Advancing Oligonucleotide Therapeutics

治疗性寡核苷酸（ThON）正迅速成为难以治疗的疾病的重要药物。它们在靶向RNA，通常是信使RNA方面是独特的。靶向RNA的能力大大增加了潜在可治疗疾病的数量，特别是那些无法通过常规小分子药物或治疗性抗体解决的疾病。12种寡核苷酸（ON）最近被FDA或EMA批准用于临床，自2018年以来超过一半。这证明了ThON的巨大临床潜力，并表明许多其他危及生命和使人衰弱的疾病可以使用相同的技术进行治疗。事实上，大量的寡核苷酸正在研究作为疾病如癌症的治疗。寡核苷酸在体内不稳定，因此修饰ThON的糖磷酸骨架对于赋予酶降解抗性是必不可少的。此外，有效的RNA靶标结合是效力所必需的，因此对ThON的修饰也是使其能够与RNA紧密结合所必需的。ThON有效性的主要障碍是它们进入细胞的吸收差;只有1%的给药剂量到达靶部位。从内体捕获中逃脱也是一个问题，我们将解决这个问题。ThON的改善的细胞摄取将具有几个益处;它将允许以较低的施用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。在这一领域的成功肯定会导致新的药物，因此迫切需要具有增强的药理学和毒理学特性的ThON的新设计。在即将结束的现有BBSRC资助的项目中，我们开发了一个新的人工寡核苷酸骨架家族，其中糖-磷酸骨架被糖修饰所取代在一个实施方案中，所述寡核苷酸是寡核苷酸（锁核酸，LNA），并且磷酸二酯骨架被酰胺或三唑键取代。与典型的核酸不同，这些人工骨架（LNA-酰胺，LNA-三唑）不带电荷，并且在体内完全难以降解。令人惊讶的是，我们发现将LNA-酰胺与硫代磷酸酯主链（PS）组合在ThON中对改善细胞摄取具有主要的协同作用。我们尚未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ThON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，我们也将研究一种相关类型的人工DNA骨架，LNA-三唑。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。这将提供一个应急的情况下，在后期阶段，我们遇到意想不到的问题与LNA-酰胺backbone.这些研究的结论应该使我们能够开发更有效的ThON和移动他们更接近临床。我们将与拥有专业知识和资源的合作者合作，将这些进展应用于临床。除了在高影响力的国际期刊上发表我们的工作，我们还打算申请专利，以保护知识产权，造福英国经济。

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.