Abstract
The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.
Highlights
The development of non-viral-based gene delivery systems constitutes an essential challenge in therapeutics
We have investigated the mechanism through which MPG promotes gene delivery into cells and have demonstrated that it is independent of the endosomal pathway
To exclude the possibility that the MPG/DNA complexes remain associated with the cell membrane in the presence of inhibitors and only enter the cell upon dilution of the inhibitor, transfection experiments were performed in the presence of inhibitors, using auorescently labelled oligonucleotide [17]
Summary
The development of non-viral-based gene delivery systems constitutes an essential challenge in therapeutics. They exhibit several advantages over viral systems, the interest of non-viral synthetic gene delivery systems for therapeutic applications remains limited by their poor ability to escape from the endosomal compartment and to translocate DNA into the nucleus [1]. Poor release from the endosomal compartment after cellular uptake constitutes one of the major limitations of nonviral gene delivery systems. Peptide carriers that combine DNA binding and membrane destabilising properties have been demonstrated to promote gene transfer into cultured cells [5,6] and living animals [7]
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