Natural and synthetic lipid nanoparticles for therapeutic RNA delivery

Abstract

RNA therapeutics can potentially revolutionize the way in which we are able to treat a disease, namely directly at its biological origin. RNA therapeutics can modulate the expression of almost any gene. However, clinical translation of RNA therapeutics has been hampered by several major bottlenecks such as RNA stability, cellular delivery, and cytosolic release of RNA therapeutics. Therefore, drug delivery systems, such as lipid nanoparticles (LNPs), extracellular vesicles (EVs) or EV-liposome hybrid nanoparticles are the cornerstone of RNA therapeutic development. By using such systems, the RNA molecule can be shielded from degradation and at the same time the system provides a vehicle for cellular uptake and cytosolic release. In this thesis, both LNPs and EV-liposome hybrids have been subject of investigation for the delivery of RNA therapeutics. The development of LNPs for both siRNA and mRNA delivery was described and several key ‘design principles’ of LNPs including the prominent role of ionizable lipids and polyethylene glycol(PEG)-lipids were discussed. In addition, multiple conventional and newly developed production methods of LNPs were summarized and compared to each other. The use of LNPs for functional delivery of both siRNA and mRNA was investigated. In one study, the effect of LNPs-siRNA mediated post-transcriptional silencing of individual enzymes of the novo ceramide synthesis pathway on liver and plasma ceramide concentrations was evaluated. Increased concentrations of ceramides are potentially involved in pathophysiology of several cardiovascular and metabolic diseases. Although we showed silencing of both of the specific enzymes in vivo, the effect on plasma ceramide concentrations varied per enzyme. In another study, the potential use of LNPs to deliver mRNA molecules to ischemic area of the heart after myocardial infarction was assessed. LNPs delivered mRNA to the ischemic area of the heart after myocardial infarction however the delivery was not specific. LNPs are generally composed of 4 different lipids: an ionizable lipid, a helper lipid, a PEG-lipid and cholesterol. Given the large amount of types of lipids available, it is possible to generate large libraries of lipid formulations. Simultaneous analysis of important in vivo characteristics of multiple, different LNP formulations could greatly reduce the number of animals required for such studies. A possible approach here is the use of unique DNA barcodes where each unique LNP formulation contains a unique DNA barcode. Simultaneous screening of DNA barcode occurrence can be used as method to analyze characteristics of multiple LNPs at the same time. This concept is not new and has published before. In this thesis, we aimed to setup and validate this highly innovative technology. EVs can potentially be used as drug delivery system. EVs might have multiple advantages over synthetic lipid based systems of which increased RNA delivery efficiency is the most appealing. Currently, loading of EVs with therapeutic RNAs represent a major drawback of this system. We developed and evaluated the generation of ‘hybrid nanoparticles’ composed of both EV and synthetic lipid components. We hypothesized that a hybrid nanoparticle would incorporate beneficial characteristics of both synthetic and biological nanoparticles for nucleic acid delivery.