Peptide-Mediated Gene Delivery: Influence of Peptide Structure on Gene Expression

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Abstract

Cationic peptides possessing a single cysteine, tryptophan, and lysine repeat were synthesized to define the minimal peptide length needed to mediate transient gene expression in mammalian cells. The N-terminal cysteine in each peptide was either alkylated or oxidatively dimerized to produce peptides possessing lysine chains of 3, 6, 8, 13, 16, 18, 26, and 36 residues. Each synthetic peptide was studied for its ability to condense plasmid DNA and compared to polylysine19 and cationic lipids to establish relative in vitro gene transfer efficiency in HepG2 and COS7 cells. Peptides with lysine repeats of 13 or more bound DNA tightly and produced condensates that decreased in mean diameter from 231 to 53 nm as lysine chain length increased. In contrast, peptides possessing 8 or fewer lysine residues were similar to polylysine19, which bound DNA weakly and produced large (0.7-3 microns) DNA condensates. The luciferase expression was elevated 1000-fold after HepG2 cells were transfected with DNA condensates prepared with alkylated Cys-Trp-Lys18 (AlkCWK18) versus polylysine19. The gene transfer efficiencies of AlkCWK18 and cationic lipids were equivalent in HepG2 cells but different by 10-fold in COS 7 cells. A 40-fold reduction in particle size and a 1000-fold amplification in transfection efficiency for AlkCWK18 DNA condensates relative to polylysine19 DNA condensates suggest a contribution from tryptophan that leads to enhanced gene transfer properties for AlkCWK18. Tryptophan-containing cationic peptides result in the formation of small DNA condensates that mediate efficient nonspecific gene transfer in mammalian cells. Due to their low toxicity, these peptides may find utility as carriers for nonspecific gene delivery or may be developed further as low molecular weight DNA condensing agents used in targeted gene delivery systems.

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