Therapeutic Potential of RNA Interference
A New Molecular Approach to Antiviral Treatment for Hepatitis C
J Viral Hepat. 2012;19(11):757-765. © 2012 Blackwell Publishing
Abstract and Introduction
Hepatitis C virus (HCV) infection remains a major cause of chronic liver disease with an estimated 170 million carriers worldwide. Current treatments have significant side effects and have met with only partial success. Therefore, alternative antiviral drugs that efficiently block virus production are needed. During recent decades, RNA interference (RNAi) technology has not only become a powerful tool for functional genomics but also represents a new therapeutic approach for treating human diseases including viral infections. RNAi is a sequence-specific and post-transcriptional gene silencing process mediated by double-stranded RNA (dsRNA). As the HCV genome is a single-stranded RNA that functions as both a messenger RNA (mRNA) and replication template, it is an attractive target for the study of RNAi-based viral therapies. In this review, we will give a brief overview about the history and current status of RNAi and focus on its potential application as a therapeutic option for treatment for HCV infection.
Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma. More than 170 million individuals are affected with this virus worldwide.
The current HCV antiviral therapy for interferon/ribavirin is successful in approximately half of the G1 cases. With the addition of the new FDA- and EMA-approved NS3 protease inhibitors, boceprevir and telaprevir, the rate of sustained virologic response in G1 has improved to 70%, still leaving an unmet clinical need. RNAi has been shown to be a naturally occurring process of sequence-specific gene silencing in plants and vertebrates.
This process is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules (dsRNA) in a cell's cytoplasm. The dsRNA can either be chemically synthesized as small inferring RNA (siRNA) then directly transfected into cells or can be produced inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by different mechanisms. To date, hundreds of miRNAs have been identified as human genome. These are 22–24 nucleotides in length and downregulate gene expression by attaching themselves to messenger RNAs (mRNAs) and preventing them from being translated into proteins.
Because of the functional similarities between miRNA and siRNA, which is involved in the inhibition of viruses and silencing of transposable elements in plants, insects, fungi and nematodes, exogenously introduction of siRNA into the target cells by various transfection methods may trigger the RNAi pathway against target gene. Many viruses, including HCV, produce a transitory double-stranded RNA during replication that can serve as RNA target for RNAi pathway. This makes HCV an attractive target for RNAi therapy. Whether it will ultimately be necessary is dependent on the outcome of current studies looking at the efficacy of interferon-free combination therapeutic regimes that include protease, polymerase and NS5A inhibitors.
A Brief History of RNAi Therapeutics
During the 1990s, a number of post-transcriptional gene silencing (PTGS) phenomena, introducing the concept or RNA silencing pathway, were discovered in plants, fungi, animals and ciliates.[5–7] Before that RNA was known for its traditional function as a conveyor of message from DNA to protein. But in addition to this role, other numerous novel roles for RNA in the regulation of gene expression have been discovered and will continue to be probed for therapeutic applications.[8–10] The RNA that interferes with the expression of a specific gene is known as RNA interference (RNAi). The discovery and characterization of the RNAi pathway has been one of the most important scientific developments of the last two decades.
RNAi pathway is a process of sequence-specific, post-transcriptional gene silencing triggered by the presence of double-stranded RNA (dsRNA). dsRNA is formed transiently during the replication of many viral genomes, but commonly is not present in noninfected cells. As a result, RNAi is believed to represent an accustom form of nucleic acid-based immunity against intracellular pathogens. The first observations of gene silencing by dsRNA derived from experiments with plants. In the late 1980s, RNAi was noted as a surprise observation by plant scientists during the process of plant transformation experiments, in which the insertion of a transgene into the genome led to the silencing of both the transgene and homologous endogens. RNAi molecular mechanism remained unclear until the late 1990s, when Nobel Prize recipients Fire and Mello work in the nematode Caenorhabditis elegans showed that RNAi is an evolutionary conserved gene silencing mechanism. They observed that injection of long dsRNA (hundreds of bps) induces degradation of mRNAs homologues to the dsRNA. In some cases, this silencing process lasted for several generations. Following this observation, Elbashir et al. provided a major breakthrough. They showed that chemically synthesized short interfering RNA (siRNA), which were designed to mimic the native siRNAs produced by RNAi in other systems could silence target mRNAs in transfected cells. As the demonstration of gene silencing process functioned by RNAi in the nematode, the use of RNAi has rapidly emerged as the technique of choice for functional genomics studies.[9,14,15]
The Mechanism of RNA Interference
Post-transcriptional gene silencing pathway mediated by RNAi is related to a natural defence against viruses and the mobilization of transposable genetic elements in plants, insects, fungi and nematodes.
The RNAi pathway functions as follows: (Fig. 1)