The two major barriers to the development of efficient non-viral gene delivery systems are the cell membrane and the nuclear membrane. In order to overcome these limitations we have designed a single peptide-based gene delivery system, MPG, which combines a hydrophobic domain derived for the fusion sequence of the HIV-1 gp41 protein with the NLS of the large T antigen of SV40 (17
). MPG can cross cell membranes in a receptor-independent fashion and can therefore be associated with the class of peptides termed cell-penetrating peptides (for a review see 33
). In the present work, we have investigated the mechanism through which MPG promotes gene delivery and have demonstrated that it is independent of the endosomal pathway. In this respect the mechanism through which MPG promotes gene delivery differs from that of other peptide-based gene delivery systems described so far (2
) and is more related to that proposed for cell-penetrating peptides such as penetratin, transportan and TAT (9
). However, recently some controversy concerning the mechanism of cell-penetrating peptides has arisen and divergent interpretations have been ascribed to differences in experimental procedures or in cell lines used (12
). Recent progress in live cell imaging microscopy should help to avoid misinterpretations. Several studies have revealed that in the case of the TAT-based gene delivery system, the size of the peptide/DNA complexes plays an essential role in the uptake mechanism. Particles smaller than 300 nm do not enter the cell through the endosomal pathway (14
), in contrast to particles of 500–700 nm, which are taken up by endocytosis (16
). Along these lines, an explanation for the cellular uptake mechanism of MPG may therefore be found within the size of the MPG/DNA particles. Indeed, we previously determined by dynamic light scattering that the size of MPG/DNA particles was ~200–300 nm (18
). In this study, we also report that small MPG/DNA particles are able to enter cells in the presence of inhibitors of the endosomal pathway, in contrast to larger MPG/DNA aggregates. One part of the controversy concerning the mechanism of cell-penetrating peptides may be due to the size of the particles, which plays a crucial role in the internalisation pathway.
Most transfection systems described so far require the nuclear membrane to break down during mitosis for DNA to get into the nucleus, and are therefore dependent on active cell cycle progression (1
). As the nucleus constitutes one of the major cellular compartments to be targeted, several strategies have been proposed to improve nuclear uptake of DNA. NLSs have been associated with gene delivery formulations, either covalently attached to DNA (34
), fused with a peptide nucleic acid (36
) or incorporated into lipid-based formulations (37
). The nuclear translocation property of TAT has been used to promote nuclear targeting of plasmids (13
). Here we have demonstrated that the NLS of MPG facilitates nuclear translocation of DNA and that MPG/DNA complexes interact directly with the nuclear import machinery though importin α. Hence, the NLS constitutes an essential and unique feature of MPG in comparison with other gene delivery systems. Moreover, we have shown that a specific mutation in the NLS impairs translocation of DNA into the nucleus and have used this property to promote delivery of siRNA into the cytoplasm, where it was more efficient in its silencing function than in the nucleus, consistent with the recently reported active site of siRNA (38
Our investigation of the mechanism underlying the ability of MPG to deliver nucleic acids into cells has revealed that MPG-mediated delivery is independent of the endosomal pathway, and in part dependent on the NLS, which is involved in electrostatic interactions with DNA as well as specific interactions with the nuclear import machinery. Moreover, we have shown that the NLS is essential to promote delivery of nucleic acids to the nucleus, but that it is not required for cytoplasmic targeting. This latter feature suggests that specific variations in the sequence of MPG may yield carriers with distinct targeting features. In conclusion, the efficiency together with the targeting diversity of MPG-like peptides supports their potential for a wide range of therapeutic applications.