Using messenger RNA (mRNA) as therapeutic nucleic acids is recently highlighted because mRNA is negligible to risk of host-genome integration and the transfection of mRNA to non-dividing cells is easier than that of DNA. These great advantages of the mRNA transfection are derived from mRNA property of expressing the coded gene in cytoplasm; however mRNA is much more fragile than DNA and quick digestion of the transfected mRNA in cytoplasm limits the longevity of gene expression. We assume that construction of mRNA carrier system having high tolerability against nuclease digestion would promote not only survival of the loaded-mRNA under physiological environment but also the survival in cytoplasm, leading to the increased and prolonged gene expression. Herein, polyplex micelles (PMs) having hydrophobic barrier compartment between hydrophilic shell and the mRNA-loaded core were designed. The PMs were prepared from mRNA and triblock copolymer composed of hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx) segment, thermo-switchable poly(2-<i>n</i>-propyl-2-oxazoline) (PnPrOx) segment and mRNA binding cationic poly(L-Lysine) (PLys) segment as follows: mixing mRNA with the triblock copolymer at temperature below LCST of PnPrOx, and subsequent incubation at physiological temperature, which is above the LCST of PnPrOx. The PnPrOx segment successfully enhanced tolerability against nuclease attack, increased survival of mRNA in cytoplasm, and improved gene expression compared to PMs prepared from diblock copolymer without PnPrOx segment. Moreover, enhancement of the PnPrOx barrier compartment was achieved by adding PnPrOx homo-polymer to the PMs solution before the incubation at the physiorogical temperature, showing further improved tolerability against nuclease and promoted gene expression. These results suggest forming hydrophobic layer is promising strategy to increase stability of PMs and the increased stability contribute to improve gene expression property of the loaded-mRNA.

Messenger RNA loaded polyplex micelles having hydrophobic core protective layer composed of thermo-switchable poly(oxazoline) for promoted gene expression

2019 Spring National Meeting

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