Are siRNAs Specific Enough to Become Therapeutics? - siRNA

Thus, the microarray patterns from two different siRNAs targeting the same mRNA may be identical, but the siRNAs could still have differential effects through translational inhibition. With all of these studies demonstrating that siRNAs may not be as specific as we once thought, why are so many investigators using RNAi?
For sure, not everyone using siRNAs observes these nonspecific effects on gene expression. After the Rosetta Inpharmactics group demonstrated a plethora of secondary effects from their siRNA transfections (Jackson et al., Nature Biotechnology, 21:6, 2003), another paper was published showing the high degree of specificity of siRNAs (Chi et al., PNAS, 100:11, 2003). Both groups utilized gene expression profiling and obtained different results. It remains unclear under which conditions these nonspecific effects may occur.
Dharmacon recently published a letter to the editor in Nature Methods (2:4, 2005) demonstrating that the majority of the genes up-regulated in response to siRNA/Lipofectamine2000 transfection were due to the cationic lipid itself.
Minimizing Non-specific Effects
Thus, it is conceivable that different delivery methods may be required to demonstrate specificity against the intended target mRNA. Further, nonspecific effects can be minimized by simply lowering the siRNA concentration; however, this strategy is possible only when the siRNA under study is highly active at subnanomolar concentrations.
Along these lines, studies from the VACT Cancer Center laboratory have identified an siRNA that inhibits 50% of thymidylate synthase expression at picomolar concentrations (Schmitz et al., Cancer Research, 64, 2004).
Another nonsequence effect that has been observed with siRNA administration is the induction of the innate immune system. While it was initially thought that siRNAs were small enough to avoid detection by the immune system, recent studies have proved otherwise.
Two recent publications in Nature have identified two nucleotide motifs that can induce interferons and interleukins (Hornung et al., Nature Medicine, 11, 2005; Judge et al., Nature Biotechnology, 23:4, 2005). Lipid-encapsulated siRNAs containing the sequences 5'-UGUGU-3' or 5'-GUCCUUCAA-3' induced IFN-a and IL-6 in the serum when injected into mice. If these siRNA sequences were mismatched, the ability of the siRNA to induce IFN-a was greatly diminished demonstrating the specificity of the immune response to these sequences. Neither the siRNAs nor the cationic delivery molecules by themselves were able to activate the immune system.
SiRNA Therapeutics has shown that while its HBV-targeted siRNA can be immunostimulatory, its chemical siRNA modifications prevented this nonspecific effect. It is likely that more immunostimulatory sequences will be identified and only careful siRNA design and chemical modifications will prevent this effect from hindering advancement of siRNA therapeutic molecules.
It remains unclear how these unintended effects of siRNAs may impact their potential therapeutic use. However, the great strides already accomplished regarding chemically stabilized siRNAs and multiple new delivery options are moving the RNAi field forward at a fast pace. As an enhanced understanding of the RNAi pathway is obtained, new selection guidelines will emerge that account for the G:U and C:A mismatches, potential miRNA-like structures, and immunostimulatory sequences.
In addition to intravenous administration, recent studies have shown the feasibility of inhaled siRNAs and topical applications (Bitko et al., Nature Medicine, 11, 2005; Jiang et al., Oligonucleotides, 14, 2005).
Several companies already have therapeutic siRNAs in clinical trials. With its Phase I trial almost completed, Acuity Pharmaceuticals is planning Phase II clinical trials later this year with its lead molecule Cand5 targeting VEGF against AMD.
Earlier this year, SiRNA Therapeutics presented interim data from its AMD Phase I trial with siRNA-027 targeting VEGFR-1 and is actively pursuing efforts to target Hepatitis C. Given the success of these trials, Alnylam has suspended further development of its own siRNA targeting VEGF and has focused its effort on filling its IND for ALN-RSV01, an siRNA that targets RSV infection.
While siRNA therapeutics is a reality, the fate of the RNAi therapeutics field will be determined based on the data from these clinical trials.