Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells

Abstract

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines in vitro was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.

Figure 1

Evaluation of the transfection efficiency of LAH peptides. Increasing amounts of transfection reagent were mixed with a constant amount of reporter plasmid [2 μg of cytomegalovirus (CMV)-Luc per duplicate]. The complexes were incubated for 3 h in serum-free medium. Luciferase activity was measured 24–43 h posttransfection. The transfection efficiency is expressed as total light units per 10 s per well, and the average of duplicates is shown. (A) Transfection efficiency of LAH peptides on HepG2 (Upper) and 293 (Lower) cells. (B) Comparison of the transfection efficiency of LAH4 vs. pLys180 (poly l-lysine with a degree of polymerization of 180) and 25-kDa PEI on four different cell lines. Only the best condition for each reagent is shown (open bars, PEI; gray bars, pLys; black bars, LAH4). (C) HepG2 cells were transfected with PEI [N/P = 13; N/P is the molar ratio of amino nitrogen (present in the transfection agent) to DNA phosphate], DOTAP (N/P = 4), or LAH4 in the presence (black bars) or absence (gray bars) of 175 nM bafilomycin A1.

Figure 2

Edmundson helical wheel diagrams. The helical wheel projections allow the visualization of the distribution of hydrophobic and polar residues with respect to the helical axis. The histidine residues are in italics.

Figure 3

Efficiency of plasmid DNA delivery. HepG2 cells were transfected with peptide/cytomegalovirus (CMV)-Luc complexes. At 7 and 25 h after transfection, cells were harvested and lysed and the low-molecular-weight DNA was recovered. At the same time, the luciferase activity was measured. The blot at Left serves as a standard curve and corresponds to the results obtained after hybridization of 25, 50, 100, and 150 ng of CMV-Luc. Graphs below the blots show the corresponding luciferase activity.

Figure 4

Antibacterial activity of LAH4. Growth of E. coli was determined at pH 7.4 (open symbols) and 5.5 (filled symbols) as a function of peptide concentration for LAH4 in the absence (circles) or presence (squares) of DNA (LAH4/DNA with a wt/wt ratio of 6/1).