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The potential of RNA interference as a tool in the management of viral hepatitis
Journal of Hepatology 42 (2005) 139–144 www.elsevier.com/locate/jhep
Review
The potential of RNA interference as a tool in the management of viral hepatitis John A. Taylor1,2, Nikolai V. Naoumov2,* 1
2
School of Biological Sciences, University of Auckland, Auckland, New Zealand Institute of Hepatology, University College London, 69–75 Chenies Mews, London WC1E 6HX, UK
1. Introduction RNA interference or RNAi is an evolutionary-conserved mechanism present in virtually all eukaryotic cell. The process is triggered by the presence of double-stranded RNA (dsRNA), which is formed transiently during the replication of many viral genomes, but generally is absent from non-infected cells. Therefore, RNAi is believed to represent an ancestral form of nucleic acid based ‘immunity’ against intracellular pathogens resulting in a sequence-specific silencing of gene expression [1,2]. Gene silencing by RNAi involves the generation of short (21–23 nucleotide) duplex RNA species from a longer precursor by an endonuclease called ‘dicer’ [3]. The resultant short dsRNAs, referred to as small interfering RNA (siRNA), are incorporated into a ribonucleoprotein complex termed RISC for RNA-inducing silencing complex, essentially, a programmable endonuclease that acquires target specificity by unwinding the strands of siRNA and retaining one as a guide to complimentary mRNA species in the cytoplasm [4]. RISC then cleaves the target mRNA in the middle of the homologous sequence and can target further molecules resulting in a processive depletion of a specific mRNA pool. In 1998, Fire and colleagues demonstrated the potent sequence-specific silencing of gene expression mediated by homologous dsRNA in the nematode Caenorhabditis elegans [5]. The observation of a similar response in * Corresponding author. Tel.: C44 20 7679 6512; fax: C44 20 7380 0405. E-mail address: [email protected] (N.V. Naoumov). Abbreviations RNAi, RNA interference; siRNA, short-interfering ribonucleic acid; dsRNA, double-stranded ribonucleic acid; RISC, RNAinduced silencing complex; shRNA, short hairpin ribonucleic acid; ORF, open reading frame; RT-PCR, reverse transcription-polymerase chain reaction.
mammalian cells was masked by the induction of the interferon response, a non-sequence specific response to dsRNA induced by many animal viruses leading to global changes in cellular transcription and translation. By restricting their length to less than 21–23 nt, the length of siRNA molecules generated by dicer, Tuschl and coworkers showed that synthetic RNA duplexes introduced into mammalian cells could induce sequence specific cleavage of mRNA by without activation of the interferon response [6]. The direct introduction of siRNA in mammalian cells bypasses the need for cleavage of a longer template by dicer while effectively priming RISC for the selective down-regulation of gene expression. Not surprisingly, this technology was quickly recognised as a practical approach to genetic analysis in mammalian cells and as a potential tool in the treatment of human diseases including those caused by virus infection.
2. Design and delivery of functional siRNA The key to successful gene silencing by RNAi lies both in the design of effective siRNA and its efficient introduction into target cells. Though design remains largely an empirical process, principles have been established that facilitate the production of siRNA that can potentially silence gene expression with high efficiency [7]. One determinant likely to influence effective silencing is the degree of RNA secondary structure in the vicinity of the target site. siRNAs that target highly structured regions of mRNA appear to function less efficiently than those whose targets are in less structured regions [8]. Failure of siRNA to effectively silence target genes has also been associated with the preferential incorporation of the sense RNA strand of the duplex into RISC, resulting in an
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.10.022
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inability to guide the complex to the target mRNA. Two recent papers, report that the bias for incorporation of the antisense strand into RISC is determined by the relative stabilities of the base pair at the 5 0 ends of the duplex [9,10]. These studies will aid further efforts to improve predictive models for the design of functional siRNA, leading to more effective gene silencing. The first demonstration of siRNA-mediated gene silencing involved transient transfection of a chemically synthesised RNA duplex [6]. Chemical synthesis has now been employed widely to silence more than 200 genes. Apart from cost, a drawback of this approach may be the relatively short half-life of siRNA duplex inside a cell, resulting in a transient effect over a few days only. To circumvent this potential problem several groups have developed an alternative approach involving the use of DNA vectors that enable the separate transcription of both strands of siRNA or, of short hairpin RNAs (shRNA) after transient or stable transfection of mammalian cells [11–14]. shRNA resembles siRNA in the length of the RNA duplex, but is formed from a single RNA molecule containing an inverted repeat sequence separated by a short hairpin loop region (Fig. 1). Plasmid vectors that express si/shRNA usually contain promoter sequences specific for RNA pol II, which generates precisely defined transcripts. The DNA template for pol II transcription is usually generated by cloning a DNA sequence, formed by annealing complimentary oligonucleotides, flanked by restriction enzyme sites into a suitable plasmid vector. Transcription of the shRNA ‘gene’ is terminated by a run of at least four T-residues at the 3 0 end, resulting in a UU sequence in the RNA. The
shRNA is processed in vivo by dicer to yield a functional siRNA. A more recent development in the vector delivery of siRNA is the availability of viral vectors capable of stably transducing a wide variety of target cells [12,15,16]. Adenoviral and lentiviral systems in particular have been used successfully to deliver shRNA to primary cell lines including lymphocytes, hepatocytes and neurons. This approach circumvents the difficulty associated with transfection of these cell types and can lead to the stable expression of shRNA after insertion of the viral sequence into the genome. For example, a lentiviral vector was used to inhibit HIV replication in cultured macrophages. When the transduced cells were infected with HIV, the yield of virus was reduced by 99% [17].
3. Inhibition of viral replication by RNAi Many studies have demonstrated the potential of RNAi to effectively silence the expression of viral genes and thus inhibit virus replication in cell culture. In addition to hepatotropic viruses, (discussed below), this approach has been applied to human immunodeficiency virus, [17–20], rotavirus [21], dengue virus [22] and influenza virus [23]. These studies have targeted a variety of virus genes and employed both chemically-synthesized, and vector-encoded forms of siRNA, introduced into different cell types including primary cell cultures. Based on these data, RNAi has emerged as a potential tool for treatment of virus infections in vivo. Instead of targeting viral genes, an
Fig. 1. Mechanism of RNAi. (1) The endonuclease dicer processes long dsRNA molecules or shRNAs to 21–23 nucleotide siRNA in the cytoplasm. Alternatively, chemically synthesised siRNA can be directly introduced by transfection. (2) The siRNA interacts with the RISC complex and the duplex is unwound by an ATP-dependant helicase activity resulting in retention of the antisense strand. (3) RISC is targeted to mRNAs with a complementary sequence. (4) The target mRNA is cleaved and RISC is guided to further mRNA molecules.
J.A. Taylor, N.V. Naoumov / Journal of Hepatology 42 (2005) 139–144
alternative strategy for antiviral treatment would be to direct siRNA against essential cellular genes, whose products are required for virus replication. For example, silencing of Tsg101, a protein required for budding of virions from infected cells, greatly reduced the release of HIV from transfected 293T cells [24]. Similarly, delivery of siRNA against the HIV co-receptor CCR5, using a lentivirus vector, inhibited the ability of virus to enter primary macrophages reducing the yield of virus by 90% [18]. This approach offers an alternative strategy to the targeting of viral genes by siRNA, the success of which in vivo may be limited by the ability of the virus to mutate rapidly, and thus escape the inhibitory effect of RNAi, as reported recently for HIV [25].
4. RNAi and hepatitis viruses 4.1. Hepatitis B virus (HBV) The extensive use of overlapping reading frames within the HBV genome suggests that this virus may be particularly susceptible to RNAi, as a single siRNA could potentially target multiple viral mRNAs. Plasmids were constructed to express shRNA that targeted sequences in the viral X and core ORFs [26]. Co-transfection of these vectors into Huh7 cells with a plasmid encoding a complete HBV genome reduced the levels of core protein with either shRNA. A greater level of suppression (89 vs. 63%) was observed using siRNA that targeted both X and core mRNAs than with siRNA targeting core alone, emphasizing the potential synergistic effect of targeting multiple viral transcripts simultaneously. These authors further demonstrated the siRNA-mediated reduction of viral transcripts, including the 3.5 kb pregenomic RNA, and reported a reduction in HBV replicative intermediates of up to 95%. Successful inhibition of HBV replication in stably transfected HepAD38 cells, using siRNA targeting a site in the core ORF, has also been reported [27]. Although the level of virus secreted into the medium was reduced by 98% 48 h after transfection, this level of suppression had declined to 40% after a further 24 h underscoring the transient nature of the RNAi effect in transfected cells. These results suggest the likely need for sustained production of siRNA in vivo. In a further study, using HepG2.2.15 cells, the successful delivery of siRNA targeting the HBV polyadenylation signal not only sustained the inhibition of HBsAg secretion, but produced an additive effect in suppressing HBV replication [28]. Several studies have demonstrated that siRNA can efficiently inhibit HBV replication in animals. Although mice are not susceptible to HBV infection, the viral genome can be stably introduced to produce HBV transgenic animals that synthesise viral mRNAs in hepatocytes and secrete mature virions. More recently, hydrodynamic injection of cloned HBV sequences into the tail vein has been used to establish a transient model of acute HBV
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infection in mice [29]. This system has presented the opportunity to investigate the potential for siRNA to suppress aspects of virus replication in the context of a functional innate and adaptive immune system present in whole animals. Co-injection of a cloned HBV sequence with a plasmid expressing shRNA against distinct viral mRNAs, resulted in an effective RNAi response against HBV replication in mice [30]. Targeting a variety of sequences in each of the four viral mRNAs, these investigators demonstrated a reduction of up to 84.5% in the level of secreted HBsAg in mouse serum and a greater than 99% reduction in the number of hepatocytes positive for HBcAg, as shown by immunohistochemical staining of liver sections. A similar reduction in serum HBsAg and HBeAg expression has been reported following injection of siRNA that targets an overlapping sequence in the S and preC/C mRNAs [31]. HBsAg levels reached only 30% of control levels, and remained reduced up to 11 days after injection, while HBeAg levels were 20% of controls, which was also sustained. 4.2. Hepatitis C virus (HCV) Several studies have explored the possibility of using RNAi as a potential therapeutic intervention against HCV infection. As a (C) sense single-stranded RNA virus that replicates via a negative sense intermediate this approach offers the possibility, at least in theory, that RNAi could eliminate viral genomic, replicative form and mRNA species from infected cells and thus resolve HCV infection. However, in the absence of a suitable system for replication of HCV in culture, these studies have been restricted to demonstrating the potential of siRNA to suppress the replication of viral replicon RNA. The effect of siRNA against various sites in the NS3 and NS5B ORFs has been shown in several studies. A 21–23 fold reduction of HCV transcript levels in cells harbouring an HCV replicon was demonstrated by quantitative RT-PCR, [32]. To address the fate of HCV RNA at a single cell level, Randall et al., [33] 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 indicated that HCV-specific siRNA could mediate the clearance, to levels below the sensitivity of detection, of viral RNA and protein in O98% of cells. Similar results were obtained following the generation of stable cell lines that producing siRNA against NS3 [34]. The introduction of a HCV replicon to these cells resulted in 70% fewer G418 resistant colonies compared with controls indicating the potential for long-term suppression of HCV by RNAi. While these results are encouraging, the high mutation rate and sequence variability between HCV genotypes and quasispecies presents a considerable challenge to the successful use of siRNA against HCV infection in vivo
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where multiple siRNAs targeting different regions of the viral genome may have to be introduced simultaneously to avoid the evolution of RNAi escape mutants. Hydrodynamic transfection of siRNA in mice has also shown the potential to limit replication of HCV using viral sequences fused to a reporter gene although only a portion of the viral genome was present and no replication occurred [35]. 4.3. Hepatitis delta virus (HDV) HDV is a subviral satellite RNA whose replication is dependant on the provision of envelope protein by its natural helper virus HBV. HDV superinfection of chronic HBV carriers results in HDV persistence that is usually associated with significant hepatic inflammation and rapidly progressive liver damage. Three distinct RNA species—the 1679 nucleotide ssRNA genome, its exact complement, and an 800 nucleotide mRNA encoding the delta antigen are each potentially susceptible to cleavage by RNAi. However, a recent study shows that only the HDV mRNA is cleaved by siRNA introduced into Huh7 cells [36]. Despite the partial double-stranded nature of the genomic and antigenomic HDV RNAs that can fold into a rod-like structure with 74% base pairing, these molecules are intrinsically resistant to the endonuclease dicer both in vitro and in vivo [37]. Thus future attempts to use siRNA therapeutically against HDV should focus on silencing of the mRNA.
5. Dissecting the host response to virus infection with RNAi The ability of RNAi to function in cells infected with HBV or HCV provides a valuable tool to investigate the role of host genes involved both in virus replication, as well as in cytokine-mediated suppression of virus replication. This approach is particularly suited to analysis of host genes that are involved in the replication of HBV by simultaneous delivery of siRNA and a cloned viral genome to cultured hepatoma cells. Obvious targets for this type of analysis would include genes that encode polypeptides that have been observed to interact directly with viral components, genes that encode components of signalling pathways activated by antiviral cytokines (ex. interferon-alpha and interferon-gamma), and genes induced following virus infection. This approach offers the potential to identify new targets for antiviral therapy by dissecting the requirements for virus replication.
6. Silencing host gene expression to modify the pathogenesis of viral hepatitis Silencing of endogenous gene expression in the liver during viral hepatitis may also represent an effective
strategy for limiting viral replication or immune-mediated damage to infected hepatocytes. The value of this approach was demonstrated in experiments that examined the ability of RNAi to protect hepatocytes from Fas-mediated apoptosis during autoimmune hepatitis in mice [38]. In these studies, mice were transfected with siRNA that targeted Fas, a key mediator of T-cell mediated apoptosis in hepatocytes during autoimmune and viral hepatitis. Introduction of Fas siRNA resulted in uptake into over 80% of hepatocytes and caused significant reduction of Fas at both RNA and protein levels. Remarkably, in vivo treatment with Fas siRNA protected mouse hepatocytes from Fas-mediated apoptosis. Most significantly of all, this approach markedly attenuated hepatocyte necrosis in a model of autoimmune hepatitis and protected 82% of animals from death by fulminant hepatitis following the injection of Fas-specific antibody. Given that damage to hepatocytes during viral hepatitis is immune-mediated, this study points to the potential value of targeting both viral and cellular targets to reduce both viral replication and hepatocellular necrosis during periods of acute liver inflammation in chronic viral hepatitis.
7. Clinical application of RNAi in the treatment of viral hepatitis The studies outlined above demonstrate the feasibility of using RNAi to effect a potent and sustained inhibition of virus replication and offer proof of concept for use of RNAi as an antiviral therapy (Table 1). One obvious attraction of this technology in the treatment of human viral infections is the higher degree of specificity of siRNA in comparison to conventional antiviral chemotherapy. The availability of genome sequence libraries permits the elimination of siRNA with the potential for undesired ‘off target’ effects on endogenous genes. As with antiviral drugs, the evolution of resistance to virus-specific siRNA by rapidly mutating viruses like HCV could potentially limit the therapeutic effectiveness of RNAi. Mutations in HIV have already been reported that confer resistance to siRNA [25,39]. Simultaneous application of multiple siRNA molecules targeting conserved viral sequences would be necessary for sustained suppression of virus replication in vivo. Despite the optimism generated by studies discussed above, the major barrier to the successful clinical use of Table 1 Advantages and limitations of RNAi in a potential clinical scenario Advantages
Limitations
Broad applicability Potencya Specificity Low toxicity
Low cellular uptake in vivo Stability in vivo Potential for off-target gene silencing Potential induction of interferon Evolution of viral resistance
a
Relative to anti-sense RNA oligonucleotides.
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siRNA remains how to introduce sufficient levels of siRNA to the liver of patients suffering viral hepatitis resulting in the sustained and specific gene silencing necessary to modify the progression of disease. Hydrodynamic transfection, while successful in mice, is unlikely to be adopted as a clinical procedure given the requirement for generation of a high intravascular pressure. Even if this were possible, the short half-life of chemically synthesised siRNA in vivo limits the silencing effect to a few days at most which may be insufficient to manifest a clinical benefit. In view of these difficulties, vectored delivery of siRNA using recombinant viruses represents a more promising means of effective longterm gene silencing in the liver although this approach will bring all the attendant limitations associated with gene therapy such as potentially oncogenic effects of random integration in the host genome. While systemic application of siRNA for the treatment of viral hepatitis may not be imminent, one attractive possibility would be to transfect hepatocytes of donor livers with siRNA prior to orthotopic liver transplantation, in order to prevent viral recurrence (e.g. HCV) in the graft, or to minimise the risk of acute rejection. In summary, RNAi is now firmly established as a powerful research tool with the potential to selectively limit the expression of almost any gene at the mRNA level. The studies so far provide an important proof of principle that antiviral activity of RNAi can be achieved in cultured cells and in the liver of animals. Having established that replication of both HBV and HCV can be inhibited by RNAi, the main challenge for clinical application of this technology is the delivery, rather than design. The rapid growth of this field over the last 2 years, and its current momentum, suggests that this challenge will not prove insurmountable and that the reality of RNAi-based therapeutics may not be too far away. References [1] Hannon GJ. RNA interference. Nature 2002;418:244–251. [2] Cullen BR. Antiviral defense and genetic tool. Nat Immunol 2002;3: 597–599. [3] Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363–366. [4] Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000;404:293–296. [5] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic influence by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–811. [6] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mamalian cells. Nature 2001;411:494–498. [7] Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002;26:199–213. [8] Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 2003;278:7108–7118.
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[9] Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003; 115:199–208. [10] Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell 2003;115:209–216. [11] Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3 0 overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002;20:497–500. [12] Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfereing RNAs in mammalian cells. Science 2002;296:550–553. [13] Sui G, Soohoo C, El Bachir A, Gay F, Shi Y, Forrester WC, et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99:5515–5520. [14] Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2003;99:6047–6052. [15] Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002;2:243–247. [16] An DS, Xie Y, Mao SH, Morizono K, Kung SK, Chen IS. Efficient lentiviral vectors for short hairpin RNA delivery into human cells. Hum Gene Ther 2002;14:1207–1212. [17] Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol 2002;76:9225–9231. [18] Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, et al. siRNA-directed inhibition of HIV-1 infection. Nat Med 2002;8:681–686. [19] Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A, et al. Expression of small interfereing RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002;20:500–505. [20] Jacque JM, Triques K, Stevenson M. Modulation of HIV replication by RNA interference. Nature 2002;418:435–438. [21] Dector MA, Romero P, Lopez S, Arias CF. Rotavirus gene silencing by small interfering RNAs. Eur Mol Biol Org Rep 2002; 3:1175–1180. [22] Adelman ZN, Sanchez-Vargas I, Travanty EA, Carlson JO, Beaty BJ, Blair CD, et al. RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J Virol 2002; 76:12925–12933. [23] Ge Q, McManus MT, Nguyen T, Shen C-H, Sharp PA, Eisen HN, et al. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA 2003;100:2718–2723. [24] Garrus JE, von Schwedler UK, Pornillos OW, Morham SG, Zavitz KH, Wang HE, et al. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 2001;107:55–65. [25] Boden D, Pusch O, Lee F, Tucker L, Ramratnam B. Human immunodeficiency virus type 1 escape from RNA interference. J Virol 2003;77:11531–11531. [26] Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology 2003;37:764–771. [27] Ying C, De Clercq E, Neyts J. Selective inhibition of hepatitis B virus replication by RNA interference. Biochem Biophys Res Commun 2003;309:482–484. [28] Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line. Hepatology 2003;38: 842–850. [29] Yang PL, Althage A, Chung J, Chisari FV. Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci USA 2002;99:13825–13830. [30] McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 2003;21:639–644.
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[31] Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes H-P, et al. Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA. Gastroenterology 2003; 125:9–18. [32] Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA 2003;100:2014–2018. [33] Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci USA 2003;100:235–240. [34] Wilson JA, Jayasena S, Khvorova A, Sabatinos S, RodrigueGervais IG, Arya S, et al. RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci USA 2003;100:2783–2788.
[35] McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature 2002;418: 38–39. [36] Chang J, Taylor JM. Susceptibility of human hepatitis delta virus RNAs to small interfering RNA action. J Virol 2003;77: 9728–9731. [37] Chang J, Provost P, Taylor JM. Resistance of human hepatitis delta virus RNAs to dicer activity. J Virol 2003;77:11910–11917. [38] Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, et al. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med 2003;9:347–351. [39] Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, et al. Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol 2004;78:2601–2605.
Review
The potential of RNA interference as a tool in the management of viral hepatitis John A. Taylor1,2, Nikolai V. Naoumov2,* 1
2
School of Biological Sciences, University of Auckland, Auckland, New Zealand Institute of Hepatology, University College London, 69–75 Chenies Mews, London WC1E 6HX, UK
1. Introduction RNA interference or RNAi is an evolutionary-conserved mechanism present in virtually all eukaryotic cell. The process is triggered by the presence of double-stranded RNA (dsRNA), which is formed transiently during the replication of many viral genomes, but generally is absent from non-infected cells. Therefore, RNAi is believed to represent an ancestral form of nucleic acid based ‘immunity’ against intracellular pathogens resulting in a sequence-specific silencing of gene expression [1,2]. Gene silencing by RNAi involves the generation of short (21–23 nucleotide) duplex RNA species from a longer precursor by an endonuclease called ‘dicer’ [3]. The resultant short dsRNAs, referred to as small interfering RNA (siRNA), are incorporated into a ribonucleoprotein complex termed RISC for RNA-inducing silencing complex, essentially, a programmable endonuclease that acquires target specificity by unwinding the strands of siRNA and retaining one as a guide to complimentary mRNA species in the cytoplasm [4]. RISC then cleaves the target mRNA in the middle of the homologous sequence and can target further molecules resulting in a processive depletion of a specific mRNA pool. In 1998, Fire and colleagues demonstrated the potent sequence-specific silencing of gene expression mediated by homologous dsRNA in the nematode Caenorhabditis elegans [5]. The observation of a similar response in * Corresponding author. Tel.: C44 20 7679 6512; fax: C44 20 7380 0405. E-mail address: [email protected] (N.V. Naoumov). Abbreviations RNAi, RNA interference; siRNA, short-interfering ribonucleic acid; dsRNA, double-stranded ribonucleic acid; RISC, RNAinduced silencing complex; shRNA, short hairpin ribonucleic acid; ORF, open reading frame; RT-PCR, reverse transcription-polymerase chain reaction.
mammalian cells was masked by the induction of the interferon response, a non-sequence specific response to dsRNA induced by many animal viruses leading to global changes in cellular transcription and translation. By restricting their length to less than 21–23 nt, the length of siRNA molecules generated by dicer, Tuschl and coworkers showed that synthetic RNA duplexes introduced into mammalian cells could induce sequence specific cleavage of mRNA by without activation of the interferon response [6]. The direct introduction of siRNA in mammalian cells bypasses the need for cleavage of a longer template by dicer while effectively priming RISC for the selective down-regulation of gene expression. Not surprisingly, this technology was quickly recognised as a practical approach to genetic analysis in mammalian cells and as a potential tool in the treatment of human diseases including those caused by virus infection.
2. Design and delivery of functional siRNA The key to successful gene silencing by RNAi lies both in the design of effective siRNA and its efficient introduction into target cells. Though design remains largely an empirical process, principles have been established that facilitate the production of siRNA that can potentially silence gene expression with high efficiency [7]. One determinant likely to influence effective silencing is the degree of RNA secondary structure in the vicinity of the target site. siRNAs that target highly structured regions of mRNA appear to function less efficiently than those whose targets are in less structured regions [8]. Failure of siRNA to effectively silence target genes has also been associated with the preferential incorporation of the sense RNA strand of the duplex into RISC, resulting in an
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jhep.2004.10.022
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inability to guide the complex to the target mRNA. Two recent papers, report that the bias for incorporation of the antisense strand into RISC is determined by the relative stabilities of the base pair at the 5 0 ends of the duplex [9,10]. These studies will aid further efforts to improve predictive models for the design of functional siRNA, leading to more effective gene silencing. The first demonstration of siRNA-mediated gene silencing involved transient transfection of a chemically synthesised RNA duplex [6]. Chemical synthesis has now been employed widely to silence more than 200 genes. Apart from cost, a drawback of this approach may be the relatively short half-life of siRNA duplex inside a cell, resulting in a transient effect over a few days only. To circumvent this potential problem several groups have developed an alternative approach involving the use of DNA vectors that enable the separate transcription of both strands of siRNA or, of short hairpin RNAs (shRNA) after transient or stable transfection of mammalian cells [11–14]. shRNA resembles siRNA in the length of the RNA duplex, but is formed from a single RNA molecule containing an inverted repeat sequence separated by a short hairpin loop region (Fig. 1). Plasmid vectors that express si/shRNA usually contain promoter sequences specific for RNA pol II, which generates precisely defined transcripts. The DNA template for pol II transcription is usually generated by cloning a DNA sequence, formed by annealing complimentary oligonucleotides, flanked by restriction enzyme sites into a suitable plasmid vector. Transcription of the shRNA ‘gene’ is terminated by a run of at least four T-residues at the 3 0 end, resulting in a UU sequence in the RNA. The
shRNA is processed in vivo by dicer to yield a functional siRNA. A more recent development in the vector delivery of siRNA is the availability of viral vectors capable of stably transducing a wide variety of target cells [12,15,16]. Adenoviral and lentiviral systems in particular have been used successfully to deliver shRNA to primary cell lines including lymphocytes, hepatocytes and neurons. This approach circumvents the difficulty associated with transfection of these cell types and can lead to the stable expression of shRNA after insertion of the viral sequence into the genome. For example, a lentiviral vector was used to inhibit HIV replication in cultured macrophages. When the transduced cells were infected with HIV, the yield of virus was reduced by 99% [17].
3. Inhibition of viral replication by RNAi Many studies have demonstrated the potential of RNAi to effectively silence the expression of viral genes and thus inhibit virus replication in cell culture. In addition to hepatotropic viruses, (discussed below), this approach has been applied to human immunodeficiency virus, [17–20], rotavirus [21], dengue virus [22] and influenza virus [23]. These studies have targeted a variety of virus genes and employed both chemically-synthesized, and vector-encoded forms of siRNA, introduced into different cell types including primary cell cultures. Based on these data, RNAi has emerged as a potential tool for treatment of virus infections in vivo. Instead of targeting viral genes, an
Fig. 1. Mechanism of RNAi. (1) The endonuclease dicer processes long dsRNA molecules or shRNAs to 21–23 nucleotide siRNA in the cytoplasm. Alternatively, chemically synthesised siRNA can be directly introduced by transfection. (2) The siRNA interacts with the RISC complex and the duplex is unwound by an ATP-dependant helicase activity resulting in retention of the antisense strand. (3) RISC is targeted to mRNAs with a complementary sequence. (4) The target mRNA is cleaved and RISC is guided to further mRNA molecules.
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alternative strategy for antiviral treatment would be to direct siRNA against essential cellular genes, whose products are required for virus replication. For example, silencing of Tsg101, a protein required for budding of virions from infected cells, greatly reduced the release of HIV from transfected 293T cells [24]. Similarly, delivery of siRNA against the HIV co-receptor CCR5, using a lentivirus vector, inhibited the ability of virus to enter primary macrophages reducing the yield of virus by 90% [18]. This approach offers an alternative strategy to the targeting of viral genes by siRNA, the success of which in vivo may be limited by the ability of the virus to mutate rapidly, and thus escape the inhibitory effect of RNAi, as reported recently for HIV [25].
4. RNAi and hepatitis viruses 4.1. Hepatitis B virus (HBV) The extensive use of overlapping reading frames within the HBV genome suggests that this virus may be particularly susceptible to RNAi, as a single siRNA could potentially target multiple viral mRNAs. Plasmids were constructed to express shRNA that targeted sequences in the viral X and core ORFs [26]. Co-transfection of these vectors into Huh7 cells with a plasmid encoding a complete HBV genome reduced the levels of core protein with either shRNA. A greater level of suppression (89 vs. 63%) was observed using siRNA that targeted both X and core mRNAs than with siRNA targeting core alone, emphasizing the potential synergistic effect of targeting multiple viral transcripts simultaneously. These authors further demonstrated the siRNA-mediated reduction of viral transcripts, including the 3.5 kb pregenomic RNA, and reported a reduction in HBV replicative intermediates of up to 95%. Successful inhibition of HBV replication in stably transfected HepAD38 cells, using siRNA targeting a site in the core ORF, has also been reported [27]. Although the level of virus secreted into the medium was reduced by 98% 48 h after transfection, this level of suppression had declined to 40% after a further 24 h underscoring the transient nature of the RNAi effect in transfected cells. These results suggest the likely need for sustained production of siRNA in vivo. In a further study, using HepG2.2.15 cells, the successful delivery of siRNA targeting the HBV polyadenylation signal not only sustained the inhibition of HBsAg secretion, but produced an additive effect in suppressing HBV replication [28]. Several studies have demonstrated that siRNA can efficiently inhibit HBV replication in animals. Although mice are not susceptible to HBV infection, the viral genome can be stably introduced to produce HBV transgenic animals that synthesise viral mRNAs in hepatocytes and secrete mature virions. More recently, hydrodynamic injection of cloned HBV sequences into the tail vein has been used to establish a transient model of acute HBV
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infection in mice [29]. This system has presented the opportunity to investigate the potential for siRNA to suppress aspects of virus replication in the context of a functional innate and adaptive immune system present in whole animals. Co-injection of a cloned HBV sequence with a plasmid expressing shRNA against distinct viral mRNAs, resulted in an effective RNAi response against HBV replication in mice [30]. Targeting a variety of sequences in each of the four viral mRNAs, these investigators demonstrated a reduction of up to 84.5% in the level of secreted HBsAg in mouse serum and a greater than 99% reduction in the number of hepatocytes positive for HBcAg, as shown by immunohistochemical staining of liver sections. A similar reduction in serum HBsAg and HBeAg expression has been reported following injection of siRNA that targets an overlapping sequence in the S and preC/C mRNAs [31]. HBsAg levels reached only 30% of control levels, and remained reduced up to 11 days after injection, while HBeAg levels were 20% of controls, which was also sustained. 4.2. Hepatitis C virus (HCV) Several studies have explored the possibility of using RNAi as a potential therapeutic intervention against HCV infection. As a (C) sense single-stranded RNA virus that replicates via a negative sense intermediate this approach offers the possibility, at least in theory, that RNAi could eliminate viral genomic, replicative form and mRNA species from infected cells and thus resolve HCV infection. However, in the absence of a suitable system for replication of HCV in culture, these studies have been restricted to demonstrating the potential of siRNA to suppress the replication of viral replicon RNA. The effect of siRNA against various sites in the NS3 and NS5B ORFs has been shown in several studies. A 21–23 fold reduction of HCV transcript levels in cells harbouring an HCV replicon was demonstrated by quantitative RT-PCR, [32]. To address the fate of HCV RNA at a single cell level, Randall et al., [33] 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 indicated that HCV-specific siRNA could mediate the clearance, to levels below the sensitivity of detection, of viral RNA and protein in O98% of cells. Similar results were obtained following the generation of stable cell lines that producing siRNA against NS3 [34]. The introduction of a HCV replicon to these cells resulted in 70% fewer G418 resistant colonies compared with controls indicating the potential for long-term suppression of HCV by RNAi. While these results are encouraging, the high mutation rate and sequence variability between HCV genotypes and quasispecies presents a considerable challenge to the successful use of siRNA against HCV infection in vivo
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where multiple siRNAs targeting different regions of the viral genome may have to be introduced simultaneously to avoid the evolution of RNAi escape mutants. Hydrodynamic transfection of siRNA in mice has also shown the potential to limit replication of HCV using viral sequences fused to a reporter gene although only a portion of the viral genome was present and no replication occurred [35]. 4.3. Hepatitis delta virus (HDV) HDV is a subviral satellite RNA whose replication is dependant on the provision of envelope protein by its natural helper virus HBV. HDV superinfection of chronic HBV carriers results in HDV persistence that is usually associated with significant hepatic inflammation and rapidly progressive liver damage. Three distinct RNA species—the 1679 nucleotide ssRNA genome, its exact complement, and an 800 nucleotide mRNA encoding the delta antigen are each potentially susceptible to cleavage by RNAi. However, a recent study shows that only the HDV mRNA is cleaved by siRNA introduced into Huh7 cells [36]. Despite the partial double-stranded nature of the genomic and antigenomic HDV RNAs that can fold into a rod-like structure with 74% base pairing, these molecules are intrinsically resistant to the endonuclease dicer both in vitro and in vivo [37]. Thus future attempts to use siRNA therapeutically against HDV should focus on silencing of the mRNA.
5. Dissecting the host response to virus infection with RNAi The ability of RNAi to function in cells infected with HBV or HCV provides a valuable tool to investigate the role of host genes involved both in virus replication, as well as in cytokine-mediated suppression of virus replication. This approach is particularly suited to analysis of host genes that are involved in the replication of HBV by simultaneous delivery of siRNA and a cloned viral genome to cultured hepatoma cells. Obvious targets for this type of analysis would include genes that encode polypeptides that have been observed to interact directly with viral components, genes that encode components of signalling pathways activated by antiviral cytokines (ex. interferon-alpha and interferon-gamma), and genes induced following virus infection. This approach offers the potential to identify new targets for antiviral therapy by dissecting the requirements for virus replication.
6. Silencing host gene expression to modify the pathogenesis of viral hepatitis Silencing of endogenous gene expression in the liver during viral hepatitis may also represent an effective
strategy for limiting viral replication or immune-mediated damage to infected hepatocytes. The value of this approach was demonstrated in experiments that examined the ability of RNAi to protect hepatocytes from Fas-mediated apoptosis during autoimmune hepatitis in mice [38]. In these studies, mice were transfected with siRNA that targeted Fas, a key mediator of T-cell mediated apoptosis in hepatocytes during autoimmune and viral hepatitis. Introduction of Fas siRNA resulted in uptake into over 80% of hepatocytes and caused significant reduction of Fas at both RNA and protein levels. Remarkably, in vivo treatment with Fas siRNA protected mouse hepatocytes from Fas-mediated apoptosis. Most significantly of all, this approach markedly attenuated hepatocyte necrosis in a model of autoimmune hepatitis and protected 82% of animals from death by fulminant hepatitis following the injection of Fas-specific antibody. Given that damage to hepatocytes during viral hepatitis is immune-mediated, this study points to the potential value of targeting both viral and cellular targets to reduce both viral replication and hepatocellular necrosis during periods of acute liver inflammation in chronic viral hepatitis.
7. Clinical application of RNAi in the treatment of viral hepatitis The studies outlined above demonstrate the feasibility of using RNAi to effect a potent and sustained inhibition of virus replication and offer proof of concept for use of RNAi as an antiviral therapy (Table 1). One obvious attraction of this technology in the treatment of human viral infections is the higher degree of specificity of siRNA in comparison to conventional antiviral chemotherapy. The availability of genome sequence libraries permits the elimination of siRNA with the potential for undesired ‘off target’ effects on endogenous genes. As with antiviral drugs, the evolution of resistance to virus-specific siRNA by rapidly mutating viruses like HCV could potentially limit the therapeutic effectiveness of RNAi. Mutations in HIV have already been reported that confer resistance to siRNA [25,39]. Simultaneous application of multiple siRNA molecules targeting conserved viral sequences would be necessary for sustained suppression of virus replication in vivo. Despite the optimism generated by studies discussed above, the major barrier to the successful clinical use of Table 1 Advantages and limitations of RNAi in a potential clinical scenario Advantages
Limitations
Broad applicability Potencya Specificity Low toxicity
Low cellular uptake in vivo Stability in vivo Potential for off-target gene silencing Potential induction of interferon Evolution of viral resistance
a
Relative to anti-sense RNA oligonucleotides.
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siRNA remains how to introduce sufficient levels of siRNA to the liver of patients suffering viral hepatitis resulting in the sustained and specific gene silencing necessary to modify the progression of disease. Hydrodynamic transfection, while successful in mice, is unlikely to be adopted as a clinical procedure given the requirement for generation of a high intravascular pressure. Even if this were possible, the short half-life of chemically synthesised siRNA in vivo limits the silencing effect to a few days at most which may be insufficient to manifest a clinical benefit. In view of these difficulties, vectored delivery of siRNA using recombinant viruses represents a more promising means of effective longterm gene silencing in the liver although this approach will bring all the attendant limitations associated with gene therapy such as potentially oncogenic effects of random integration in the host genome. While systemic application of siRNA for the treatment of viral hepatitis may not be imminent, one attractive possibility would be to transfect hepatocytes of donor livers with siRNA prior to orthotopic liver transplantation, in order to prevent viral recurrence (e.g. HCV) in the graft, or to minimise the risk of acute rejection. In summary, RNAi is now firmly established as a powerful research tool with the potential to selectively limit the expression of almost any gene at the mRNA level. The studies so far provide an important proof of principle that antiviral activity of RNAi can be achieved in cultured cells and in the liver of animals. Having established that replication of both HBV and HCV can be inhibited by RNAi, the main challenge for clinical application of this technology is the delivery, rather than design. The rapid growth of this field over the last 2 years, and its current momentum, suggests that this challenge will not prove insurmountable and that the reality of RNAi-based therapeutics may not be too far away. References [1] Hannon GJ. RNA interference. Nature 2002;418:244–251. [2] Cullen BR. Antiviral defense and genetic tool. Nat Immunol 2002;3: 597–599. [3] Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363–366. [4] Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 2000;404:293–296. [5] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic influence by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–811. [6] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mamalian cells. Nature 2001;411:494–498. [7] Elbashir SM, Harborth J, Weber K, Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 2002;26:199–213. [8] Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem 2003;278:7108–7118.
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