An optimized amphiphilic cationic peptide as an efficient non-viral gene delivery vector

Background Due to their chemical definition and reduced size, the use of pe ptides as gene delivery systems is gaining interest as compared to the more common polymeric non-viral vectors. To achieve gene transfer efficiencies that would make peptides a realistic alternative to existing methods, we ha ve evaluated and attempted to concert those properties with a direct impact on the activity of the system. These considerations have led to the design , synthesis and characterization of a 23-residue cationic peptide which we term RAWA. Methods We have characterized RAWA biophysically and functionally. Biophysi cal studies include evaluation of DNA condensation and membrane perturbing activities. DNA transfer activity has been evaluated in cell culture at con trolled DNA-to-peptide stoichiometries, using a luciferase gene as reporter . Requirements for additional effecters such as chloroquine and peptide cof actors have also been considered. Results RAWA displays in vitro DNA condensing activity similar to that of p rotamines, reaching maximum effect at a peptide-to-DNA molar charge ratio ( CR) of 4 (+/-). The reduced membrane perturbing activity diminishes its cyt otoxic potential. In COS-7 cells, transfection efficiency with RAWA peptipl exes, compares favorably with well-recognized systems, including Lipofectam ine Plus, Superfect, GenePorter and FuGene. The peptide-associated activity between free and DNA-bound species has been mapped by analyzing dependency on chloroquine treatment. The lack of significant serum inhibition and low toxicity make this system advantageous for potential in vivo application. A ternary complex including the acid-triggered fusogenic JTS-1 peptide is p resented as a potential strategy for further in vivo studies. Conclusions We have developed a gene delivery system based on an amphipathi c cationic peptide with improved DNA condensation ability and reduced cytot oxicity, which maintains membrane binding and perturbing activities. Observ ed efficiency with this molecule is very high and compares favorably with c urrently available transfection systems. Copyright (C) 2000. John Wiley & S ons, Ltd.