Investigation of charge-neutral backbone modified oligonucleotides for antisense applications

Thesis

Antisense oligonucleotides (ASOs) are short strands of DNA or RNA that bind to complementary pre-mRNA and alter gene expression. The oligonucleotides can prevent inclusion of mutated sites and restore expression or induce mRNA degradation to silence it. Antisense oligonucleotides hold tremendous promise as therapeutic agents for genetic disorders and other diseases that are not treatable by small molecule medicines. Eleven antisense oligonucleotides have been approved by the US Food and Drug Administration but a major need still exists for the improvement of their therapeutic properties including more efficient cell uptake and targeting of specific tissues.

In this work, new chemical modifications and methods for introducing them into oligonucleotides were explored. Solid phase chemistry was used to evaluate conditions for replacing the OH group at the 5´-position of a ribose sugar with an azide. This allows the synthesis of various internucleoside triazole backbones by copper-catalysed click chemistry (CuAAC). The backbones were then tested in splice switching oligonucleotides in a HeLa cell luciferase reporter assay and compared to phosphorothioate and alkyl phosphonate backbones.

As the charge-neutral phosphonate and triazole linkages reduce the overall negative charge of the oligonucleotide, they have the potential to increase cellular uptake. In particular it was shown that a combination of a locked nucleic acid sugar and the alkyl phosphonate linkage improves the splice switching efficiency of an oligonucleotide compared to a standard 2´-O-methyl phosphorothioate control oligonucleotide. Our findings highlight the need to understand the origins of the improved activity, including the efficiency of oligonucleotide uptake into cells and intracellular trafficking and localisation.

Assay development was then started to find a method to allow for simple and effective screening of new antisense modifications. Initial results show potential pitfalls with current techniques that employ large dye molecules attached to oligonucleotides. These molecules allow precise visualisation but were shown to influence the activity of the oligonucleotides. Method development done in this thesis gives an alternative to visualise oligonucleotides using an azide installed during solid-phase synthesis as explored in earlier sections of this thesis as handle for in-situ CuAAC ligation of dyes in fixed cells.

The work discussed in this thesis begins with development of modifications with potential applications in ASOs. It then tests the efficacy of these and other modifications in exon-skipping oligonucleotides, revealing patterns of use for developing more effective molecules. Finally, it advances techniques to explore the reasons behind these patterns that avoid problems associated with some current practices. These factors can help to create high performing ASO therapeutic molecules in future.

Authors: Lagan, BJ

• DOI: 10.5287/ora-zgmovpdeq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Antisense Oligonucleotide (ASO)
• Subjects: Molecular biology; Chemistry, Organic; Chemical biology