HVJ-E/importin-ß hybrid vector for overcoming cytoplasmic and nuclear membranes as double barrier for non-viral gene delivery.

In order to enhance the nuclear import of the transgene, we prepared plasmid DNA/importin-ß conjugates consisting of biotinylated poly(ethylenimine)s and recombinant streptavidin-fused importin-ß. Hemagglutinating virus of Japan-envelope vector containing the PEI polyplex/importin-ß conjugate showed high transfection efficiency not only in vitro but also in vivo. We showed that novel HVJ-E/importin-ß-conjugated PEI polyplex hybrid vector could overcome plasma and nuclear membrane barriers to achieve effective transfection.

Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique.

Among the well-studied polypeptide-type gene carriers, transfection efficiency is empirically known to be higher for poly(L-arginine) (PR) than poly(L-lysine) (PK). The big difference between PR and PK should be determined at one of the intracellular trafficking steps based on the different charge densities, structures or PKa values. However, the endosomal escape and the intranuclear transcription efficiency in living cells have not been clarified yet. In this study, a novel method for quantifying the intranuclear transcription efficiency and the nuclear transport of the polyplex is established based on the nuclear and the cytosolic microinjection technique, and the results for PK and PR with different molecular weights (MWs) are compared in living cells. The intranuclear transcription efficiency is the same in PR and PK and it decreases rapidly with increasing MW, in spite of the commonly measured transfection efficiency. The transcription efficiency is strongly suppressed at high MW and strongly correlates with the polyplex forming ability expressed as a critical ratio of the number of polypeptide cationic groups to the number of pDNA anionic groups. When considered with the results of the cellular uptake and in vitro transfection with or without chloroquine, the rate-limiting step for their gene transfer is the buffering effect-independent endosomal escape.

Enhanced nuclear import and transfection efficiency of plasmid DNA using streptavidin-fused importin-beta.

In order to enhance the nuclear import of exogenous genes, novel plasmid DNA/importin-beta conjugates, which consist of a biotinylated plasmid DNA and a recombinant streptavidin-fused importin-beta, were prepared. The spacer length between plasmid DNA and biotin and the number of introduced biotin were adjusted. The microinjection of plasmid DNA/importin-beta conjugates into the cytoplasm of NIH3T3 cells resulted in the nuclear localization of conjugates and the higher expression efficiency, compared to intact plasmid DNA alone. These results indicate that plasmid DNA/importin-beta conjugates would be an important tool to enhance the nuclear localization of exogenous DNA in non-viral gene delivery system.