The effectors of the RNAi pathway are small RNA (RNA) molecules that can be classified into at least three categories based on their origin and function: microRNAs (miRNA), short interfering RNAs (siRNA), and short hairpin RNAs (shRNA).

MiRNAs represent endogenously encoded small RNAs, about 22 nucleotides long, which function to post-transcriptionally repress gene expression by binding to target mRNAs in a sequence specific manner. Though discovered relatively recently, miRNA are now recognized as key regulators of gene expression in plants and animals. The genes encoding miRNAs are transcribed by RNA polymerase II in a long primary miRNA that is cleaved in the nucleus by Drosha/DGCR8 into a precursor miRNA (pre-miRNA). The pre-miRNA consists of about 70 base pairs with a single-stranded hairpin loop. Pre-miRNA is exported from the nucleus via exportin-5, and is cleaved by Dicer in the cytoplasm to generate a mature miRNA about 22 nucleotides long. This mature miRNA is incorporated into a complex known as the RNA-induced silencing complex (RISC) where it binds to its target mRNA though complementary base pairing. If complementarity is perfect, mRNA degradation typically occurs, whereas if complementarity is partial, translational repression typically occurs [1].

The shRNAs are dsRNAs engineered with a similar structure as miRNA, and are either tranfected into cells directly or a vector encoding the shRNA is introduced into the cells and transcribed.

SiRNA molecules are synthetic dsRNAs ranging in size from about 19–30 nucleotides that function to cause mRNA degradation through the perfect pairing of complementary sequences [2]. These molecules have become invaluable research tools to study the functions of genes and an alternative to knock-out mice.

One advantage of using shRNAs is that they assimilate into the endogenous RNAi pathway, and are therefore more efficient than exogenously introduced siRNAs [3]. Using a viral vector-based delivery system for shRNAs increases transfection efficiency and allows harder-to-transfect cells such as non-dividing neuronal cells to be targeted. However, there are obvious safety concerns associated with the use of viral expression vectors in humans. Other delivery strategies for shRNA include coupling with monoclonal antibodies, peptides, or aptamers that recognize specific cell surface molecules, but each of these strategies are associated with increased toxicity [4,5,6,7].

In recent years, differential expression of miRNA has been associated with a wide variety of human diseases ranging from many types of cancer to autoimmune diseases and everything in between. This revelation makes them an attractive target for therapies. The premise is that if we can elucidate reliable techniques to alter miRNA levels in specific cells/tissues, perhaps we can ameliorate disease symptoms or cure diseases altogether. There are two main strategies that can be employed to do this. The first is the use of synthetic miRNA mimics to restore the expression level of a particular miRNA back to normal levels [8]. This strategy presents many of the same challenges faced by siRNA therapies to be discussed later, such as stability and delivery. In addition, most miRNAs are pleiotropic hence altering levels of any given miRNA may result in unintended consequences. The second strategy is the use of miRNA “sponges” to block the action of over-expressed miRNAs [8]. Again, many of the same challenges apply to the use of sponges including design, delivery, and the pleiotropic nature of most miRNAs.

One study set out to develop a siRNA-based therapeutic targeting Sjögren’s syndrome (SjS), an autoimmune disease characterized by lymphocytic infiltration in the salivary and lacrimal glands resulting is severe dry mouth and dry eye symptoms due in part to apoptotic cell death of secretory acinar cells in the glands [19]. Currently, the only course of treatment available to SjS patients is immunosuppressive therapy and secretagogues, a class of drugs that stimulate secretion. Pauley et al. designed a conjugate composed of a siRNA molecule targeting caspase-3 and a muscarinic receptor agonist, carbachol [19]. Carbachol binds to muscarinic receptors inducing secretion and is endocytosed via receptor-mediated endocytosis. Linking a siRNA molecule to carbachol allows cellular uptake of the siRNA through this receptor-mediated endocytic pathway and specific delivery of the siRNA since only cells expressing muscarinic receptors including salivary acinar cells would be targeted. Pauley et al. showed that both the siRNA and carbachol remained functional after conjugation and HSG cells treated with conjugate (in the absence of transfection reagent) exhibited significant reduction in caspase-3 gene/protein expression, indicating that the conjugate is taken up by cells via muscarinic receptor-endocytosis. More importantly, inflammatory cytokine-induced apoptosis of HSG cells was inhibited by conjugate treatment [19]. These data suggest that this strategy may be useful to prevent apoptotic cell death of secretory acinar cells in SjS patients, thus maintaining secretory function to improve quality of life for the patients. Further studies are needed to continue developing this conjugate for in vivo use.

Several studies have examined the potential use of siRNA therapy in rheumatoid arthritis (RA). In 2005, Schiffelers et al. investigated the use of localized electroporation for in vivo delivery of siRNA into joint tissue of arthritic mice [20]. It was demonstrated that electroporation of anti-TNFα siRNA resulted in inhibition of joint inflammation in collagen-induced arthritis. A later study by Khoury et al. in 2006 utilized a DNA molecule for complexation and a cationic liposome for the intravenous delivery of anti-TNFα siRNA in collagen-induced arthritic mice [21]. When administered weekly, a 50%–70% reduction of TNF-α and an almost complete ablation of arthritic symptoms was observed in siRNA-treated mice. However, this systemic delivery approach resulted in uptake of siRNA by multiple organs including brain, lungs, liver and spleen.

A more recent study extended this work by testing the efficacy of a “wraposome” to systemically deliver siRNA to arthritic joints in collagen-induced arthritic mice [22]. A wraposome consists of a core composed of a cationic lipid bilayer and siRNA complex enveloped in a neutrak lipid bilayer with polyethylene glycol on the surface. This design results in a neutral surface charge and prevents the non-specific binding of the liposome to plasma proteins after intravenous injection. Collagen-induced arthritic mice receiving siRNA targeting TNF-α via this systemic wraposome delivery exhibited significant decreases in the severity of arthritis coupled with reduction of TNF-α in the joints. Interestingly, the siRNA was mainly incorporated into macrophages and neutrophils in the inflamed synovium [22]. These findings support the potential therapeutic effects of suppressing inflammatory molecules via siRNA in rheumatoid arthritis.

Yet another study examining the potential use of RNAi therapy in an arthritis animal model used a different strategy. Chen et al. used a lenti-viral vector to deliver short hairpin RNA targeting TLR7 in collagen-induced arthritic mice [23]. Again, severity of arthritis symptoms and expression of inflammatory mediators in the joints were reduced in treated mice compared to untreated mice.

In addition to siRNA therapeutic potential in RA, certain miRNAs, particularly miR-146a, have been found to be differentially expressed in RA and other autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus (SLE) [24,25,26,27,28,29,30,31,32]. One study demonstrated the potential therapeutic application of miR-146a for RA by demonstrating the inhibitory effect of miR-146a on osteoclastogenesis in vitro [33]. Furthermore, Nakasa et al. were able to demonstrate that intravenous administration of miR-146a resulted in the suppression of cartilage and bone destruction in a collagen-induced arthritis mouse model [33]. These findings support the therapeutic potential of miRNA modulation in autoimmune diseases. However, further studies are needed to identify and understand differentially expressed miRNAs in autoimmune disease patients.