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  • Title: Intracellular small interfering RNA delivery using genetically engineered double-stranded RNA binding protein domain.
    Author: Kim J, Lee SH, Choe J, Park TG.
    Journal: J Gene Med; 2009 Sep; 11(9):804-12. PubMed ID: 19569061.
    Abstract:
    BACKGROUND: A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double-stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery. METHODS: A double-stranded RNA binding domain (dsRBD) of human double-stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines. RESULTS: The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down-regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation. CONCLUSIONS: The results suggest that dsRBD-based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity.
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