Mechanism of lipid mediated siRNA delivery

Cancer is one of the most lethal diseases worldwide and accounts for nearly 10 million deaths each year. Multiple treatments have been developed but in the case of generalized disease, effective treatments are often lacking. New precision-based treatments that can address specific genetic changes with less adverse effects would be of great value.

RNA interference (RNAi) is an endogenous conserved cellular mechanism that utilizes micro RNAs (miRNAs) to downregulate and adjust gene expression through sequence complementarity. This mechanism can be exploited pharmacologically by delivering synthetic short interfering RNAs (siRNAs) that target specific genes. Because of their high gene specificity and simple design, RNAi would be an ideal precision-based cancer treatment. Due to its molecular properties, however, native siRNA is a poor therapeutic in the sense that it cannot diffuse across the cellular membrane and is highly unstable and immunogenic. Thus, for over two decades, extensive work has been devoted to improve siRNA as a therapeutic modality. In addition to chemical modifications that protects siRNA against enzymatic degradation and mask it from the immune system, major advancements have been made to enhance delivery of siRNA into target cells, usually by combining the siRNA with a strategy that facilitates cellular uptake. While promising, intracellular delivery of siRNA is challenging, and a series of barriers must be circumvented to achieve a therapeutic effect. A frequently mentioned bottleneck is the necessary step for siRNA to escape the endosomal-lysosomal system to reach the cytosol were the siRNA acts. With current delivery strategies, only a fraction of the delivered RNA reaches the cytosol, while the rest remain inactive within enclosing endosomes. Thus, the potential for improvement is significant. Additionally, there are other therapeutic RNA applications and gene editing approaches that would benefit from this. These include potential protein replacement therapies, splice-correcting therapies, CRISPR mediated gene editing, or the presently most well-known application of RNA therapies, mRNA-based vaccines, to mention a few.

Presently, lipid nanoparticle (LNP) mediated delivery is the most clinically advanced delivery strategy for RNA, utilized in Pfizer’s and Moderna’s mRNA-based vaccines against SARS-CoV-2, and also in the first clinically approved siRNA therapeutic, patisiran. While proven functional, because of a lack of suitable analytical methods to study RNA delivery at therapeutically relevant doses and conditions, little is known about the actual effect of LNPs at a subcellular level. Thus, the aim of this thesis project is to develop methods to characterize and determine the efficiency and mechanism of lipid-based RNA delivery strategies. In particular, the thesis will focus on a detailed characterization of the endosomal escape of RNA, the key limiting step during delivery.