Abstract by Rikke Dueholm

Over the past 25 years, oligonucleotides (ONs) have gained considerable interest as potential therapeutic agents due to their precise ability to upregulate or downregulate gene expression. This specificity makes them highly promising candidates for treating a wide range of diseases. However, ONs only function within the cytosol of the cell, significantly challenging their development into viable drugs. Among the ONs, small-interfering RNAs (siRNAs) have emerged as a particularly promising class, as they can target specific mRNA sequences and inhibit protein translation. This thesis aimed at investigating potential drug delivery methods for siRNA therapeutics. Three distinct delivery forms were developed and evaluated:

(i) Exploring the potential of using cell-penetrating peptides (CPPs) as non-specific, systemic delivery vehicles for siRNA. A method for interaction characterization between peptides and siRNA was established, to systematically evaluate CPPs as potential conjugation partners. Promising CPPs were identified as potential conjugation partners based on their non-siRNA-interacting behaviour.

(ii) Optimizing the linker moiety in divalent siRNAs for local delivery to the central nervous system (CNS). A synthesis protocol was established for the linear synthesis of divalent siRNAs, and different linkers were investigated. In vivo experiments identified a highly promising linker that facilitated efficient gene silencing after CNS delivery to mice.

(iii) Investigate the systemic, tissue-targeted potential of divalent siRNAs. A specific divalent siRNA structure with high adipocyte specificity was identified. Its tissue-targeting behaviour was evaluated in vivo after systemic delivery. Remarkably, this divalent siRNA effectively silenced genes in various adipocyte deposits in mice while exerting minimal impact on other tissues.

In summary, this thesis presents three promising siRNA delivery technologies for both systemic and local, as well as targeted and non-targeted delivery. The findings advance our understanding of how different delivery strategies can be harnessed to develop new and effective siRNA therapeutics. These advancements have the potential to significantly contribute to the development of future siRNA-based treatments for various diseases.