Advancing Oligonucleotide Therapeutics

Lay Summary
Therapeutic 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.

Therapeutic oligonucleotides (ThONs) are important agents for hard to treat diseases. They target RNA, and they vastly increase the number of potentially treatable illnesses. Twelve oligonucleotides (ONs) have recently been approved by the FDA or EMA for clinical use, over half since 2018, demonstrating their enormous clinical potential. A large number are currently under investigation as treatments for diseases such as cancer. Modifications to the sugar phosphate backbone of ThONs are essential to confer resistance to degradation in vivo and to ensure efficient RNA target binding. Despite recent successes the field is still in its infancy; a major barrier to the efficacy of ThONs is their poor uptake into cells (as little as 1% reaches the target site). Improved cell uptake would allow lower clinical doses and less frequent administration, reduce toxicity and cost, and encourage further developments in the field. Hence, there is an urgent need for new designs of ThONs with enhanced pharmacological and toxicological properties.

We have developed a new family of artificial oligonucleotide backbones in which the sugar is replaced by locked nucleic acid (LNA) and the phosphodiester backbone is replaced by an amide or triazole linkage. Unlike canonical nucleic acids, these LNA-amide and LNA-triazole backbones are uncharged and refractory to degradation in vivo. We have discovered that combining LNA-amide with phosphorothioate backbones (PS) has a major synergistic effect on improving productive cell uptake of ThONs. The molecular basis of this must be elucidated, and we will conduct biochemical and biological experiments to achieve this. Cell culture and mouse studies on multiple targets will allow us to identify optimum combinations of LNA-amide and PS backbones and enable us to develop even more effective ThONs. We will also collaborate with experts in the biomedical aspects of the field to bring our technology towards a clinical setting.