| In the past few years, scientists have discovered a new mechanism for the reduction of gene expression in mammalian cells via sequence-specific cleavage of a particular messenger RNA (mRNA); this phenomenon is known as RNA interference (RNAi). Since RNAi is triggered by nucleic acids (small interfering RNA (siRNA) duplexes), I hypothesized that cyclodextrn-containing polycations (CDPs), previously developed in my laboratory for delivery of plasmid DNA, may be suitable vectors for the delivery of siRNA.; Because some report suggest siRNA duplexes may be immunogenic, I first examined the safety of synthetic siRNA duplexes both in cultured cells and in vivo. Using a number of different siRNA sequences, two different strains of mice, and three different methods of administration, I fail to observe any siRNA-dependent cytokine (IL-12 or IFN-alpha) responses, morphological changes, or alterations in complete blood counts (CBCs) or liver enzyme levels.; I hypothesized that cyclodextrn-containing polycations (CDPs), previously developed in my laboratory for delivery of plasmid DNA, may be suitable vectors for the delivery of siRNA. I demonstrate that CDP/siRNA complexes can be formed that are small enough to be endocytosed, can be modified to ensure stability in physiological fluid, and protect the siRNA payload from serum nuclease degradation.; Finally, down-regulation of specific target genes, including genes implicated in disease, is shown in vitro and in mice. An endogenous reporter gene (luciferase) in the livers of transgenic mice is down-regulated by galactosylated CDP/siRNA formulations that target hepatocytes. The level of a chimeric oncogene, EWS-Fli1, is reduced by polyplex formulations in cultured Ewing's sarcoma cells and by transferrin-targeted formulations in tumor-bearing mice; this in vivo down-regulation corresponds to an inhibition of tumor growth. These results suggest that CDP-containing siRNA formulations have strong potential for development into therapeutics. |
