A New Potent Secondary Amphipathic Cell-Penetrating Peptide For Sirna Delivery Into Mammalian Cells

RNA interference constitutes a powerful tool for biological studies, but has also
become one of the most challenging therapeutic strategies. However, small
interfering RNA (siRNA)-based strategies suffer from their poor delivery and
biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the
intracellular delivery of various biologically active molecules into living cells
and have more recently been applied to siRNA delivery. To improve cellular uptake
of siRNA into challenging cell lines, we have designed a secondary amphipathic
peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine
residues. CADY adopts a helical conformation within cell membranes, thereby
exposing charged residues on one side, and Trp groups that favor cellular uptake
on the other. We show that CADY forms stable complexes with siRNA, thereby
increasing their stability and improving their delivery into a wide variety of
cell lines, including suspension and primary cell lines. CADY-mediated delivery
of subnanomolar concentrations of siRNA leads to significant knockdown of the
target gene at both the mRNA and protein levels. Moreover, we demonstrate that
CADY is not toxic and enters cells through a mechanism which is independent of
the major endosomal pathway. Given its biological properties, we propose that
CADY-based technology will have a significant effect on the development of
fundamental and therapeutic siRNA-based applications.