Endosomal escape of RNA therapeutics

RNA therapeutics is a new class of targeted therapies that has entered the clinic in the last decade. Lipid nanoparticles (LNPs) and conjugation to N-acetylgalactosamine have proven to be efficient strategies to deliver RNA payload to the liver. Successful delivery of sensitive RNA molecules to other target tissues or tumors remains one of the key challenges with the development of new RNA-based treatments. Extrahepatic delivery is still poor, limiting the therapeutic efficacy. One of the central hurdles to delivery is the intracellular accumulation of RNA therapeutics and delivery vehicles in endocytic vesicles following uptake, without an effective way to exit into the cytoplasm and engage with the therapeutic targets. Knowledge of this rate-limiting process have remained poor, hampering rational efforts to overcome it.

This thesis has aimed to devise and leverage techniques to study the endosomal escape of small interfering RNA (siRNA) and messenger RNA (mRNA) from endosomal compartments into the cytosol — to advance the understanding of the escape process, the interactions between RNA delivery vehicles and endosomes that potentially trigger membrane disruption, and the dose-response relationship between the small amount of siRNA molecules released and the biological response.

The sensitive membrane-damage sensor Galectin-9 was used to probe individual release events, showing that endosomal escape of both siRNA and mRNA formulated in LNPs is very inefficient. Only a fraction of LNPs triggered endosomal damage, only a minority of damage events led to productive release of RNA payload, and most of the payload remained trapped in the damaged endosomes. The endosomal release of lipid- modified siRNA was enhanced by simultaneous treatment with membrane-damaging small molecule drugs, that promoted efficient escape of siRNA payload on a single- vesicle level. In addition, a microscopy-based approach was developed to quantify the number of siRNA molecules delivered to the cytosol while also monitoring the resulting knockdown of a reporter gene. From experimental data combined with mathematical modelling, the cytosolic IC50 of two siRNA sequences with a known difference in potency was determined to be ~970 and ~6,700 molecules, respectively. Finally, a novel dual-labeled LNP was developed, composed of fluorescently labeled ionizable lipid (BODIPY-MC3) and RNA. Dual-labeled LNPs were used to visualize the interaction between ionizable lipid from the LNP and the endosomal membrane, resulting in damage to the lipid bilayer.

This thesis presents detailed investigations of the endosomal escape of RNA therapeutics, providing new insights into the cellular and biophysical barriers limiting payload release and how they could potentially be overcome. The work is of high relevance for ongoing efforts to develop new RNA-based therapies with rational strategies.