The nuclear pore complex is involved in nuclear transfer of plasmid DNA condensed with an oligolysine-RGD peptide containing nuclear localisation properties

One of the major barriers to efficient gene transfer and expression of nonv iral vectors for gene therapy is passage across the nuclear envelope. We ha ve previously shown that an oligolysine-RGD peptide that condenses plasmid DNA and binds to cell surface integrins can mediate increased internalisati on of plasmid DNA into cells and synergistic enhancement of gene expression when complexed to a cationic lipid. In this report, we show that this enha ncement is due to increased nuclear transfer of the plasmid DNA. We have ap plied the digitonin-permeabilised cell system that has been well establishe d for the study of the nuclear transport of proteins to examine the nuclear transfer of plasmid DNA. Nuclear transfer of plasmid DNA complexed to an o ligolysine-RGD peptide and lipofectamine appears to be an energy-dependent process involving the nuclear pore complex, since it is inhibited at 4 degr eesC and by treatment with wheat germ agglutinin or with an antibody to the nuclear pore complex which all block nuclear protein import. In accordance with active nuclear transport, we have shown that all these treatments inh ibit expression of a luciferase reporter plasmid in permeabilised cells. Nu clear transfer of pDNA is enhanced in mitotic cells, but cell division is n ot a prerequisite for transfer. We propose that the oligolysine-RGD peptide acts as a nuclear localisation signal and that the cationic lipid is more important for cell entry and endosome destabilisation than nuclear transfer .