Non-viral gene therapy uses engineered nanoparticles in the virus size range for the cell-targeted delivery of therapeutic nucleic acids. A diverse range of macromolecules are suitable for constructing such 'artificial viruses'. However, proteins, either man-made or from natural sources, are especially convenient for mimicking the viral functions critical for gene transfer. Cell penetration is a critical step for the delivery of nucleic acids in sufficient amounts and hence for reaching satisfactory transgene expression levels. Membrane-active peptides have shown great promise because of their positive role in cross-membrane transport and intracellular trafficking, and they have been incorporated into different artificial viruses. In this review, we will discuss the biological properties of these peptides together with the newest rational approaches designed to optimize their application. © 2008 Elsevier Ltd. All rights reserved.