RNA interference (RNAi) is a catalytic mechanism of gene-specific silencing in eukaryotic organisms with profound implications for biology and medicine. However, the inducers of such a powerful mechanism of gene-targeting are very different in mammalian cells and in non-mammalian cells.
Long double-strand RNAs (dsRNAs) triggered potent sequence-specific gene silencing in C. elegans and Drosophila Melanogaster. In contrast, long dsRNAs induce sequence-nonspecific response in mammalian cells due to activation of interferon related pathways. RNAi mechanism was considered non-functional in mammalian cells until siRNA duplexes were discovered.
The use of short interfering RNA (siRNA) to selectively target messenger RNA (mRNA) for degradation results in the silencing or knock-down of the gene expressed through the degradation of target mRNA. siRNA are typically double-strand RNA (dsRNA) of 20-25 nucleotide long with a few unpaired overhang bases on each strand. Based on this model, molecular biology techniques using siRNA have emerged both as a research tool and a candidate for therapeutics (Dykxhoorn, Novina & Sharp. Nat. Rev. Mol. Cell Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184 (2007); de Fougerolle s, et al. Nature Rev. Drug Discov. 6:443-453 (2007)).
While being used widely for gene silencing in mammalian cells, siRNA has posed numerous problems that limit the potential of RNAi in biomedical research and development of RNA-therapeutics. Unlike long dsRNA in non-mammlain cells, designing siRNA has proven difficult. First, within a gene sequence, only a certain sequence motifs can serve as template for siRNA. Identification of such motifs has been nearly a process of trials and errors despite of numerous algorithms developed over the years. Second, unlike long dsRNA in non-mammalian cells, siRNA has by and large low gene-silencing efficiency in mammalian cells (Reynolds A et al. Nature Biotech 22:326, 2004; A. de Fougerolles et al, Nat Rev Drug Discov 6, 443 (2007).). Finding a highly efficacious siRNA motif along an mRNA is difficult, and can be impossible for some mRNAs, due to nearly unlimited possible motifs of 20-25 nt contained in an mRNA. Third, after an siRNA is designed, delivery of negatively charged siRNA into mammalian cells has proven a daunting challenge (Li C X, et al. Cell Cycle 5:2103-2109 (2006)).
It would be highly desirable if a technology can enable the use of long dsRNA in mammalian cells. There is so far no reported success on introducing exogenous long dsRNA directly into the target mammalian cells due to strong cellular innate immune response, such as nonspecific interferon (IFN) responses. (Yang, S. et al. Mol. Cell. Biol. 21: 7807-7816 (2001)).