A new class of peptide gene delivery agents were developed by inserting mul
tiple cysteine residues into short (dp 20) synthetic peptides, Substitution
of one to four cysteine residues for lysine residues in Cys-Trp-Lys(18) re
sulted in low molecular weight DNA condensing peptides that spontaneously o
xidize after binding to plasmid DNA to form interpeptide disulfide bonds. T
he stability of cross-linked peptide DNA condensates increased in proportio
n to the number of cysteines incorporated into the peptide. Disulfide bond
formation led to a decrease in particle size relative to control peptide DN
A condensates and prevented dissociation of peptide DNA condensates in conc
entrated sodium chloride. Cross-linked peptide DNA condensates were 5-60-fo
ld more potent at mediating gene expression in HepG2 and COS 7 cells relati
ve to uncross-linked peptide DNA condensates. The enhanced gene expression
was dependent on the number of cysteine residues incorporated, with a pepti
de containing two cysteines mediating maximal gene expression. Cross-Linkin
g peptides caused elevated gene expression without increasing DNA uptake by
cells, suggesting a mechanism involving intracellular release of DNA trigg
ered by disulfide bond reduction. The results establish cross-linking pepti
des as a novel class of potent gene delivery agents that enhance gene expre
ssion through a new mechanism of action.