Expanding the Chemical Diversity of Therapeutic Oligonucleotides

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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.