Alternative scaffolds for systemic delivery of small interfering RNAs

Small interfering RNAs (siRNAs) are a promising class of RNA directed therapeutics. While exerting their gene-silencing activity similar to microRNAs (miRNAs) by recruiting the RNA induced silencing complex (RISC) toward messenger RNA (mRNA) targets, siRNAs are capable of inducing Argonaute-2 (AGO2) mediated target RNA cleavage. Despite the recent approval of patisiran, the first siRNA drug, systemic delivery remains a challenge and is currently limited to the liver. Whereas patisiran is dependent on lipid nanoparticle formulation (LNP), potent gene silencing has been achieved through naked administration of N-acetylgalactosamine (GalNAc) conjugated siRNAs. However, LNP-independent delivery requires the use of fully chemically modified siRNAs, which have raised concerns with respect to the formation of potentially toxic metabolites. Therefore, the goal of this work was to investigate alternative siRNA scaffolds which do not require a complex mixture of non-natural building blocks for systemic delivery. In the first approach, we investigated the use of fully phosphorothioate (PS) modified siRNAs. Full PS modification is commonly used in the design of single stranded antisense oligonucleotides (ASOs) and was shown to increase their binding to serum proteins. However, application of PS modifications to siRNA therapeutics has been restricted to the terminal positions due to conflicting reports on the activity and toxicity of full PS siRNAs. Our group recently reported the development of stereochemically biased PS siRNAs and demonstrated a higher activity of Rp- than Sp-enriched PS siRNAs in mammalian cells. Building on these results, we scaled up our synthesis protocol for PS siRNAs in order to characterize the biodistribution profile and the in vivo activity of a PS siRNA directed against the oncogene Lin28B. In addition, we investigated the albumin-binding capability and metabolic stability of various siRNA formats in biological fluids. In a second project, we adopted the previously described convertible nucleoside approach to introduce various chemical modifications into the major groove of siRNAs. We then evaluated polyamine conjugation as a means to increase the cellular uptake and nuclease stability of siRNAs under cell culture conditions. In a third project, we focused on the development of a new methodology to uncover the targetome of individual miRNAs. For this purpose, we synthesized 2’, 3’-cyclic phosphate terminated miRNA mimics (miRNA>p) which can be ligated to their RNA targets through the action of both an endogenous and an exogenously added RNA>p ligase. In addition, we contributed to several projects in the field of CRISPR-Cas genome editing and the development of a method to characterize surface-bound RNAs in a microarray-like setup