Abstract
Background
Non‐viral gene delivery vectors are multi‐component systems reflecting various functionalities required for effective cell transfection, including DNA condensation, promotion of cell membrane interactions and provision for subcellular targeting through endosomal escape and/or nuclear delivery. Elements mediating these functions will clearly display inter‐dependency. In this study we sought to explore the relationship within non‐viral vectors of condensation and nuclear localisation.
Methods
Binary, tertiary and quaternary vectors were prepared with combinations of pDNA, DOTAP lipid, the polycation peptide protamine and either SV40 nuclear localisation sequence peptide (‘SV40 NLS’) or a one amino acid substituted mutant of SV40 NLS (‘mutant sequence’). The efficiency of pDNA condensation was determined by gel electrophoresis and quantitative fluorescence spectroscopy. Transfection efficiency was examined in mammalian cells in vitro using standard methods, by electroporation to bypass the plasma membrane barrier and in cells arrested in G~0~/G~1~ cell cycle phase to examine the effect of cell division and nuclear membrane disruption.
Results
Small NLS peptide sequences, despite possessing a significant proportion of basic amino acids, display minimal pDNA‐condensing ability when compared to larger polycations such as protamine. In standard in vitro cell adherent transfection studies the predominant elements affording enhanced gene expression were effective pDNA condensation and lipid enhancement of cell membrane interactions. These features conversely hinder efficient gene expression in cells that have undergone electroporation. The benefit of SV40 NLS was only apparent when used in non‐dividing cell populations.
Conclusions
Whilst effective levels of non‐viral‐mediated gene expression generally rely on efficient condensation of pDNA and enhanced interactions with cellular membranes, non‐covalently associated NLS within a multi‐component non‐viral gene vector appears to contribute benefit in sustaining gene expression in non‐dividing cells. Copyright © 2007 John Wiley & Sons, Ltd.