[en] BUSINESS ROLE IN WATERSHEDS: MOTIVATIONS, BENEFITS AND LIMITATIONS

Gene silencing, via RNA interference (RNAi) technologies using small interfering RNA (siRNA), has been developed as an important tool for target identification and validation in drug discovery and has huge therapeutic potential. However, effective delivery into cells presents a major challenge to the use of siRNA. pH responsive cell-penetrating peptides have attracted considerable attention in recent years as delivery vectors due to their ability to transport their cargos across the biological membrane and/or to promote endosomal escape and prevent lyososomal degradation. To evaluate the in vitro transfection efficiency of the pH responsive peptide-based siRNA delivery system, the western blotting technique is commonly employed. This method offers a simple, efficient and economical way to study the gene silencing effect of the siRNA by analysing the protein of interest in a sample with minimum equipment requirement. This chapter provides a description of siRNA delivery and analysis by western blotting protocols for qualitatively and quantitatively assessing gene silencing efficiency and selectivity

Histidine-Rich Cationic Cell-Penetrating Peptides for Plasmid DNA and siRNA Delivery

International audienceAmphipathic, pH-responsive, membrane-active peptides such as LAH4 and derivatives thereof have the ability to effectively deliver genes and small interfering RNA (siRNA) into mammalian cells. Their ability to bind and protect nucleic acids and then disrupt membranes when activated at low pH enables them to harness the endocytic machinery to deliver cargo efficiently and with low associated toxicity. This chapter describes protocols for the chemical synthesis of transfection peptides of the LAH4 family, complex formation with nucleic acids, and their use for the in vitro delivery of either plasmid DNA or siRNA into mammalian cell lines

Lipid interactions of LAH4, a peptide with antimicrobial and nucleic acid transfection activities

The cationic amphipathic designer peptide LAH4 exhibits potent antimicrobial, nucleic acid transfection and cell penetration activities. Closely related derivatives have been developed to enhance viral transduction for gene therapeutic assays. LAH4 contains four histidines and, consequently, its overall charge and membrane topology in lipid bilayers are strongly pH dependent. In order to better understand the differential interactions of this amphipathic peptide with negatively-charged membranes its interactions, topologies, and penetration depth were investigated in the presence of lipid bilayers as a function of pH, buffer, phospholipid head group, and fatty acyl chain composition using a combination of oriented synchrotron radiation circular dichroism spectroscopy as well as oriented and non-oriented solid-state NMR spectroscopy. This combination of methods indicates that in the presence of lipids with phosphatidylglycerol head groups, the topological equilibria of LAH4 is shifted towards more in-plane configurations even at neutral pH. In contrast, a transmembrane alignment is promoted when LAH4 interacts with membranes made of dimyristoyl phospholipids rather than palmitoyl-oleoyl-phospholipids. Finally, the addition of citrate buffer favours LAH4 transmembrane alignments, even at low pH, probably by complex formation with the cationic charges of the peptide. In summary, this study has revealed that the membrane topology of this peptide is readily modulated by the environmental conditions