NINDS, we have identified two classes of therapeutic siRNA that show robust distribution and efficacy in the central nervous system (CNS): di-valent siRNAs and lipophilic conjugates. This discovery has resulted in transformative therapeutic candidates advancing towards formal clinic investigation. Nevertheless, efficient, uniform, long-term, allele-selective, non-toxic delivery remains a significant hurdle in expanding oligonucleotide drugs to treat neurodegenerative disorders. This proposal aims to develop and characterize innovative chemistries that enable uniform distribution, multi-target silencing, and intra-nuclear gene modulation in the CNS in vivo. This proposal describes a class of multivalent siRNA compounds that exhibit complete metabolic stability, robust distribution in the spinal cord and brain (rodents, sheep, NHPs) when infused via cerebrospinal fluid (CSF), efficient uptake by neurons, and potent and durable silencing without toxicity for at least six months after a single injection. While developing CNS-active di-valent siRNAs, we developed stabilizing backbone chemistry called exNA. Here, we propose structure-function studies to investigate how valency (above two), linker, and exNA-based stabilization affect therapeutic activity. Remarkably, increased valency further reduces the rate of CSF clearance, while exNA- based stabilization enhances the duration of effect. In addition, modulating the chemical architecture can shift the intracellular distribution of siRNA from the cytoplasmic to the nucleus to target nuclear-localized mRNA. Finally, the use of orthogonal chemistry to synthesize multi-targeting compounds is essential for treating diseases with complex etiology. Completion of this proposal will: (i) optimize multivalent, ultra-stable configuration that supports uniform, potent, specific, and durable multi-target silencing (longer than six months) in the central nervous system; and (ii) establish a developmental path toward novel combinatorial treatments for HD and ALS. In addition, this proposal seeks to establish a technology platform to directly target any gene or combination of genes expressed in any region of the central nervous system. Successful completion of this work will therefore enable studies of gene function in the central nervous system and the development of novel oligonucleotide-based therapies for genetically defined neurodegenerative diseases.