Novel drug delivery systems for gene-targeted RNA interference

RNA interference refers to sequence-specific messenger RNA degradation initiated by introduction of short RNA duplexes. Since this event results in target-specific gene silencing, siRNAs are expected to serve as potential therapeutic agents to silence disease-causing genes. However, a substantial barrier to the therapeutic application is the delivery of siRNA duplexes into cells.; Cyanocobalamin is one of the essential vitamins needed for the division of all cells. Cancer cells especially require abnormally high amounts of cobalamins due to rapid cell division. As a result, the cobalamin receptor density on the surface of the cell is over-expressed. Therefore, a 21 nucleotide long bcr-abl gene-specific siRNA was conjugated to cyanocobalamin, and its uptake was investigated in K562 human chronic myelogenous leukemia cells by confocal fluorescence microscopy.; There was no uptake of fluorescein in control cells incubated with a fluorescently modified sense strand. Cells incubated with the cyanocobalamin siRNA bioconjugate demonstrated significant uptake of the conjugate due to cobalamin-dependent endocytosis. These results clearly show that the delivery of siRNA into cells is successfully achieved by chemically modifying an essential vitamin that is sufficiently recognized by cancer cells.; Suppression of bcr-abl specific genes results in the inhibition of bcr-abl oncoprotein production. Silencing bcr-abl mRNA with the corresponding siRNA in K562 cells is achieved by exact sequence-specific manner. Therefore, the cyanocobalamin siRNA conjugate was incubated in K562 cells for 48 hours and the inhibition of the bcr-abl oncoprotein was examined by SDS-PAGE and Western blot.; The cyanocobalamin siRNA conjugate silences bcr-abl gene-specific mRNA that results in a total suppression of the oncoprotein production, whereas the scrambled cyanocobalamin siRNA conjugate completely lost its silencing effect. When the cyanocobalamin siRNA conjugate was incubated in K562 cells for 48 hours or 72 hours, inhibition of cell proliferation was also observed that was consistent with suppression of the bcr-abl gene specific oncoprotein production.; These findings open a door for the potential use of cobalamin as a novel carrier of siRNA for gene-targeted RNA interference.