Protein-based vehicle for siRNA delivery

Abstract

For the efficient and specific delivery of siRNA, protein-based vehicles were produced and characterized. A double-stranded RNA binding domain (dsRBD) of human dsRNA activated protein kinase R (PKR) was utilized to complex small interfering RNA (siRNA) to deliver into mammalian cells. The recombinantly produced polypeptide dsRBD exhibited its native binding activity for siRNA without sequence specificity and the complex protected siRNA from its degradation by ribonuclease. The resultant siRNA/dsRBD complexes were transfected to mammalian cells for target gene inhibition. GFP fluorescence inhibition assay showed prominent down-regulation of a target gene when the endosomal escape function was supplemented by addition of fusogenic peptide, KALA. These results suggest that dsRBD based carrier could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition, without showing any cytotoxicity. To accomplish multi-functionality such as binding affinity to siRNA, specific cell recognition and efficient endosome escape with a single molecule of protein vehicle, a multi-domain siRNA delivery carrier was constructed by using streptavidin and Pseudomonas Exotoxin A (ETA) fusion protein with cell membrane binding moiety. The resultant protein carrier showed binding affinity for various biotinylated molecules such as fluorescein (FITC), siRNA and biotinylated poly (ethylene glycol) (PEG) grafted poly ($_L$-lysine) (PLL). When the epidermal growth factor (EGF) receptor ligand was engineered to the protein carrier construct, efficient intracellular delivery of biotinylated FITC into EGF receptor overexpressing cell was observed by confocal microscopy. For GFP fluorescence inhibition assay, cell penetrating peptide, HPH, was introduced to N terminal region of ETA-STR protein construct. The evident down-regulation of GFP expression was shown with cell line dependency when transfected with complexes of the multidomain protein based siRNA de...