Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells.

Abstract

A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic pepffde gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of -galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phasecompetent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.Advances in gene therapy depend to a large degree on the development of delivery systems capable of efficiently introducing DNA into the target cell. Although significant progress has been made with retroviruses (1) or adenoviruses (2) for gene delivery, concern about possible recombination with endogenous virus, oncogenic effects, and immunologic hostresponse reactions has encouraged a search for nonviral DNA transfection techniques (3). Nonviral techniques, including cationic or pH-sensitive liposomes (4,5), polylysine conjugates (6, 7), and direct injection of DNA (8) overcome some of the problems of the viral systems. However, there remains a need for improved transfection efficiency in the nonviral systems.Since the efficiency of the viral systems is in large part due to fusogenic amino acid sequences on viral membrane proteins, we examined whether membrane-permeabilizing peptides could be used to create systems that efficiently transfect mammalian cells. The system developed here takes advantage of the DNA binding ability and the membrane destabilization properties of gramicidin S, an amphipathic cyclic decapeptide that bears two positive charges from ornithine residues on one side of the ring and the side chains of the hydrophobic residues arrayed on the opposite face ofthe ring (9). Gramicidin S, a membrane binding peptide (10), strongly interacts with nucleotides (11) and nucleic acids (12) by charge interactions and mediates phase transfer of these molecules into organic solvents. Gramicidin S also can destabilize membranes causing an increase in the permeability of liposomal (13) qTo whom reprint requests should be addressed.-893The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Intracellular trafficking of nonviral vectors

“…In contrast, another cyclic amphipathic peptide, gramicidin S, also condenses DNA, and, when combined with a DNA/DOPE complex, enables gene transfer by an apparently pH independent mechanism. 139 While JTS-1, KALA, and gramicidin S all apparently enhance gene transfer, presumably by inducing endosomolysis, it remains unclear as to whether these peptides form pores or disrupt membranes via mechanisms similar to GALA and melittin. Pore formation by both GALA and melittin peptides, on the other hand, may accelerate an additional mechanism of membrane penetration by phospholipid 'flip-flop', as described next.…”

Gene delivery by negatively charged ternary complexes of DNA, cationic liposomes and transferrin or fusigenic peptides

“…We should note that the results in Figures 1 and 2 may be directly compared since they were obtained with the same batch of cells. Several studies have found that DOPE is the most efficient helper lipid for in vitro gene transfection, 31,44,45 most likely because of its tendency to form nonbilayer phases and to facilitate membrane fusion. 46,47 Our results with the control liposomes, ie in the absence of transferrin (Figures 1 and 2), also indicate that DOTAP:DOPE liposomes mediated higher transfection activity than pure DOTAP liposomes.…”

“…44,51,52 To test whether DOTAP:DOPE/DNA complexes associated with transferrin or GALA were also effective in other cell lines we performed experiments with COS-7 cells which have been used in previous transfection studies ( Figure 4). 51 As observed for HeLa cells, the association of transferrin or the GALA peptide with the lipid/DNA complexes resulted in a significant enhancement of the levels of luciferase expression in COS-7 cells.…”

Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential

“…10,11 However, these systems often require a large amount of peptides per pDNA complex in order to be effective, partially explaining their limited reports of successes in vivo. 12 Viruses are not the only species that evolved and refined the ability to carry their genomic contents across the membrane barrier of host cells.…”