Short interfering RNAs (siRNA) are double stranded RNA particles, thought to have evolved to repress the expression of harmful RNA such as transposons and RNA viruses (Kovar, Ban & Pospisilova 2003). Three different types of siRNA have been identified to date, and these include; endogenous-siRNA, miRNA and piRNA. The siRNAs are classed according to their length, how they are made and their mechanisms of action (see table 1 below) (Kim, Han & Siomi 2009). The siRNA inhibit post transcriptional gene expression by interfering with the translation of messenger RNA (mRNA) into the corresponding protein products in a process referred to as RNA interference (RNAi) (Reischl, Zimmer 2009). The observed RNAi is due to: silencing of gene expression mediated by siRNA directed heterochromatin formation, inhibition of mRNA translation, mRNA destruction or mRNA destabilization.
Table 1: Showing siRNA's length, origins and mechanisms of action:
Short RNA class
short RNA length
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Mechanism of action
Repetitive intergenic DNA sequences, endogenous siRNA clusters pseudogenes
Form of heterochromatin, cleave mRNA, down regulate translation and degrade mRNA.
DNA Transposons, piRNA clusters
Direct DNA methylation in the formation of heterochromatin.
Data obtained from: (Kim, Han & Siomi 2009)
The first time RNAi was observed was in 1989, when Richard Jorgensen was attempting to make petunias a deeper shade of purple. To achieve this Richard Jorgensen added extra copies of trans chalone synthase genes to the petunias (the gene that codes for the purple pigment) under the influence of a strong promoter. However, instead of the plants becoming deeper shades of purple, they were either variegated or white as expression of the pigment gene was abrogated. This phenomenon did not only cause repression of the transgene, it also affected the expression of natural, endogenous gene for cholane synthase in a phenomenon that is now referred to as co-suppression (Watson et al, 2008). Co-suppression was later found to be due to the ability of the transgene that had been added, to induce degradation of mRNA coding for cholane synthase (De Paoli et al. 2009).
In October 2006, Andrew Fire and Craig Mello received a Nobel Prize for a paper they published in 1998, which successfully characterized how mRNA expression could be suppressed by siRNA. In this paper which was published in the Nature Journal, Fire and Mello described how injecting double stranded RNA (dsRNA) for the unc-22 gene, into the nematode Caenorhabditis elegans (C. Elegans) resulted in the emergence of a twitching phenotype associated with the loss or silencing of the unc-22 gene. Thus the study successfully demonstrated how the addition of dsRNA corresponding to the unc-22 gene silenced the expression of this gene in the nematodes (Fire et al. 1998).
Overview of the siRNA therapy for disease:
Because of the efficacy siRNA and the ubiquitous nature of RNAi machinery in most eukaryotic organisms, siRNA presents a new approach to disease therapy. An obstacle which stood in the way of the effective application of siRNA for therapeutic use was how to administer exo-siRNAs without triggering cytotoxic events in vivo. For instance, administration of dsRNAs which are more than 30nt bases, results in interferon responses in the cells which cause inhibition of protein production and subsequent apoptotic cell death (Gantier, Williams 2007, Elbashir et al. 2001). However, further studies were able to demonstrate that it is possible to counter the aberrant interferon responses and achieve RNAi with increased potency and efficacy by administering exogenous short RNA duplexes which are only 27-nt long (Kim et al. 2005).
Another obstacle that has to be overcome before exo-siRNA can be used for effective therapy is how it can be delivered into the cytoplasm of the target cells in order to achieve the desired therapeutic effects in patients. Studies using animal models have demonstrated how delivery of exo-siRNA can be achieved in mucosal tissues such as the lungs in the absence of transfecting agents (Dykxhoorn, Palliser & Lieberman 2006). For instance, one study demonstrated the efficacy of intranassally administered siRNA directed at RSV and PIV in BALB/c mice infected with the viruses which are known to cause pulmonary infections such as pneumonia or bronchiolitis after infection with RSV and PIV viruses (Dykxhoorn, Palliser & Lieberman 2006, Bitko et al. 2004). Having determined the benefits of intranasal administration of siRNA in animal models, these findings can now applied to develop therapies for pulmonary conditions which could benefit from this route of drug administration such as asthma and cystic fibrosis (Akhtar, Benter 2007).
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The findings of the effects of siRNA therapy from animal studies have prompted the development of ALN-RSV01, siRNA designed to treat RSV infections. This virus infects approximately 70% of babies before they turn one and it may result in the onset of pnuemonia in immune-compromised individuals and the elderly. Treatments currently available for RSV are marginally effective and ribavirin the only antiviral approved for RSV therapy cannot be used to treat babies due to its teratogenic effects (DeVincenzo et al. 2008). Findings from the phase 2 clinical trials with ALN-RSV01 provided information regarding its safety and efficacy in human beings when administered intranasally, and now phase 3 trials are currently underway (DeVincenzo et al. 2008).
Researchers have found that local delivery of siRNA to the eyes can be achieved using intravetreal injection (Behlke 2008). Such studies have enabled research which could contribute to and improve existing therapies for diseases that affect the eyes, such as macular degeneration. Macular degeneration is one of the leading causes of blindness caused by leaking of weak new blood vessels produced under the influence of vascular endothelial growth factor (VEGF). Several studies have been carried out to determine the efficacy of siRNA directed against VEGF using primate and murine models with induced age-related macular degeneration (Akhtar, Benter 2007). One such study using mice demonstrated how siRNA for VEGF (hVEGF5) administered by subretinal injection inhibited the expression of VEGF in the mice (Reich et al. 2003).
There are already a few siRNA based therapies available for treatment of ocular diseases which have been approved by the FDA, such as formivirsen and macugen (Behlke 2008). However, several other siRNAs therapies for macular degeneration are still in clinical trials including Sirna-027 and bevasiranib (cand5). Bevasiranib has already been phase 1 and 2 clinical trials which were able to demonstrate its safety and efficacy, thus phase 3 clinical trials are currently underway for this drug (Behlke 2008).
Relevance of siRNA therapy to cancer chemotherapy:
Cancers are caused by aberrant gene expression due to several initiating factors including the accumulation genomic mutations, abnormal chromatin modifications and/or expression of viral oncoproteins. These anomalies eventually lead to increased expression of oncogenes and reduced expression of tumor suppressor genes which subsequently leads to tumor progression. Successfully delivering siRNA systematically could revolutionize drug therapy for diseases caused by aberrant gene expression due to the potent regulatory effects of siRNA on gene expression thus greatly benefitting patients with local and metastatic cancers. Unfortunately, systemic delivery of siRNA is not as straight forward as delivery to mucosal tissue or to localized regions (Behlke 2008).
Apart from targeting cancer susceptibility genes, siRNA could also be used to counter drug resistance in cancer patients. For instance, the onset of resistance to chemotherapeutic drugs such as tamoxifen in breast cancer and gemcitabine in pancreatic cancer therapy currently presents a problem. For instance, development of resistance to the pancreatic cancer chemotherapeutic drug gemcitabine has been associated with the low survival rates of pancreatic cancer patients. Only about 3% of all pancreatic cancer patients survive for up to five years while the majority has an average survival period of approximately 6 months (Réjiba et al. 2009). Resistance to gemcitabine in pancreatic cancer chemotherapy has been attributed to inactivating polymorphisms in the genes coding for enzymes, responsible for the activation of the pro-drug gemcitabine (Bergman, Pinedo & Peters 2002).
Application of siRNA in HPV to combat cervical cancer:
About 80% of all women are infected by the human papillomavirus (HPV) in their life time and a proportion of these women go on to develop cervical intraepithelial neoplasia (CIN) which may or may not progress to cervical cancer. HPV has a role in the etiology of cervical cancer due to its life cycle and it is detected in about 99% of all cervical cancer cases (Longworth, Laimins 2004). Despite this knowledge, current treatments that are available for HPV associated cervical cancer such as cisplatin do not target the carcinogenic HPV virus. The recently approved HPV vaccines such as gardasil are effective from a prophylactic point of view, but they do not offer any therapeutic benefit to women already infected with the HPV virus. Thus the novel therapeutic approaches to HPV associated cervical cancer using siRNA is an exiting new development.
When cells become infected with HPV, they start producing a range of viral proteins, but only high risk HPV (hr-HPV) is associated with production of E6 and E7 oncoproteins. The E6 and E7 oncoproteins down regulate the expression of the retinoblastoma (Rb) and p53 genes (Liu et al. 2008). Rb has a crucial role in regulating cell progression through the cell cycle, while the p53 gene, also known as the guardian of the genome has, is responsible for maintaining the integrity of the genome by repairing damaged DNA or inducing apoptosis when damage is irreparable (Kinzler, Vogelstein 1997). The two viral oncoproteins E6 and E7 have also been found to be able to up regulate telomerase activity, resulting in reduced rates of cell death and these factors put together lead to subsequent cancer progression (Longworth, Laimins 2004).
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Recent studies have been able to demonstrate how treating hr-HPV with siRNA directed at E6 and E7 proteins results in increased expression of functional Rb and p53 proteins and up regulated apoptotic cell death in cervical cancer tumors. For instance, tests carried out with NOD/SCID mice demonstrated how hr- HPV 16+ tumors gradually shrank after the mice had been treated with siRNA corresponding to E6 and E7 oncoproteins(Bharti et al. 2009).
The use of siRNA that targets the E6 and E7 proteins has several advantages including the fact that E6 and E7 are viral proteins that are not normally expressed in human cells. Thus siRNA directed at these proteins in not likely to cause off target effects as the viral protein targets E6 and E7 are not found in normal cells (Bharti et al. 2009). SiRNA can also be used in combination with conventional therapies for cervical cancer therapy. For instance, studies have demonstrated how co-treating cervical cancer cells with siRNA for E6 and E7 along with cisplatin increases the cytotoxic effects of cisplatin 4 fold (Putral et al. 2005).
The application of siRNA for therapy of human disease could potentially revolutionize medicine, particularly cancer chemotherapy. Realization of how endogenous gene expression can be manipulated by exo-siRNA has prompted researchers to investigate how these small RNAs can be designed, produced and delivered to target tissues to regulate gene expression in vivo to achieve therapeutic effects.
One of the biggest obstacles for siRNA therapy at the moment is how to achieve systemic delivery of the siRNAs to the cytoplasm of the target cells and without inducing an adverse reaction. For instance, delivery of siRNA to cells such as hematopoetic stem cells, dendritic cells and lymphocytes could prove to be very difficult as these cells are refractory to delivery of will siRNA particles attached to transfecting agents (Dykxhoorn, Palliser & Lieberman 2006). The methods that have been used to achieve systemic delivery of siRNA in animal models such as hydrodynamic delivery have been associated with transient failure of the right side of the heart. However, other methods are currently analysed to assess their ability to deliver siRNA systematically. These include immunoliposome, liposomes and receptor mediated methods to introduce siRNA into the cytoplasm (Dykxhoorn, Palliser & Lieberman 2006).
Another drawback of siRNA therapy is the off target effects. This is when RNAi by siRNA results in the down regulation of the target gene as well as genes with homologous mRNA sequences. Strategies are currently being developed to enable the development of more specific siRNA (Behlke 2008). However increasing specificity of siRNA could also impact on efficacy of the siRNA in some cases. For instance, HIV is an RNA virus that lacks genome stability due to lack of proof reading equipment, meaning the virus can and will mutate easily. Highly specific siRNA could fail to form complementary base pairs with newly formed HIV mutants. Thus the highly specific siRNA could in fact select for mutated viral particles, which could lead to the emergence of a more virulent strain of HIV (Bitko et al. 2004).