Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid

We recently described a basic technology to efficiently combine compacted D NA with phospholipids and hydrophobic peptides, to produce homogenous compl exes that are completely resistant to nuclease. We have developed this tech nology further to form gene delivery complexes that transfect cells effecti vely in vitro. In addition to plasmid DNA, the complexes contained two basi c components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidytet hanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge e quivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dim yristoleoylphosphatidylethanolamine/mug DNA produced the highest luciferase gene expression, exceeding 1 x 10(9) relative light units/s/mg protein (>3 mug luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polye thylenimine or lipofectamine complexes. With green fluorescent protein repo rter gene, > 50% of HeLa cells were positive 30 h after addition of these c omplexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal esc ape. Our results indicated that self-assembling complexes of plasmid DNA, a mphiphilic peptide and phosphatidylethanolamine are highly effective non-vi ral gene delivery systems.