Gene knockdown by RNA-induced gene silencing is presently believed to implicate a minimum of three different levels of control: (i) transcription inactivation (siRNA-guided DNA and histone methylation); (ii) small interfering RNA (siRNA)-induced mRNA degradation; and (iii) siRNA-induced transcriptional attenuation. The RNA interference (RNAi) generated by siRNA can be long lasting and effective over multiple cell divisions. Therefore, RNAi represents a potentially valuable tool that can be useful in gene function analysis, drug target validation, pathway analysis, and disease therapeutics.
Recent studies into the mechanism of RNAi-mediated transcript degradation pathway have revealed a number of key components in this pathway. A Type III RNase called Dicer processes long ds RNA into siRNA (19-23 bp duplexes) that subsequently partner with the RNA Interfering Silencing Complex (RISC) to mediate the degradation of target transcripts in a sequence specific manner. This phenomenon has been observed in a diverse group of organisms. Unfortunately, initial attempts to use long dsRNA to induce RNAi in mammalian cells met with only limited success due to induction of the interferon response, which results in a general, as opposed to targeted, inhibition of protein synthesis.
More recently, it has been shown that when short synthetic siRNAs are introduced into mammalian cells in culture, sequence-specific degradation of target mRNA can be achieved without inducing an interferon response. These short duplexes, can act catalytically at sub-molar concentrations to cleave greater than 95% of the target mRNA in a cell. A description of the mechanisms for siRNA activity, as well as some of its applications is provided in Provost et al., Ribonuclease Activity and RNA Binding of Recombinant Human Dicer, E.M.B.O.J, 2002 Nov., 1, 21(21): 5864-5874; Tabara et al., The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1 and a DexH-box Helicase to Direct RNAi in C. elegans, Cell 2002, Jun. 28, 109(7):861-71; Ketting et al., Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans, Genes and Development, 2001, 15(20):2654-9; and Martinez et al., Single-Stranded Antisense siRNAs Guide Target RNA Cleavage in RNAi, Cell 2002, Sep. 6, 110(5):563.
Despite the promise of RNAi, four main issues including functionality, specificity, delivery methods, and stability, must be addressed when working with siRNA. Specificity refers to the ability of a particular siRNA to silence a desired target without altering the expression of other genes, and recent studies have shown that “off-targeting” (i.e., the knockdown of targets other than the intended target) is much more extensive in RNAi than originally predicted (see Jackson, A. L. et al. (2003) “Expression profiling reveals off-target gene regulation by RNAi” Nature Biotechnology 21:635-7).
As off-target effects can induce undesirable phenotypes, new methods and compositions that minimize, alter, or eliminate off-target effects are considered indispensable for siRNA to become an efficacious research and therapeutic tool. The present invention addresses the issue of specificity by providing modifications to siRNA that can either increase or alter siRNA specificity.