Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics

RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.