Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser; D. Woschée; N. Mehrotra; R. Krzyszton; H. H. Strey; J. O. Rädler

Gene based therapies advance the delivery of exogenous nucleic acids with the intent to modulate the expression of disease related genes. However, clinical applications are still limited due to the difficulties inherent in delivery of nucleic acids in vivo (1, 2) . Over the last years, intense research is dedicated to develop non-viral vectors that are capable to safely and efficiently package, transport and systemically deliver nucleic acid to cells (3) (4) (5) . In particular for therapeutic purposes, there is need to deliver DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA). The molecular and cellular processes of nucleic acid nanoparticles share common structural features and entry mechanisms. Some of the major challenges associated with current cationic lipid vectors are the interaction with blood serum components, impaired intracellular uptake, limited release into the cytosol and immunological response (6, 7) . Each of these hurdles are independently affected by variations in the lipid formulations and transfection conditions. Lipid-encapsulated RNA is taken up by various, endosomal or phagocytotic, uptake mechanisms and subsequent intracellular pathways. It appears however, that nucleic acids nanocarriers are largely trapped inside the endosomes or lysosomes and that delivery by lipid nanoparticles is limited by endocytic recycling (8) . In timeresolved fluorescence microscopy studies, the uptake and fate of delivery particles has been visualized at the single-cell level (9) (10) (11) (12) (13) . Recently, it was shown that siRNA release occurred during a narrow 'window of opportunity' showing that timing and efficiency are linked (12) . However, the uptake pathways and factors that control endosomal release of lipid-based nanocarriers are not fully resolved. In particular, the interdependence of timing and efficiency of gene delivery and the role of extracellular factors on the kinetics of cellular uptake and endosomal release are poorly understood aspects in the delivery process.