Intracellular delivery of RNA therapeutics with lipid nanoparticles

Therapeutic RNA molecules such as siRNAs and antisense oligonucleotides have great potential to be used as future medicines in human patients, because they allow the modulation of virtually any protein in the body. Unfortunately, their application is hampered by their physicochemical properties that do not make them very ‘drug-like’. For example, these molecules are 10-25 times higher in molecular weight than conventional small molecule drugs and are highly negatively charged, which prevents their access to the intracellular site of action. Furthermore, they cannot be administered via the oral route because they would be degraded in the gastro-intestinal tract, but also after intravenous administration they are vulnerable to degradation in the bloodstream or rapidly excreted via the kidneys. All these factors together have made the application in human patients a significant challenge whereas in pre-clinical research and biomedical science in general, siRNAs and related molecules have made a huge impact. To also enable this technology for therapeutic applications, intracellular delivery systems have to be developed. In this PhD thesis, lipid nanoparticles (LNPs) have been investigated for this purpose. These LNPs consist of lipid-like molecules that form small vesicles of approximately 100 nanometer in diameter, that encapsulate the RNA therapeutics in their core. This protects the RNA cargo against degradation by nucleases in the bloodstream and prevents rapid excretion. The goal of this research project was to improve lipid nanoparticles for intracellular delivery of RNA therapeutics after intravenous administration. Part of this work involved the modification of the surface of the LNPs with peptides that improve the therapeutic index of the LNPs, for example to improve their interaction with, and uptake by their target cells. Another example is the application of peptides that increase the fraction of the dose that eventually reaches the part within the cell where the drugs needs to exert its function. To this end, different types of coupling chemistries were explored, including the first ever report of copper-free click chemistry on the surface of a liposome and a novel type or pH-responsive conjugation that reacts to a spatiotemporal trigger inside the cell. Furthermore, variations in the composition of the lipid nanoparticles were investigated and how this could influence their ability to deliver therapeutic RNA molecules to different target tissues and after different routes of administration. Finally, the CRISPR-Cas9 gene editing system was described, which could have even greater therapeutic impact than RNA editing/silencing systems, but seems to suffer from the same limitations as its counterparts. The lessons learned from the delivery of RNA therapeutics could also be applied to the delivery of CRISPR-Cas9, as it has already become clear that it cannot be translated to the clinic without a sophisticated delivery system and lipid nanoparticles could also play an important role here