New Basic Membrane-Destabilizing Peptides For Plasmid-Based Gene Delivery In Vitro And In Vivo

We have designed new basic amphiphilic peptides, ppTG1 and ppTG20 (20 amino
acids), and evaluated their efficiencies in vitro and in vivo as single-component
gene transfer vectors. ppTG1 and ppTG20 bind to nucleic acids and destabilize
liposomes consisting of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and
cholesterol (3:1 mol/mol) at pH 5 and pH 7. Complexes of plasmid DNA and ppTG1
gave rise to efficient transfection in a variety of human and murine cell lines
at low charge ratios ([+/-] between 1 and 2). In cell culture experiments, such
vectors were superior to the membrane-destabilizing peptide KALA. In comparison
with cationic lipid-, dendrimer-, and polymer-based transfection agents like
Superfect, polyethylenimine (PEI), and Lipofectin, ppTG1 vectors showed good
transfection efficiencies, especially at low DNA doses. Moreover, we demonstrated
for the first time successful gene transfer in living animals with a
single-component peptide vector. In the mouse, intravenous injection of a
luciferase expression plasmid complexed with ppTG1 or ppTG20 led to significant
gene expression in the lung 24 hours after injection. Structure-function studies
with ppTG1, ppTG20, and sequence variants suggest that the high gene transfer
activity of these peptides is correlated with their propensity to exist in
alpha-helical conformation, which seems to be strongly influenced by the nature
of the hydrophobic amino acids.