Advancing Oligonucleotide Therapeutics

治疗寡核苷酸(Ton)正迅速成为治疗难治性疾病的重要药物。它们在靶向RNA方面是独一无二的，通常是信使RNA。靶向RNA的能力极大地增加了潜在可治疗疾病的数量，特别是那些无法通过传统小分子药物或治疗性抗体解决的疾病。12种寡核苷酸(ON)最近已被FDA或EMA批准用于临床，其中一半以上是自2018年以来的。这表明了Thons的巨大临床潜力，并表明许多其他威胁生命和虚弱的疾病可以使用相同的技术进行治疗。事实上，人们正在研究大量的寡核苷酸作为癌症等疾病的治疗方法。寡核苷酸在体内不稳定，因此对Ton的糖磷酸骨架进行修饰对于抵抗酶降解是必不可少的。此外，有效的RNA靶标结合是有效的，因此对Ton的修饰也是必要的，以使它们能够与RNA紧密结合。Ton疗效的一个主要障碍是它们对细胞的摄取很差；只有1%的给药剂量到达目标部位。逃脱内体困住也是一个问题，我们将解决这个问题。改善细胞对thons的摄取将有几个好处；它将允许以较少的频率使用较低的临床剂量，减少与毒性和成本相关的问题。这些因素对于鼓励该领域的进一步发展至关重要。这一领域的成功必然会带来新的药物，因此迫切需要具有增强药理和毒理学特性的新的Ton设计。在一个由BBSRC资助的现有项目中，我们开发了一种新的人工寡核苷酸骨架家族，其中糖-磷酸骨架被糖修饰(锁定核酸，LNA)取代，磷酸二酯骨架被酰胺或三唑键取代。与经典核酸不同，这些人工骨架(LNA-酰胺、LNA-三氮唑)不带电荷，在体内完全不能降解。令人惊讶的是，我们发现将LNA-酰胺与Ton中的硫代磷酸骨架(PS)结合在一起，在改善细胞摄取方面具有重大的协同效应。我们还没有确定改善细胞摄取的分子基础，因此我们建议进行生化和生物学实验来确定这一点。我们将研究LNA-酰胺和PS骨架在Ton中的各种组合，旨在解决细胞培养和小鼠研究中的多个靶点。同时，我们还将研究一种相关类型的人工DNA骨架，LNA-三氮唑。这个主链也是不带电荷的，但它的化学结构与酰胺键有很大的不同。它将提供一种应急措施，以防我们在后期遇到LNA-酰胺骨骼的意外问题。这些研究的结论应该能够使我们开发出更有效的Ton，并使它们更接近临床。我们将与拥有将这些进展转化为临床背景的专业知识和资源的合作者合作。除了在高影响力的国际期刊上发表我们的研究成果外，我们还打算申请专利来保护知识产权，以造福于英国经济。

Lay SummaryTherapeutic oligonucleotides (ThONs) are rapidly emerging as important agents for hard to treat diseases. They are unique in targeting RNA, usually messenger RNA. The ability to target RNA vastly increases the number of potentially treatable illnesses, particularly those that cannot be addressed by conventional small molecule drugs or therapeutic antibodies. Twelve oligonucleotides (ONs) have recently been approved by the FDA or EMA for clinical use, over half since 2018. This demonstrates the enormous clinical potential of ThONs and suggests that many other life-threatening and debilitating diseases could be treated using the same technology. Indeed, a large number of oligonucleotides are under investigation as treatments for diseases such as cancer. Oligonucleotides are not stable in vivo, so modifications to the sugar phosphate backbone of ThONs are essential to confer resistance to enzymatic degradation. In addition, efficient RNA target binding is required for potency, so modifications to ThONs are also necessary to enable them to bind tightly to RNA. A major barrier to the efficacy of ThONs is their poor uptake into cells; as little as 1% of the administered dose reaches the target site. Escape from endosomal entrapment is also an issue and we will address this. Improved cell uptake of ThONs would have several benefits; it would allow lower clinical doses to be used with less frequent administration, reducing problems associated with toxicity and cost. These factors are crucial in encouraging further developments in the field. Success in this area will certainly lead to new drugs, and there is therefore an urgent need for new designs of ThONs with enhanced pharmacological and toxicological properties.In an existing BBSRC-funded project, shortly to come to an end, we have developed a new family of artificial oligonucleotide backbones in which the sugar-phosphate backbone is replaced by a sugar modification (locked nucleic acid, LNA) and the phosphodiester backbone is replaced by an amide or triazole linkage. Unlike canonical nucleic acids, these artificial backbones (LNA-amide, LNA-triazole) do not carry a charge, and are completely refractory to degradation in vivo. Surprisingly we discovered that combining LNA-amide with phosphorothioate backbones (PS) in ThONs has a major synergistic effect on improving cell uptake. We have not yet determined the molecular basis of the improved cell uptake and we therefore propose to carry out biochemical and biological experiments to determine this. We will investigate various combinations of LNA-amide and PS backbones in ThONs designed to address multiple targets in cell culture and mouse studies. In parallel we will also investigate a related type of artificial DNA backbone, LNA-triazole. This backbone is also uncharged but its chemical structure is quite different from the amide linkage. It will provide a contingency in case at a late stage we encounter unexpected problems with LNA-amide backbone.The conclusions from these studies should enable us to develop more effective ThONs and move them closer to the clinic. We will work with collaborators who have the expertise and resources to take these advances into a clinical context. As well as publishing our work in high-impact international journals we also intend to file patents to protect the IP for the benefit of the UK economy.