Kinetics of RNA-LNP delivery and protein expression

Lipid nanoparticles (LNPs) employing ionizable lipids are the most advanced technology for delivery of RNA, most notably mRNA, to cells. LNPs represent well-defined core-shell particles with efficient nucleic acid encapsulation, low immunogenicity and enhanced efficacy. While much is known about the structure and activity of LNPs, less attention is given to the timing of LNP uptake, cytosolic transfer and protein expression. However, LNP kinetics is a key factor determining delivery efficiency. Hence quantitative insight into the multi-cascaded pathway of LNPs is of interest to elucidate the mechanism of delivery. Here, we review experiments as well as theoretical modeling of the timing of LNP uptake, mRNA-release and protein expression. We describe LNP delivery as a sequence of stochastic transfer processes and review a mathematical model of subsequent protein translation from mRNA. We compile probabilities and numbers obtained from time resolved microscopy. Specifically, live-cell imaging on single cell arrays (LISCA) allows for high-throughput acquisition of thousands of individual GFP reporter expression time courses. The traces yield the distribution of mRNA life-times, expression rates and expression onset. Correlation analysis reveals an inverse dependence of gene expression efficiency and transfection onset-times. Finally, we discuss why timing of mRNA release is critical in the context of codelivery of multiple nucleic acid species as in the case of mRNA co-expression or CRISPR/Cas gene editing.