RNA interference as a tool for treating disease

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Recent advancements in the studies of gene expression regulation demonstrating their potential use in treating myriad kinds of diseases have attracted considerable attention from researchers worldwide. With the completion of the Human Genome Project, there is now an exponentially better insight to the molecular aetiology of most common diseases and it has been postulated that this information may prove useful to control the expression of the pathogenic genes by gene silencing (Stoneking, 1997). The RNA interference (RNAi) technology is one such novel tool that is currently being developed clinically as a therapeutic modality to suppress gene expression in various diseases. The RNAi phenomenon was first unveiled in plants over a decade ago. Soon after the initial experiment with Caenorhabditis elegans, it came to light that the RNAi mechanism can be triggered in cells or organisms through the delivery of homologous double-stranded RNA (dsRNA) (Fire et al., 1998). Since then the RNAi techniques have been intensely exploited in the field of functional genomics to elucidate the functions of mammalian genes in both ex vivo and in vivo contexts (Elbashir et al., 2001a; Nakade et al., 2010). The hallmark of RNAi, which is defined by its selective gene silencing mechanism in response to the dsRNA cues, not only makes it a valuable research tool for studying and investigating gene expression but also shows a promising prospect to revolutionise the field of medicine in future.

Growing evidence indicates that the short interfering RNAs (siRNAs), also known as silencing RNAs, are the key players in RNAi pathway to selectively silence target genes. The siRNAs are chemically synthesized molecules consisting of 20-25 nucleotides (Elbashir et al., 2001b). Several studies have shown that this class of dsRNA plays important roles in various biological processes. The inhibitory effects of the siRNAs have been reported in numerous cell-based studies looking at pathways and alterations in the expression of genes that are involved in cellular processes, including endocytosis, signal transduction, apoptosis, and cell cycle regulation and also ingenes associated with neurodegenerative diseases and xenotransplantation (Miyagawa et al., 2005; Sudarsana et al., 2006; Collinet et al., 2010). In animals and plants, siRNAs are derived from cytoplasmic processing of long dsRNA by RNAse-III type enzyme termed Dicer, and its associated proteins such as transactivating region binding protein (TRBP) (Figure 1). Dicer cleaves long dsRNA into 21 nucleotides siRNA duplexes that consist of 2-nucleotide 3'-overhangs with 5'-phosphate and 3'-hydoxyl termini (Zhang et al., 2004). One of the strands, known as the guide strand/sense strand, is integrated into an RNA-induced silencing complex (RISC). The other strand,known as the passenger strand/antisense strand, is degraded. However in 2006, a group of researchers postulated that this antisense strand might play a role in directing histone methylation and transcriptional gene silencing in human cells (Weinberg et al., 2006). The siRNA guide strand, by complete complementation, will trigger the degradation of target mRNA in the RISC through its core component Argonaute 2 (Ago2) (Meister et al., 2004). This sequence-specific gene-silencing feature makes the siRNA an effective method to investigate functional roles of individual genes related to certain diseases in the mammalian cells, and potentially permits its possible use against human diseases.

siRNA therapy has its distinct advantages over traditional pharmaceutical drugs. Most importantly, its highly selective inhibitory effect makes it a significantly better therapeutic modality as compared to the development of new chemicals. Furthermore, the siRNAs are relatively easy to be synthesized and produced on a large scale, highlighting their huge potential in silencing therapeutics. Although current biological drugs such as monoclonal antibodies are also able to specifically target diseased cells in the body, they appear to have some pitfalls. Biological drugs seem to have fewer side effects compared to traditional chemical drugs, it can lead to development of some rare side effects which may eventually lead to severe and life-threatening diseases (Bumcot et al., 2006; Salvana and Salata, 2009). Such limitations have resulted in the rapid acceleration of development and optimization of siRNA-based gene therapy. Besides, compared to the use of monoclonal antibodies, siRNA-based therapeutic requires only very low concentrations; only a few siRNA molecules per cell are needed to induce its therapeutic effect. siRNA-based drugs are capable of targeting any mRNAs of interest regardless of their cellular location, by harnessing the endogenous biological pathway. Reports also show that the siRNA molecules are able to block specific gene expressions in different mammalian cell lines (Reich et al., 2003; Singh et al, 2007; Hao et al., 2009). This body of evidence attests to the potential role of siRNA as a key target molecule in biomedical research and in the development of innovative medicines. The RNAi-based gene therapy holds great therapeutic potential in various genetic diseases, especially those that are due to dominant genetic effect (Lewin et al., 2005; Leachman et al., 2008). At present, RNAi technology is in clinical use to treat diseases including cancer, infectious diseases, and also respiratory diseases.

Utility Potential of siRNA in Diseases

siRNA in cancer

RNAi-based gene therapy has high prospective in treating one of the most genetically complicated disease, cancer. Cancer has been, previously, considered as an incurable disease. However, this perception has changed considerably with the current development of siRNA as an innovative medicine. The siRNA can be used directly as a therapeutic modality to specifically silence genes that are involved in cancer development and growth (Dong et al., 2009). Apart from that, it can also be implemented in large-scale screening to identify cancer-related genes, which could ultimately lead to the invention of curative genetic treatments against these cancers. Thus far, the RNAi-based therapeutics has pledged a propitious future in treating cervical cancer. The oncogenes of the human papilloma virus (HPV) E6 and E7 have been identified as good targets for the RNAi-based therapeutics against cervical cancer. The siRNAs targeting E6/E7 have been observed to significantly inhibit tumor growth in mouse model (Jonson et al., 2008). In another study, an atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 has been reported to induce the suppression of tumor growth in a cervical cancer xenograft mice model (Fujii et al., 2006). The atelocollagen-siRNA complex is a non-viral delivery system which has been showed to have potential uses in studying cancer metastasis, drug discovery and functional screening of genes (Honma et al., 2007), which presents us with a novel therapeutic approach that could be extended to treat other cancers as well. In addition to that, knockdown of Akt isoforms 2 and 3 in uterine cancer using siRNA technology showed significant reduction in the resistance of the endometrial KLE cells to cisplatin-induced apoptosis (Gagnon et al., 2004). Other than that, another group of researchers demonstrated that stable suppression of MDR-1 gene by inducing the endogenous expression of hairpin siRNA. Their result shown siRNA is able to reverse chemo-resistance in a human uterine sarcoma cell line (Huaa et al., 2005).

siRNA therapy is being reported in DNA repair mechanisms. Endo-exonuclease is overexpressed in a variety of cancer cells as well as involved in the nucleolytic processing of DNA (Chow and Choudhury, 2005). siRNA is being used to identify endo-exonuclease in cancer cells, and that endo-exonuclease is a potential yet promising anticancer target (Chow et al., 2004). Besides that, signaling molecules are being reported to play vital roles in some cancers. B-RAF is the protein that encodes serine/threonine-specific protein kinase that is mutated in ~70% of human melanomas. B-RAF linked extracellular signal-regulated protein kinase (ERK) pathway promotes cell proliferation and cell growth (Wellbrock et al., 2004). A number of siRNA delivery systems are being developed for cancer cell treatment particularly those derived from signaling molecules. The delivery of siRNA targeted at signaling peptide of secretory clusterin is specifically carried by nontoxic carriers such as copolymers of PEI and PEG (PEI-g-PEG) (Malek et al., 2008). These complexes reduced the secretion of clusterin and increased the ionizing radiation lethality in human MCF-7 breast cancer cells (Sutton et al., 2006). Recently, a team of researchers and doctors from the California Institute of Technology (Caltech, USA) have conducted the first in-human phase 1 clinical trial in melanoma patients using a siRNA targeted nanoparticle delivery system. They demonstrated that delivery of siRNA systemically can produce a specific gene inhibition (reduction in mRNA and protein) (Davis et al., 2010). During the clinical trials, the presence of an mRNA fragment that demonstrates that siRNA-mediated mRNA cleavage occurs specifically at the site predicted for an RNAi mechanism from a patient who received the highest dose of the nanoparticles (Davis et al., 2010).

siRNA in Infectious Diseases

Considering the specificity and potentiality of RNAi triggering activity, RNAi phenomenon have been applied in gene silencing research to combat various virus-causing diseases including human immunodeficiency virus (HIV), severe acute respiratory syndrome (SARS)-associated coronavirus, human amoebiasis hepatitis parasite and the influenza virus (Ge et al., 2003; Leung and Whittaker, 2005). Researchers believe RNAi can be exploited for treating various viral diseases as it has been shown to be capable of inhibiting the expression of viral surface antigens, suppressing the transcription of viral genomes in host cells and hindering the assembly of viral particles (Zhang et al., 2010). RNAi also displays its roles in virus-host interactions (Peng et al., 2009). HIV is a highly lethal lentivirus (a member of the retrovirus family) that leads to the most fearsome of all diseases, the acquired immunodeficiency syndrome (AIDS). It is a condition where the immune system cannot function properly, causing life-threatening opportunistic infections in humans. In HIV, knockdown of the primary HIV coreceptor chemokine (C-C motif) receptor 5 (CCR) by siRNA resulted in the prevention of viral entry into human peripheral blood lymphocytes primary haematopoietic stem cells (Qin et al., 2003). Another group of researcher demonstrated that HIV infection can be suppressed with the treatment of anti-CCR5 (viral coreceptor) and antiviral siRNAs complexed to scFvCD7-9R in HIV-infected Hu-PBL mice (Kumar et al., 2008). This has attested the potential of siRNA therapy particularly in HIV infection and has shown its feasibility in preclinical animal model.

In the case of SARS, it has recently been shown that SARS-associated coronavirus replication can be efficiently inhibited using vector-derived siRNA-mediated RNAi in Vero cells. The generation of the plasmid-mediated siRNAs exemplifies a powerful means in inhibiting viral replication as shown by titer assays and by examining the viral RNA and protein levels (Wang et al., 2004). The potency of siRNA inhibitors of SARS coronavirus in vitro were further evaluated by a group of researcher for efficacy and safety in a rhesus macaque (Macaca mulatta) SARS model. Throughout their hard work, a significant result shows that siRNA does not have any symptoms on inducing toxicity and thus it can fulfill the need for targeted therapeutic agents (Li et al., 2005). As for human amoebiasis, a common infection of the human gastrointestinal tract caused by the protozoan parasite, Entamoeba histolytica and it is the second leading cause of death worldwide due to protozoan infection (Vayssie et al., 2004). Once inside its host, E. histolytica invades the intestinal mucosa, causing dysentery, spreads in the bloodstream and to the liver. In the liver, E. histolytica produces liver abscesses and eventually leads to liver damage (Petri, 2002). E. hystolytica exerts its pathogenic effects on its host by employing γ-tubulin, which plays vital roles in microtubule nucleation allowing it to take over control of nucleating microtubules in the host. The microtubule nucleation and cycling of the parasite can be knocked down by RNAi approach as demonstrated by Vayssie et al. (2004). The viral amino acid sequence is homologous (46%) but not identical to humans. Therefore, there are suggestions that specific siRNAs can be developed in order to destroy the parasite's γ-tubulin while leaving the host's counterpart untouched.

siRNA in Respiratory diseases

RNAi is a popular method of controlling gene expression in various respiratory diseases. With the treatment development, siRNA could be used to screen potential therapeutic targets. The molecular mechanisms underlying the pathogenesis of most respiratory diseases such as COPD, asthma and cystic fibrosis include oxidative stress and apoptotic cell death of the structural cells of the lungs (Wixted et al., 2009). To this end, a cell-based apoptosis assay was devised to identify genes that modulate cellular response to oxidative stress by screening the druggable genome siRNA library. Numerous potential therapeutic targets such as FLT3LG and CES4 have been identified, and this presents researchers with a vast starting point for development of treatments, illustrating the immense usefulness of siRNA in treatment development. A direct use of siRNA in treating COPD could be achieved with the silencing of matrix metalloproteinase-12 (MMP-12). MMP-12, a well-documented player in the development of lung diseases, causes elastin degradation and lung parenchyma disorganization when activated (Garbacki et al., 2009). By targeting MMP-12, siRNA has been shown to silence its expression and thus could potentially be a therapeutic candidate to treat COPD. Another potential siRNA-COPD therapy could also be developed by knocking down transforming growth factor (TGF)-α production in NCI-H292 human airway epithelial cells (Shao et al., 2004). As a consequence, epidermal growth factor receptor (EGFR) expression is significantly reduced, leading to diminishing mucus production.

siRNA delivery for asthma has already been developed and has demonstrated its promising results as biopharmaceutical therapeutics in animal models (Darcan-Nicolaisen et al., 2009; Walker et al., 2009). One potential therapeutic target to treat asthma is by silencing the expression of STAT6, a transcription factor which orchestrates the immune response central in asthma development. The silencing was demonstrated by Walker and colleagues, who further found suppressive effects of the downstream inflammatory chemokines such as CCL26, supporting the feasibility of developing this model as a therapeutic target to treat asthma (Walker et al., 2008). Delivery of RNase P-associated external guide sequence (EGS) targeting IL-4, a cytokine which activates STAT6, into pulmonary tissues was also found to have ameliorative effects on asthma (Dreyfus et al., 2004). Delivery of the RNase P-associated external guide sequence (EGS) into pulmonary tissues was found beneficial in asthma. EGS posited its usefulness in the pulmonary delivery of catalytic RNA oligonucleotides by targeting specific gene expression through site-specific cleavage of mRNA mechanism (Dreyfus et al., 2004). siRNA has also shown potential in treating cystic fibrosis. It is a lethal disorder that is due to mutations in the genes encoding the cAMP-activated anion CF transmembrane conductance regulator (CFTR) channel. With the insertion of single complementary-strand oligonucleotide into cultured cystic fibrosis transmembrane conductance regulator cell line, the cystic fibrosis phenotype has been shown to be reversed (Zamecnik et al., 2004). Another study showed a therapeutic potential of siRNA targeting the Hop protein, which causes the maturation of the defective CFTR channel and thus correcting cystic fibrosis (Marozkina et al., 2010). The body of evidence presented here represents only a fraction of the studies that have been carried out to develop siRNA-based gene therapies, demonstrating the tremendous potential of siRNA-based gene therapies in treating respiratory diseases.

Drawbacks of siRNA technology

The promising outcomes of siRNA-based strategies for gene therapy in in vitro studies have accelerated the pace of the in vivo studies. For several years, quite a number of pharmaceutical companies have ventured into the siRNA-based therapeutics. However there are some hurdles that need to be overcome before the siRNA can be widely utilized in clinical practice. siRNAs are found to be easily degraded by RNases and interact with blood components in vivo. It has also been reported that boranophosphate modification of siRNAs has assisted in reducing the nuclease degradation problems (Hall et al., 2004; Hall et al., 2006). Nevertheless siRNAs which are longer than 30 nucleotides have been found to stimulate the immune system in specialized sensitive cell lines and cause toxicity at high concentration. The high levels of siRNAs have been demonstrated to produce interferon and inflammatory cytokines (Persengiev et al., 2004). To overcome these problems, chemical modifications of the siRNAs have been tried out and which has helped. The modification in the designing siRNAs such as the generation of blunt 19 mer duplexes with 2'O methyl modifications and 25 mer duplexes with 2'O methyl modifications in the sense strand have been found beneficial in reducing interferon response (Czauderna et al., 2003; Chen et al., 2005). Other than that, small molecular mass of the siRNA leads to fast elimination of siRNA by kidney filtration. However the delivery of the short sequence of siRNA makes it useful in mammalian cell without producing any interferon response (Reynolds et al., 2006).

Perhaps the most challenging aspect in RNAi therapeutics currently is the lack of an appropriate delivery system and also the problem of low affinity of siRNA towards their target RNAs. Unmodified naked siRNAs are unable to penetrate through the cell membrane because of their strong anionic charge on the backbone and electric repulsion from the anionic cell membrane (Reischl and Zimmer, 2009). In order to overcome these hurdles, chemical modification of siRNAs has been sorted with some encouraging results and the use of carriers of the siRNAs both viral and non-viral have been implemented (Aigner 2006; Behlke, 2006). Non-viral carriers have proven to be somewhat better when compared to their viral counterparts as they are not only protect the siRNAs from nucleases and also do not elicit interferon induction. Furthermore, in-vivo administered of siRNAs which resulted in low transfection efficiency and poor tissue penetration has indirectly delayed their therapeutic potentiality. Thus, this has raised some concerns among researcher regarding the efficiency and safety of siRNA-based therapeutics. The low affinity of siRNA can be due to secondary structures of the target mRNAs. This problem can be solved if the high GC rich regions are avoidable on the target mRNAs while designing siRNAs. In addition, the poor pharmacokinetic properties of siRNA are to be taken into account when considering their application in in vivo therapy. Hence careful designing and preliminary testing are an essential part before proceed to the administration of these siRNA in vivo.

Concluding Remarks

In this review, we have highlighted the utility potential of RNAi therapeutics and shown how this appealing approach has revolutionized the field of medicine. There are numerous studies that have been done to evaluate the potential use of RNAi therapeutics and accumulating evidence ascertains that almost all of genes can be potently suppressed by siRNA in vitro. However, the systemic delivery of siRNA remains a major obstacle that needs to be overcome before the RNAi drugs are transferred from bench to bedside. To date, the progress in developing systemic delivery systems to effectively deliver siRNA in vivo is advancing significantly. Yet, knowledge gaps pertaining to the molecular machinery of RNA and RNAi still need to be addressed as the systemic delivery system remains a crucial component in realization of the full potential of this promising technology.