A New Molecular Approach to Antiviral Treatment for Hepatitis C

Journal of Viral Hepatitis

Therapeutic Potential of RNA Interference

A New Molecular Approach to Antiviral Treatment for Hepatitis C

M. Motavaf, S. Safari, S. M. Alavian
Nov 09, 2012
Authors & Disclosures
J Viral Hepat. 2012;19(11):757-765. © 2012 Blackwell Publishing

Abstract and Introduction
Abstract

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.

Introduction
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.[1]

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.[2]

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.[3] 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.[4]

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.[11]

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.[12] 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.[6] 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.[2] Following this observation, Elbashir et al.[13] 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,[2] 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)

Figure 1.
Mechanism of RNA interference.

Click To Enlarge




Firstly, a trigger dsRNA is introduced into the cell's cytoplasm. Following that, an RNaseIII-like enzyme called dicer with endoribonuclease activity identifies dsRNA and cleaves this dsRNA into 21-bp to 23-bp fragments, with 5' phosphate groups and two nucleotides 3' over-hanged, called small interfering (siRNA). Dicer usually contains a helicase/ATPase domain, a PAZ domain (Piwi-Argonaute-Zwille), two RNaseIII-like domains and a dsRNA-binding domain. The 3' overhangs are a common feature produced by RNaseIII activity. The distance between the PAZ and the RNaseIII domains determines the size of produced siRNAs. Dicer then delivers the siRNA to an Argonaut-containing RNA-induced silencing complex (RISC). RISC undergoes an ATP-dependent step that activates the unwinding of the double-stranded siRNAs, retaining one strand to act as a co-factor. This single-strand RNA, complexed with RISC, identifies its complementary mRNAs in cell's cytoplasm. RISC complex contains an endonuclease activity, which is attributed to the Argonaut subunit, causes a single-site cleavage of the target mRNA. The resulting fragments of target mRNA is thus destabilized fully degraded through natural endogenous mechanism. Activated RISC is capable of degrading of thousands of target mRNA molecules.
       
Several studies of antiviral RNAi therapies have been corroborated in animal models, for instance, influenza,,[16–18] respiratory syncytial virus (RSV),[19–20] parainfluenza virus,[19] coxsackie virus B,[21–22] SARS-associated corona virus[23,24] and herpes simplex virus (HSV-2), using a topical application.[25,26] Based on these data, RNAi has emerged as a potential tool for treatment for viral infections in vivo. The fundamental principle for RNAi-based therapy is to trigger PTGS using mimics of siRNA as Dicer substrates. This siRNA can be synthesized chemically or expressed virally. Either of these two types of siRNAs can be delivered to Dicer in the cytosol by a variety of delivery approaches.[11,27]

Different strategies such as hydrodynamic injection of naked siRNA or siRNA conjugates,[28] electroporation[29–31] and the use of cationic carriers[32–36] have been applied for in vivo delivery of synthetic siRNA in laboratory animals. Production of intracellular siRNAs has been achieved through the delivery of plasmids or viral vectors containing expression units for short-hairpin RNAs (shRNAs) in which siRNA is produced from one of the stems of the hairpin.[37] Adenoviral and lentiviral systems have been used successfully to deliver shRNA to primary cell lines including lymphocytes, hepatocytes and neuron.[38,39]

Each type of viral vector has specific characteristics that need to be determined for the specific target. The adenovirus and adeno-associated virus (AAV)–derived vectors provide an efficient delivery vehicle for transient shRNA expression.[40] Ad-gutless vector, which supports transgene expression for many months, is used for liver-directed systemic delivery,[41] while a conditionally replicating adenovirus (CRAd) is designed to replicate and kill tumour cells. On the other hand, by long-term expression, retroviruses provide major advantage of incorporating the transgenic siRNA genes into the host cell genome for longer-term therapy.[42] Both types of siRNA techniques offer advantages and suffer from limitations. For example, the use of siRNAs theoretically allows one to deliver a desired quantity of these effector molecules to cells. Other advantage of siRNA over shRNA is that siRNAs do not need to be taken up by the nucleus to exert an effect. As the persistence of unmodified siRNAs is limited, frequent dosing will likely be needed to maintain efficacy in a therapeutic setting. Even though certain RNA modifications have been shown to increase the stability of siRNA, some of these modifications result in loss of siRNA function.[37] Therefore, the ideal modifications for therapeutic siRNAs need to balance stability with efficacy.
       
The advantage of shRNA expression plasmids or viral vectors over siRNA is that shRNAs do not suffer from similar stability problems and they can be intracellularly expressed in large quantities over extended periods of time. In addition, a single expression vector can be designed to encode multiple shRNAs, thereby increasing the potency of the product. Although, multiple chemically synthesized siRNAs can theoretically be used together, but testing of individual siRNAs in clinical trials and their interactions before using them simultaneously is necessary.[37]

RNAi Against Hepatitis C Virus
Chronic hepatitis C virus (HCV) infection affects a large number of patients worldwide. Current combination therapy with pegylated interferon (IFN-α) and ribavirin has improved the clinical outcome, but less than half of the patients with chronic hepatitis C can be expected to respond to these available agents. These presently available therapies are expensive, prolonged and associated significant adverse effect. As such, there is a clear need for the development of additional agents that act through alternate mechanisms.[43,44] RNAi appears to be an effective nucleic acid-based gene silencing tool to target highly conserved or functionally important regions within the HCV genome.[45] Several studies have shown that HCV replication is exquisitely sensitive to either chemically synthesized siRNAs or shRNA expression targeting HCV RNA.[45–47] HCV is a member of Flaviviridae viruses, and the liver is the major site of its replication. As the HCV genome is a single-stranded RNA that functions also as a messenger RNA, it is an attractive target for developing RNAi-based therapies. Its genome is a positive, single-strand RNA molecule that includes a large open reading frame encoding for a 3010 ± 20 amino acid polyprotein and two untranslated regions at the 5' and 3' ends. The enzymes encoded by host and virus process this polyprotein into structural and nonstructural proteins post-translationally. The structural proteins, encoded in the N-terminal region, include the core protein followed by two envelope glycosylated proteins: E1 and E2. The nonstructural domain encodes for six proteins: NS2, 3, 4A, 4B, 5A and 5B.[1,48]

It was reported that siRNAs directly targeting HCV replicon RNAs, specifically at the 5' untranslated region (UTR), core, NS3, NS4B or NS5A, were effective at reducing viral replication.[5,46,47,49–51] Also, it has been shown that synthetic dsRNA can inhibit HCV RNA replication in cell culture. In one study designed by Kapadia et al.,[15] after testing seven different HCV-specific siRNAs by real-time RT-PCR of HCV RNA, they selected two siRNAs, NS3–1948 and NS5B-6133 (named on the basis of their location and nucleotide start site in the HCV subgenome), as they had the highest specific effect on HCV RNA replication. They observed siRNAs NS3–1948 and NS5B-6133 significantly inhibited HCV transcript levels.[15]

In other study, Randall et al. examined the effect of siRNA against NS5B by immunofluorescence microscopy and formation of G418-resistant colonies in cells expressing neomycin phosphotransferase from replicon HCV RNAs. Both approaches showed that HCV-specific siRNA could mediate the clearance of viral RNA and protein in more than 98% of cells.[46] In Wilson et al. study, electroporation of siRNA against NS5A and NS4B demonstrated significant reduction in HCV RNA levels 72 h post-transfection. By using RT_PCR, the dramatic reduction in HCV replicon RNA levels was demonstrated, with a 99% and 94% decrease in replicon RNA levels after treatment with NS4B_siRNA and NS5A_siRNA. In this study, electroporation of negative control siRNAs containing six mismatched nucleotides did not have any effect on the levels of HCV replicon RNA or HCV nonstructural proteins. These results indicate that the effects of the siRNAs on HCV RNA levels and replicon protein were sequence-specific and did not mediate by induction of nonspecific host-defence pathways. Thus, the effects of siRNAs on HCV replicon protein and RNA levels seem to be the result of siRNA-directed degradation of the HCV replicon RNA mediated by RISC.[49]
In the study by Yokota et al., they engineered siRNAs and siRNA-expressing vectors to target HCV RNA and evaluated the effects on viral replication using an HCV replicon system. They showed that siRNA targeted the HCV 5' UTR efficiently and cleaved the target specifically. In addition, they indicated that the cleavage of HCV RNA not only suppressed viral protein synthesis, but also blocked the replication of viral RNA. They presented the effectiveness of vector-derived dsRNAs and synthetic siRNAs. More than 80% suppression was obtained using a siRNA concentration of only 2.5 nm.[50] The effect of all 5'-UTR-directed siRNAs was not equal. Among the siRNAs tested, siRNA 331, which is directed against a region upstream of the start codon, was the most efficient, whereas siRNA 82, which is directed against helix II, had almost no effect on viral genome expression. These results may be due to the highly folded structure of the 5' UTR, which may leave few single-stranded gaps that siRNAs can reach to. Seo et al.[51] used a version of the HCV RNA subgenomic replicon that has a Neomycin resistance gene for selection and a luciferase gene for monitoring the levels of replicon expression. In cells transfected with siRNAs specific for either the 5'–UTR or the luciferase, reduction (85–90%) in the levels of luciferase was observed. Whereas nonspecific control siRNAs or siRNAs with three nucleotide mismatch to the luciferase target did not show any reduction.

HCV Targets for RNAi
Hepatitis C virus RNA was predicted to be highly susceptible to RNAi because replication occurs in the cytoplasm and the viral genome resembles an mRNA. The replicon system has been used to evaluate siRNA-targeting the 5'UTR,[46, 50–52] core[46] E[53] NS3,[15, 54, 55] NS4B,[46] NS5A,[56] NS5B.[46, 49, 57] These results indicate that most regions of HCV RNA are accessible to RNAi machinery that leads to decrease in HCV protein expression and decline in both genomic and antigenomic HCV RNA (Table 1).

5' and 3' Noncoding Regions
The 5'UTR and the extreme end of the 3'UTR are the most conserved regions of HCV RNA in terms of primary sequence and secondary structures. The relatively conserved nature of these regions signifies their functional importance in the viral life cycle. The 5'UTR is a 341-nucleotide (nt) sequence that is highly conserved even between the most distantly related HCV subtypes.[58] For example, the sequences between 30 and 170 nt appear to be very conserved between different quasispecies of HCV 1b genotype.[59] A combination of computational, phylogenetic and mutational analyses of the HCV 5'UTR has identified four major structural domains (domains I–IV), most of which are also conserved among HCV genotypes.[60–62]

As siRNA is highly sequence-specific and any sequence mismatches between the siRNA and the target affect the efficiency of RNAi, the 5'UTR would seem to be an ideal target for siRNA.[50] For example, in the study by Seo et al.,[51] siRNA duplexes that were directed against different regions of the 5'UTR of the HCV genome and significant decline in HCV replication protein expression were observed.
       
The 3'UTR of HCV varies between 200 and 235 nt long, which typically consists of three different regions, in the 5' to 3' direction, a variable region, a poly(U) and/or poly(UC) stretch and a highly conserved 98-nt X region.[63–65] The variable region is located immediately after the termination codon of the HCV polyprotein and is variable in length (ranging from 27 to 70 nt) and structure among different genotypes. However, it is highly conserved among viral strains of the same genotype.[63,66] Examination of the 3' terminal sequences of the HCV genome has indicated that most HCV RNAs contain identical 3' ends with no extra sequence downstream of the X tail.[64] Even though there is no poly (A) sequence in the 3' UTR, the 3' UTR sequence, particularly the X region, is involved in the regulation of translation, in the same way as the poly (A) sequence in the mRNAs of other RNA viruses. Thus, it is involved in the replication, stabilization and also packaging of viral RNA.[67] The essential role of this sequence offers a high effective target site for RNAi. It is noteworthy to mention that even though these two regions are much conserved, some of the siRNA targets appear to be more efficient than others. This difference could be due to the complex secondary structure of the UTRs and the attachment of cellular proteins to these regions that make it difficult for siRNAs to efficiently hybridize to some sequences in the transfected cells.[68]

Ns3 Serine Protease and Ns3 Helicase
NS3 is a bifunctional protein. The amino-terminal domain is a serine protease responsible for releasing the individual proteins from the NS3- NS5B polyprotein. The carboxyl-terminal domain is an RNA helicase. The NS3 serine protease is a cytoplasmic protease and is involved in HCV polyprotein maturation in which it cleaves an initially synthesized polyprotein into functional proteins.[69,70] This proteolytic characteristic belongs to the N-terminal region of NS3. This protease domain constitutes a noncovalent complex with NS4A, which is a 54-amino acid activator of NS3 protease.[69,71] NS3 offers several target sites to small antiviral molecules, for instance, the catalytic site or the substrate-binding site.[54, 72, 73] Helicase activity of NS3 belongs to its C-terminal region.[74] The NS3 helicase primary function is to unwind the viral genomic RNA during replication. Studies probing the mechanism of polynucleotide unwinding have led to observations that may suggest high effective target site for RNAi.[75,76]

NS5B RNA-dependent RNA Polymerase
The NS5B protein is an RNA-dependent RNA polymerase (RdRP) and is the essential component in HCV replication. It has been shown that NS5B associates with NS3 and NS4A to produce a negative-strand copy of the RNA genome, which in turn can produce several positive-strand RNA copies. Viral heterogeneity results from high error rate of NS5B gene-coded RNA-dependent RNA polymerase, which gives significant advantage to HCV survival.[11]

Hepatitis C virus NS5B RNA-dependent RNA polymerase is currently pursued as the most popular target to develop safe anti-HCV agents, as it is not expressed in uninfected cells.[77] McCaffrey et al. was the first to demonstrate feasibility of siRNA-targeting HCV NS5B in vivo. By co-expression of an NS5B-luciferase fusion gene with an anti-NS5B siRNA expression plasmid, they found a significant reduction in luciferase expression in the mouse liver indicating selective degradation by the NS5B siRNA.[57] Additionally, several other groups have observed suppression of HCV replicon by siRNA-mediated targeting either NS5B..[54,78] The result of Trejo-Ávila et al.[79] study indicated that the NS5B-siRNAs used in their study can specifically inhibit HCV RNA replication and protein expression (more than 90%) compared with control cells.

NS5A
NS5A is a pleiotropic protein with key roles in both viral RNA replication and modulation of the physiology of the host cell. This protein exerts a wide range of effects on cellular pathways and processes, including innate immunity and host cell growth and proliferation. NS5A plays an important role in the establishment of high-level HCV RNA replication.[80–82] Study of Sen A et al.[56] demonstrated that siRNAs targeted against NS5A of HCV genotype 1a specifically inhibit NS5A RNA and protein expression in a human hepatoma (HepG2) cell line. 

Advantages and Limitations of RNAi Therapeutics
As HCV is a major public health issue and the current HCV therapy for IFN and ribavirin is successful in only half of treated patients, there are many alternative therapeutics under development. Most of these are small molecule approaches inhibiting the function of viral replication. RNAi-based antiviral strategies have a number of advantages and disadvantages as compared with these therapies. One of the primary advantages is that because several guides and algorithms exist to aid in the choice of siRNA, the siRNAs are easy to design and synthesize, unlike small molecule inhibitors. Furthermore, the availability of genome sequence libraries permits the design of highly specific siRNAs, which will reduce undesired 'off-target' effects on nonhomologues genes sequence. In addition to its easy design, compared with other antisense strategies such as antisense DNA oligonucletides and ribozymes, RNAi effector functions at much lower concentrations, which is an important factor in a therapeutic process.[83] Because nonspecific effects are dependent on siRNA concentration, optimization of target sequence will increase siRNA efficacy and decrease nonspecific side effects. Therefore, the design of a highly effective siRNA target site is necessary for specificity of gene silencing.[47,84]

Furthermore, as Dicer and the RISC function in the cytoplasm, the cytoplasmic location of RNAi machinery makes it technically easier than other methods in which the silencer needs to be taken up by the nucleus to exert an effect.[85] Other advantage of RNAi-based therapies is that they mimic a natural process and effect existing protein complexes; thereby, they do not cause any toxicity that can result from introducing a foreign compound into the body.[86] Moreover, siRNA is able to affect multiple steps in the viral life cycle, which is a significant progress in available treatments. It makes siRNA able to reduce the levels of viral transcripts and proteins even in the absence of active viral replication that resides in the liver cell of chronically infected patients. The gene silencing effect of siRNA typically lasts for approximately 3–7 days before they naturally disappear.[87] This short transitional effect lowers the chance of side effects as with other therapeutic methods such as IFNa and ribavirin. Despite this fact, one major concern regarding the use of RNAi-based therapies against virus infections, particularly RNA viruses, is the development of escape mutations. Many RNA viruses encode polymerase enzymes that lack proofreading abilities and as a result have high rates errors during viral replication. Thus, there is a high probability that viruses with resistance to RNAi evolve during the replication.[88] There are some approaches that may reduce the chance of resistance mutant development. One approach to delay viral mutation might be based on the choice of the target site. Some certain regions of the viral genome, such as The IRES region in the HCV 5' UTR, are highly conserved. These maintained sequences are essential for viral replication; as a result, point mutations in these structures might lead to loss of function. Considering this fact, targeting these regions by RNAi might prevent viral escape. Other alternative approach is using a pool of siRNAs to simultaneously target multiple sites in the viral genome.[46,89] Clinical trials with RNAi have now begun for several disorders, but challenges such as off-target effects, toxicity and safe and efficient delivery methods have to be overcome before the widespread use of RNAi as a gene-based therapy.[90] Unintended activation of the innate immune response is another complication of RNAi-based therapies. Some siRNAs have been shown to trigger innate immunity leading to cytokine and interferon production. For example, dsRNAs smaller than 30 nts activate protein kinase PKR and 2',5'-oligoadenylate synthetase, but shorter siRNA duplexes minimize this activation. Despite the disadvantages mentioned, several RNAi-based therapies have successfully made it to clinical trials. Like many other potential therapeutic, issues of unintended consequences, such as toxicity, should be evaluated in standard pharmacokinetic and phase I safety studies.

Conclusion
Currently, it is estimated there are about 170 million people infected with HCV worldwide, but despite current advances in treatment options, more effective and safer antiviral agents for hepatitis C are clearly still needed. RNAi is thought to be a natural defence mechanism that evolved to protect organisms from RNA viruses, such as hepatitis C virus. Through this fact, RNAi-based therapies represent an exciting new technology that could have therapeutic applications for viral infections. Many pioneer studies have demonstrated the effectiveness of using siRNAs for treating HCV infection.
       
Even though the results obtained demonstrate the potential of RNAi therapies against viral hepatitis, RNAi is still a developing field of biology, and its practical applications are still to be established. Whether it will ultimately be necessary in HCV infection 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.

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