1
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A Novel Approach of Antiviral Drugs Targeting Viral Genomes. Microorganisms 2022; 10:microorganisms10081552. [PMID: 36013970 PMCID: PMC9414836 DOI: 10.3390/microorganisms10081552] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 07/25/2022] [Accepted: 07/29/2022] [Indexed: 11/16/2022] Open
Abstract
Outbreaks of viral diseases, which cause morbidity and mortality in animals and humans, are increasing annually worldwide. Vaccines, antiviral drugs, and antibody therapeutics are the most effective tools for combating viral infection. The ongoing coronavirus disease 2019 pandemic, in particular, raises an urgent need for the development of rapid and broad-spectrum therapeutics. Current antiviral drugs and antiviral antibodies, which are mostly specific at protein levels, have encountered difficulties because the rapid evolution of mutant viral strains resulted in drug resistance. Therefore, degrading viral genomes is considered a novel approach for developing antiviral drugs. The current article highlights all potent candidates that exhibit antiviral activity by digesting viral genomes such as RNases, RNA interference, interferon-stimulated genes 20, and CRISPR/Cas systems. Besides that, we introduce a potential single-chain variable fragment (scFv) that presents antiviral activity against various DNA and RNA viruses due to its unique nucleic acid hydrolyzing characteristic, promoting it as a promising candidate for broad-spectrum antiviral therapeutics.
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2
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Khan IW, Dad Ullah MU, Choudhry M, Ali MJ, Ali MA, Lam SLK, Shah PA, Kaur SP, Lau DTY. Novel Therapies of Hepatitis B and D. Microorganisms 2021; 9:2607. [PMID: 34946209 PMCID: PMC8707465 DOI: 10.3390/microorganisms9122607] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2021] [Revised: 12/03/2021] [Accepted: 12/07/2021] [Indexed: 02/05/2023] Open
Abstract
Hepatitis B virus (HBV) infection is a global public health issue and is a major cause of cirrhosis and hepatocellular carcinoma (HCC). Hepatitis D virus (HDV) requires the hepatitis B surface antigen (HBsAg) to replicate. The eradication of HBV, therefore, can also cure HDV. The current therapies for chronic hepatitis B and D are suboptimal and cannot definitely cure the viruses. In order to achieve functional or complete cure of these infections, novel therapeutic agents that target the various sites of the viral replicative cycle are necessary. Furthermore, novel immunomodulatory agents are also essential to achieve viral clearance. Many of these new promising compounds such as entry inhibitors, covalently closed circular DNA (cccDNA) inhibitors, small interfering RNAs (siRNAs), capsid assembly modulators and nucleic acid polymers are in various stages of clinical developments. In this review article, we provided a comprehensive overview of the structure and lifecycle of HBV, the limitations of the current therapies and a summary of the novel therapeutic agents for both HDV and HBV infection.
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Affiliation(s)
- Iman Waheed Khan
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Mati Ullah Dad Ullah
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Mina Choudhry
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Mukarram Jamat Ali
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Muhammad Ashar Ali
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Sam L. K. Lam
- Liver Center, Department of Medicine, Department of Pharmacy, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA;
| | - Pir Ahmad Shah
- Department of Internal Medicine, University of Texas, San Antonio, TX 78229, USA;
| | - Satinder Pal Kaur
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
| | - Daryl T. Y. Lau
- Liver Center, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA; (I.W.K.); (M.U.D.U.); (M.C.); (M.J.A.); (M.A.A.); (S.P.K.)
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3
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Kim H, Ko C, Lee JY, Kim M. Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure, Mode-of-Action and Efficacy. Molecules 2021; 26:molecules26247420. [PMID: 34946502 PMCID: PMC8705634 DOI: 10.3390/molecules26247420] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2021] [Revised: 11/24/2021] [Accepted: 12/02/2021] [Indexed: 12/15/2022] Open
Abstract
Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.
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Affiliation(s)
- Hyejin Kim
- Correspondence: (H.K.); (M.K.); Tel.: +82-42-860-7130 (H.K.); +82-42-860-7540 (M.K.)
| | | | | | - Meehyein Kim
- Correspondence: (H.K.); (M.K.); Tel.: +82-42-860-7130 (H.K.); +82-42-860-7540 (M.K.)
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4
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Qu B, Brown RJP. Strategies to Inhibit Hepatitis B Virus at the Transcript Level. Viruses 2021; 13:v13071327. [PMID: 34372533 PMCID: PMC8310268 DOI: 10.3390/v13071327] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Revised: 07/02/2021] [Accepted: 07/06/2021] [Indexed: 12/11/2022] Open
Abstract
Approximately 240 million people are chronically infected with hepatitis B virus (HBV), despite four decades of effective HBV vaccination. During chronic infection, HBV forms two distinct templates responsible for viral transcription: (1) episomal covalently closed circular (ccc)DNA and (2) host genome-integrated viral templates. Multiple ubiquitous and liver-specific transcription factors are recruited onto these templates and modulate viral gene transcription. This review details the latest developments in antivirals that inhibit HBV gene transcription or destabilize viral transcripts. Notably, nuclear receptor agonists exhibit potent inhibition of viral gene transcription from cccDNA. Small molecule inhibitors repress HBV X protein-mediated transcription from cccDNA, while small interfering RNAs and single-stranded oligonucleotides result in transcript degradation from both cccDNA and integrated templates. These antivirals mediate their effects by reducing viral transcripts abundance, some leading to a loss of surface antigen expression, and they can potentially be added to the arsenal of drugs with demonstrable anti-HBV activity. Thus, these candidates deserve special attention for future repurposing or further development as anti-HBV therapeutics.
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Affiliation(s)
- Bingqian Qu
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- European Virus Bioinformatics Center, 07743 Jena, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
| | - Richard J. P. Brown
- Division of Veterinary Medicine, Paul Ehrlich Institute, 63225 Langen, Germany
- Correspondence: (B.Q.); (R.J.P.B.)
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van den Berg F, Limani SW, Mnyandu N, Maepa MB, Ely A, Arbuthnot P. Advances with RNAi-Based Therapy for Hepatitis B Virus Infection. Viruses 2020; 12:E851. [PMID: 32759756 PMCID: PMC7472220 DOI: 10.3390/v12080851] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 07/22/2020] [Accepted: 07/29/2020] [Indexed: 02/06/2023] Open
Abstract
Infection with hepatitis B virus (HBV) remains a global health challenge. Approximately 292 million people worldwide are chronically infected with HBV and the annual mortality from the infection is approaching 900,000. Despite the availability of an effective prophylactic vaccine, millions of individuals are at risk of potentially fatal complicating cirrhosis and hepatocellular carcinoma. Current drug treatments can suppress viral replication, slow the progression of liver fibrosis, and reduce infectivity, but can rarely clear the viral covalently closed circular DNA (cccDNA) that is responsible for HBV persistence. Alternative therapeutic strategies, including those based on viral gene silencing by harnessing the RNA interference (RNAi) pathway, effectively suppress HBV replication and thus hold promise. RNAi-based silencing of certain viral genes may even lead to disabling of cccDNA during chronic infection. This review summarizes different RNAi activators that have been tested against HBV, the advances with vectors used to deliver artificial potentially therapeutic RNAi sequences to the liver, and the current status of preclinical and clinical investigation.
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Affiliation(s)
| | | | | | | | | | - Patrick Arbuthnot
- Wits/SAMRC Antiviral Gene Therapy Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2050, South Africa; (F.v.d.B.); (S.W.L.); (N.M.); (M.B.M.); (A.E.)
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Maillard PV, van der Veen AG, Poirier EZ, Reis e Sousa C. Slicing and dicing viruses: antiviral RNA interference in mammals. EMBO J 2019; 38:e100941. [PMID: 30872283 PMCID: PMC6463209 DOI: 10.15252/embj.2018100941] [Citation(s) in RCA: 86] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Revised: 01/17/2019] [Accepted: 01/25/2019] [Indexed: 12/15/2022] Open
Abstract
To protect against the harmful consequences of viral infections, organisms are equipped with sophisticated antiviral mechanisms, including cell-intrinsic means to restrict viral replication and propagation. Plant and invertebrate cells utilise mostly RNA interference (RNAi), an RNA-based mechanism, for cell-intrinsic immunity to viruses while vertebrates rely on the protein-based interferon (IFN)-driven innate immune system for the same purpose. The RNAi machinery is conserved in vertebrate cells, yet whether antiviral RNAi is still active in mammals and functionally relevant to mammalian antiviral defence is intensely debated. Here, we discuss cellular and viral factors that impact on antiviral RNAi and the contexts in which this system might be at play in mammalian resistance to viral infection.
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Affiliation(s)
- Pierre V Maillard
- Division of Infection and Immunity, University College London, London, UK
| | | | - Enzo Z Poirier
- Immunobiology Laboratory, The Francis Crick Institute, London, UK
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Mouse models for hepatitis B virus research. Lab Anim Res 2018; 34:85-91. [PMID: 30310404 PMCID: PMC6170223 DOI: 10.5625/lar.2018.34.3.85] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2018] [Revised: 09/13/2018] [Accepted: 09/13/2018] [Indexed: 12/16/2022] Open
Abstract
Hepatitis B virus (HBV) infection remains a major global health problem; indeed, there are 250 million carriers worldwide. The host range of HBV is narrow; therefore, few primates are susceptible to HBV infection. However, ethical constraints, high cost, and large size limit the use of primates as suitable animal models. Thus, in vivo testing of therapies that target HBV has been hampered by the lack of an appropriate in vivo research model. To address this, mouse model systems of HBV are being developed and several are used for studying HBV in vivo. In this review, we summarize the currently available mouse models, including HBV transgenic mice, hydrodynamic injection-mediated HBV replicon delivery systems, adeno-associated virus-mediated HBV replicon delivery systems, and human liver chimeric mouse models. These developed (or being developed) mouse model systems are promising and should be useful tools for studying HBV.
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Abstract
With high morbidity and mortality worldwide, there is great interest in effective therapies for chronic hepatitis B (CHB) virus. There are currently several dozen investigational agents being developed for treatment of CHB. They can be broadly divided into two categories: (1) direct-acting antivirals (DAAs) that interfere with a specific step in viral replication; and (2) host-targeting agents that inhibit viral replication by modifying host cell function, with the latter group further divided into the subcategories of immune modulators and agents that target other host functions. Included among the DAAs being developed are RNA interference therapies, covalently closed circular DNA (cccDNA) formation and transcription inhibitors, core/capsid inhibitors, reverse transcriptase inhibitors, hepatitis B surface antigen (HBsAg) release inhibitors, antisense oligonucleotides, and helioxanthin analogues. Included among the host-targeting agents are entry inhibitors, cyclophilin inhibitors, and multiple immunomodulatory agents, including Toll-like receptor agonists, immune checkpoint inhibitors, therapeutic vaccines, engineered T cells, and several cytokine agents, including recombinant human interleukin-7 (CYT107) and SB 9200, a novel therapy that is believed to both have direct antiviral properties and to induce endogenous interferon. In this review we discuss agents that are currently in the clinical stage of development for CHB treatment as well as strategies and agents currently at the evaluation and discovery phase and potential future targets. Effective approaches to CHB may require suppression of viral replication combined with one or more host-targeting agents. Some of the recent research advances have led to the hope that with such a combined approach we may have a functional cure for CHB in the not distant future.
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Affiliation(s)
- Altaf Dawood
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Syed Abdul Basit
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Mahendran Jayaraj
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA
| | - Robert G Gish
- Department of Internal Medicine, Section of Gastroenterology, University of Nevada School of Medicine, Las Vegas, NV, USA.
- Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University Medical Center, Stanford, CA, USA.
- Hepatitis B Foundation, Doylestown, PA, USA.
- Asian Pacific Health Foundation, San Diego, CA, USA.
- National Viral Hepatitis Roundtable, Washington, DC, USA.
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Qureshi A, Tantray VG, Kirmani AR, Ahangar AG. A review on current status of antiviral siRNA. Rev Med Virol 2018; 28:e1976. [PMID: 29656441 PMCID: PMC7169094 DOI: 10.1002/rmv.1976] [Citation(s) in RCA: 68] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2017] [Revised: 01/18/2018] [Accepted: 02/12/2018] [Indexed: 01/12/2023]
Abstract
Viral diseases like influenza, AIDS, hepatitis, and Ebola cause severe epidemics worldwide. Along with their resistant strains, new pathogenic viruses continue to be discovered so creating an ongoing need for new antiviral treatments. RNA interference is a cellular gene‐silencing phenomenon in which sequence‐specific degradation of target mRNA is achieved by means of complementary short interfering RNA (siRNA) molecules. Short interfering RNA technology affords a potential tractable strategy to combat viral pathogenesis because siRNAs are specific, easy to design, and can be directed against multiple strains of a virus by targeting their conserved gene regions. In this review, we briefly summarize the current status of siRNA therapy for representative examples from different virus families. In addition, other aspects like their design, delivery, medical significance, bioinformatics resources, and limitations are also discussed.
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Affiliation(s)
- Abid Qureshi
- Biomedical Informatics Center, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, India
| | - Vaqar Gani Tantray
- Biomedical Informatics Center, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, India
| | - Altaf Rehman Kirmani
- Biomedical Informatics Center, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, India
| | - Abdul Ghani Ahangar
- Biomedical Informatics Center, Sher-i-Kashmir Institute of Medical Sciences (SKIMS), Srinagar, India
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Zhu C, Zhu H, Song H, Xu L, Li L, Liu F, Liu X. Hepatitis B virus inhibits the in vivo and in vitro synthesis and secretion of apolipoprotein C3. Lipids Health Dis 2017; 16:213. [PMID: 29132372 PMCID: PMC5683573 DOI: 10.1186/s12944-017-0607-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2017] [Accepted: 11/05/2017] [Indexed: 01/17/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) infection in the body can damage liver cells and cause disorders in blood lipid metabolism. Apolipoprotein C3 (ApoC3) plays an important role in the regulation of lipid metabolism, but no study on the HBV regulation of ApoC3 has been reported. This purpose of this study was to investigate the effect of HBV on ApoC3 expression and its regulatory mechanism. METHODS The expression levels of ApoC3 mRNA and protein in the human hepatoma cell lines HepG2 and HepG2.2.15 were determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. The HepG2 cells were co-transfected with the ApoC3 gene promoter and either HBV-infected clone pHBV1.3 or its individual genes. The changes in luciferase activity were assayed. The expression levels of ApoC3 mRNA and protein were determined using RT-qPCR and Western blot. The content of ApoC3 in the supernatant of the cultured cells was determined using an enzyme-linked immunosorbent assay (ELISA). The sera were collected from 149 patients with HBV infection and 102 healthy subjects at physical examination as the normal controls. The serological levels of ApoC3 in the HBV group and the normal control group were determined using ELISA. The contents of serum triglyceride (TG) and very-low-density lipoprotein (VLDL) in the HBV patients and the normal control were determined using an automatic biochemical analyser. RESULTS The expression levels of ApoC3 mRNA and protein were lower in the HepG2.2.15 cells than in the HepG2 cells. pHBV1.3 and its X gene could inhibit the activity of the ApoC3 promoter and its mRNA and protein expression. The serum levels of ApoC3, VLDL and TG were 65.39 ± 7.48 μg/ml, 1.24 ± 0.49 mmol/L, and 0.46 ± 0.10 mmol/L in the HBV patients and 41.02 ± 6.88 μg/ml, 0.76 ± 0.21 mmol/L, 0.29 ± 0.05 mmol/L in the normal controls, respectively, statistical analysis revealed significantly lower serum levels of ApoC3, VLDL and TG in HBV patients than in the normal controls (P < 0.05). CONCLUSION HBV can inhibit the in vivo and in vitro synthesis and secretion of ApoC3.
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Affiliation(s)
- Chengliang Zhu
- Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, Hubei, 430060, People's Republic of China
| | - Hengcheng Zhu
- Department of Clinical Laboratory, Renmin Hospital of Wuhan University, Wuhan, Hubei, 430060, People's Republic of China
| | - Hui Song
- Department of Clinical Laboratory, Shanghai Gongli Hospital, the Second Military Medical University, Pudong New Area, Shanghai, 200135, China
| | - Limin Xu
- Department of Clinical Laboratory, Shanghai Gongli Hospital, the Second Military Medical University, Pudong New Area, Shanghai, 200135, China
| | - Longxuan Li
- Department of Neurology, Shanghai Gongli Hospital, the Second Military Medical University, Pudong New Area, Shanghai, 200135, China
| | - Fang Liu
- The State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei, 430072, People's Republic of China
| | - Xinghui Liu
- Department of Clinical Laboratory, Shanghai Gongli Hospital, the Second Military Medical University, Pudong New Area, Shanghai, 200135, China.
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Enhanced antiviral and antifibrotic effects of short hairpin RNAs targeting HBV and TGF-β in HBV-persistent mice. Sci Rep 2017. [PMID: 28634402 PMCID: PMC5478661 DOI: 10.1038/s41598-017-04170-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The hepatitis B virus (HBV) causes acute and chronic liver infection, which may lead to liver cirrhosis and hepatocellular carcinoma. Current treatments including interferons and nucleotide analogs, have limited therapeutic effects, underscoring the need to identify effective therapeutic options to inhibit HBV replication and prevent complications. Previous animal models mimicking chronic HBV infection do not faithfully reflect disease progression in humans. Here, we used our established HBV-persistent mouse line with liver fibrosis to evaluate the efficacy of novel therapies. The combination of two short hairpin RNAs (dual-shRNA) against different coding regions of HBV delivered by a self-complementary AAV vector showed better antiviral effects than single shRNA both in vitro and in HBV-persistent mice. The dual-shRNA also exhibited stronger antifibrotic activity in vivo. Vector carrying shRNA against TGF-β, though did not inhibit HBV replication alone, enhanced the antiviral and antifibrotic activities of single and dual HBV shRNAs. Co-administration of TGF-β shRNA and HBV dual-shRNA decreased HBV DNA, HBV RNA, HBsAg, HBeAg, and liver fibrosis markers in serum and tissues, and improved liver morphology more effectively than single treatments. Our results suggest that the combination of shRNAs against HBV and TGF-β could be developed into a viable treatment for human HBV infection.
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Park J, Park J, Pei Y, Xu J, Yeo Y. Pharmacokinetics and biodistribution of recently-developed siRNA nanomedicines. Adv Drug Deliv Rev 2016; 104:93-109. [PMID: 26686832 DOI: 10.1016/j.addr.2015.12.004] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Revised: 10/26/2015] [Accepted: 12/03/2015] [Indexed: 02/07/2023]
Abstract
Small interfering RNA (siRNA) is a promising drug candidate, expected to have broad therapeutic potentials toward various diseases including viral infections and cancer. With recent advances in bioconjugate chemistry and carrier technology, several siRNA-based drugs have advanced to clinical trials. However, most cases address local applications or diseases in the filtering organs, reflecting remaining challenges in systemic delivery of siRNA. The difficulty in siRNA delivery is in large part due to poor circulation stability and unfavorable pharmacokinetics and biodistribution profiles of siRNA. This review describes the pharmacokinetics and biodistribution of siRNA nanomedicines, focusing on those reported in the past 5years, and their pharmacological effects in selected disease models such as hepatocellular carcinoma, liver infections, and respiratory diseases. The examples discussed here will provide an insight into the current status of the art and unmet needs in siRNA delivery.
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13
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Zhang X, Lv L, Ouyang X, Zhang S, Fang J, Cai L, Li D. Association of TIP30 expression and prognosis of hepatocellular carcinoma in patients with HBV infection. Cancer Med 2016; 5:2180-9. [PMID: 27418384 PMCID: PMC5055146 DOI: 10.1002/cam4.728] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 03/09/2016] [Accepted: 03/12/2016] [Indexed: 12/30/2022] Open
Abstract
Altered expression of TIP30, a tumor suppressor, has been observed in many cancers. In this study, we have evaluated the expression of TIP30 in the tissues of 209 hepatocellular carcinomas (HCC) and their adjacent tissues by using a high‐density tissue microarray, and analyzed its correlation with the clinical pathological parameters of the patients. The results revealed negative or weak expression of TIP30 in 43.5% (91/209) of the HCC tissues, and in only 27% (56/209) of the adjacent tissues. The expression level of TIP30 in HCC was inversely correlated with serum alpha‐fetoprotein (AFP) levels, HBV infection, and tumor differentiation. Multivariate analysis for survival indicated that serum HBV infection was the most significant predictor of poor prognosis in HCC (P = 0.0023), and TIP30 expression and tumor differentiation were also independent indicators in this respect (P = 0.0364 and P = 0.0397, respectively). Patients with medium or high expression levels of TIP30 (TIP30++/+++) had a better 5‐year overall survival rate than those with low/negative (TIP30+/−) expression (P < 0.001). TIP30+/−/HBV+ patients had the worst 5‐year overall survival rate, whereas TIP30++/+++/HBV− patients had the best. To further explore the correlation between TIP30 and HBV infection in HCC, HBV+ hepatoblastoma cell‐line HepG2 2.2.15 and HCC cell‐line Hep3B were used. Upon silencing of HBV, we observed an upregulation of TIP30 and decreased cell proliferation. In the in vivo studies, we found that the mice inoculated with HepG2 2.2.15 cells with HBV silencing had a prolonged tumor latency and a longer life span, as compared to the control mice inoculated with untreated control cells. In conclusion, the results suggest that downregulation of TIP30 may result from HBV infection, and subsequently promotes the progression of HCC.
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Affiliation(s)
- Xia Zhang
- Department of Hepatology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Lizhi Lv
- Department of Hepatobiliary Surgery, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Xuenong Ouyang
- Department of Oncology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Shi'an Zhang
- Department of Hepatology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Jian Fang
- Department of Hepatology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Lirong Cai
- Department of Hepatology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China
| | - Dongliang Li
- Department of Hepatology, Fuzhou General Hospital, Nanjing Command, Fuzhou 350025, China.
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14
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Li G, Fu L, Jiang J, Ping Y, Huang Y, Wang Y. siRNA combinations mediate greater suppression of hepatitis B virus replication in mice. Cell Biochem Biophys 2015; 69:641-7. [PMID: 24549857 DOI: 10.1007/s12013-014-9846-2] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Hepatitis B virus (HBV) infection is a major world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome shortcomings of established drugs. In this study, the inhibitory effect mediated by combination of siRNAs targeting different sites of HBV in transgenic mice was analyzed. HBsAg and HBeAg in the sera of the mice were analyzed by enzyme-linked immunoadsorbent assay, HBV DNA by real-time PCR and HBV mRNA by RT-PCR. Our data demonstrated that all the three siRNAs employed showed marked anti-HBV effects. The expression of HBsAg and the replication of HBV DNA could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, combination of siRNAs compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA used for therapy was the same. Secreted HBsAg and HBeAg in the serum of mice treated with siRNA combination were reduced by 96.7 and 96.6 %, respectively. Immunohistochemical detection of liver tissue revealed 91 % reduction of HBsAg-positive cells in the combination therapy group. The combination of siRNAs caused a greater inhibition in the levels of viral mRNA and DNA (90 and 87.7 %) relative to the control group. It was noted that the siRNA3 showed stronger inhibition of cccDNA (78.6 %). Our results revealed that combination of siRNAs mediated a stronger inhibition of viral replication and antigen expression in transgenic mice than single siRNAs.
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Affiliation(s)
- Guiqiu Li
- Department of Clinical Laboratory, the Affiliated First Hospital of Harbin Medical University, Harbin, 150001, China
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15
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Recent advances in use of gene therapy to treat hepatitis B virus infection. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2015; 848:31-49. [PMID: 25757614 DOI: 10.1007/978-1-4939-2432-5_2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Chronic infection with hepatitis B virus (HBV) occurs in approximately 5 % of the world's human population and persistence of the virus is associated with serious complications of cirrhosis and liver cancer. Currently available treatments are modestly effective and advancing novel therapeutic strategies is a medical priority. Stability of the viral cccDNA replication intermediate is a major factor that has impeded the development of therapies that are capable of eliminating chronic infection. Recent advances that employ gene therapy strategies offer useful advantages over current therapeutics. Silencing of HBV gene expression by harnessing the RNA interference pathway has been shown to be highly effective in cell culture and in vivo. However, a potential limitation of this approach is that the post-transcriptional mechanism of gene silencing does not disable cccDNA. Early results using designer transcription activator-like effector nucleases (TALENs) and repressor TALEs (rTALEs) have shown potential as a mode of inactivating cccDNA. In this article, we review the recent advances that have been made in HBV gene therapy, with a particular emphasis on the potential anti-HBV therapeutic utility of designed sequence-specific DNA binding proteins and their derivatives.
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Mollaie HR, Monavari SHR, Arabzadeh SAM, Shamsi-Shahrabadi M, Fazlalipour M, Afshar RM. RNAi and miRNA in viral infections and cancers. Asian Pac J Cancer Prev 2015; 14:7045-56. [PMID: 24460249 DOI: 10.7314/apjcp.2013.14.12.7045] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Since the first report of RNA interference (RNAi) less than a decade ago, this type of molecular intervention has been introduced to repress gene expression in vitro and also for in vivo studies in mammals. Understanding the mechanisms of action of synthetic small interfering RNAs (siRNAs) underlies use as therapeutic agents in the areas of cancer and viral infection. Recent studies have also promoted different theories about cell-specific targeting of siRNAs. Design and delivery strategies for successful treatment of human diseases are becomingmore established and relationships between miRNA and RNAi pathways have been revealed as virus-host cell interactions. Although both are well conserved in plants, invertebrates and mammals, there is also variabilityand a more complete understanding of differences will be needed for optimal application. RNA interference (RNAi) is rapid, cheap and selective in complex biological systems and has created new insight sin fields of cancer research, genetic disorders, virology and drug design. Our knowledge about the role of miRNAs and siRNAs pathways in virus-host cell interactions in virus infected cells is incomplete. There are different viral diseases but few antiviral drugs are available. For example, acyclovir for herpes viruses, alpha-interferon for hepatitis C and B viruses and anti-retroviral for HIV are accessible. Also cancer is obviously an important target for siRNA-based therapies, but the main problem in cancer therapy is targeting metastatic cells which spread from the original tumor. There are also other possible reservations and problems that might delay or even hinder siRNA-based therapies for the treatment of certain conditions; however, this remains the most promising approach for a wide range of diseases. Clearly, more studies must be done to allow efficient delivery and better understanding of unwanted side effects of siRNA-based therapies. In this review miRNA and RNAi biology, experimental design, anti-viral and anti-cancer effects are discussed.
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Affiliation(s)
- Hamid Reza Mollaie
- Department of Virology, Iran University of Medical Sciences, Tehran, Iran E-mail :
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Marimani M, Hean J, Bloom K, Ely A, Arbuthnot P. Recent advances in developing nucleic acid-based HBV therapy. Future Microbiol 2014; 8:1489-504. [PMID: 24199806 DOI: 10.2217/fmb.13.87] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
Chronic HBV infection remains an important public health problem and currently licensed therapies rarely prevent complications of viral persistence. Silencing HBV gene expression using gene therapy, particularly with exogenous activators of RNAi, holds promise for developing an HBV gene therapy. However, immune stimulation, off-targeting effects and inefficient delivery of RNAi activators remain problematic. Several new approaches have recently been employed to address these issues. Chemical modifications to anti-HBV synthetic siRNAs have been investigated and a variety of vectors are being developed for delivery of RNAi effectors. In this article, we review the potential utility of gene therapy for treating HBV infection.
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Affiliation(s)
- Musa Marimani
- Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
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Chen X, Qian Y, Yan F, Tu J, Yang X, Xing Y, Chen Z. 5'-triphosphate-siRNA activates RIG-I-dependent type I interferon production and enhances inhibition of hepatitis B virus replication in HepG2.2.15 cells. Eur J Pharmacol 2013; 721:86-95. [PMID: 24099962 DOI: 10.1016/j.ejphar.2013.09.050] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2013] [Revised: 09/06/2013] [Accepted: 09/22/2013] [Indexed: 12/25/2022]
Abstract
Hepatitis B virus (HBV) infection often results in acute or chronic viral hepatitis and other liver diseases including cirrhosis and hepatocellular carcinoma. Current therapies for HBV usually have severe side effects and can cause development of drug-resistant mutants. An alternative and safe immunotherapeutic approach for HBV infection is urgently needed for effective anti-HBV therapy. In this study, we propose a new strategy for anti-HBV therapy that activates type-I interferon (IFN) antiviral innate immunity through stimulating pattern-recognition receptors with RNA interference (RNAi) using a 5'-end triphosphate-modified small interfering RNA (3p-siRNA). We designed and generated a 3p-siRNA targeting overlapping region of S gene and P gene of the HBV genome at the 5'-end of pregenomic HBV RNA. Our results demonstrated that 3p-siRNA induced a RIG-I-dependent antiviral type-I IFN response when transfected into HepG2.2.15 cells that support HBV replication. The 3p-siRNA significantly inhibited HBsAg and HBeAg secretion from HepG2.2.15 cells in a RIG-I-dependent manner, and the antiviral effect of 3p-siRNA was superior to that of siRNA. Furthermore, 3p-siRNA had more pronounced inhibition effects on the replication of HBV DNA and the transcription of mRNA than that of siRNA. Finally, 3p-siRNA displayed antiviral activity with long-term suppression of HBV replication. In conclusion, our findings suggest that 3p-siRNA could act as a powerful bifunctional antiviral molecule with potential for developing a promising therapeutic against chronic HBV infection.
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Affiliation(s)
- Xiaojuan Chen
- Division of Infection and Immunity, Department of Electromagnetic and Laser Biology, Beijing Institute of Radiation Medicine, 27 Taiping Rd, Beijing 100850, China
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Meng Z, Zhang X, Wu J, Pei R, Xu Y, Yang D, Roggendorf M, Lu M. RNAi induces innate immunity through multiple cellular signaling pathways. PLoS One 2013; 8:e64708. [PMID: 23700487 PMCID: PMC3659100 DOI: 10.1371/journal.pone.0064708] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2012] [Accepted: 04/17/2013] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND AIMS Our previous results showed that the knockdown of woodchuck hepatitis virus (WHV) by RNA interference (RNAi) led to upregulation of interferon stimulated genes (ISGs) in primary hepatocytes. In the present study, we tested the hypothesis that the cellular signaling pathways recognizing RNA molecules may be involved the ISG stimulation by RNAi. METHODS Primary murine hepatocytes (PMHs) from wild type mice and WHV transgenic (Tg) mice were prepared and treated with defined siRNAs. The mRNA levels of target genes and ISGs were detected by real-time RT-PCR. The involvement of the signaling pathways including RIG-I/MDA5, PKR, and TLR3/7/8/9 was examined by specific inhibition and the analysis of their activation by Western blotting. RESULTS In PMHs from WHV Tg mice, specific siRNAs targeting WHV, mouse β-actin, and GAPDH reduced the levels of targeted mRNAs and increased the mRNA expression of IFN-β, MxA, and IP-10. The enhanced ISG expression by siRNA transfection were abolished by siRNA-specific 2'-O-methyl antisense RNA and the inhibitors 2-AP and chloroquine blocking PKR and other TLR-mediated signaling pathways. Furthermore, Western blotting revealed that RNAi results in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-êB, indicating the possible role of IRF3 in the RNAi-directed induction of ISGs. In contrast, silencing of RIG-I and MDA5 failed to block RNAi-mediated MxA induction. CONCLUSIONS RNAi is capable of enhancing innate immune responses through the PKR- and TLR-dependent signaling pathways in primary hepatocytes. The immune stimulation by RNAi may contribute to the antiviral activity of siRNAs in vivo.
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Affiliation(s)
- Zhongji Meng
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
- Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Shiyan, China
| | - Xiaoyong Zhang
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
| | - Jun Wu
- Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Rongjuan Pei
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
| | - Yang Xu
- Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Dongliang Yang
- Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Michael Roggendorf
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
| | - Mengji Lu
- Institute of Virology, University Hospital of Essen, University Duisburg-Essen, Essen, Germany
- * E-mail:
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Yang S, Chen Y, Ahmadie R, Ho EA. Advancements in the field of intravaginal siRNA delivery. J Control Release 2013; 167:29-39. [PMID: 23298612 DOI: 10.1016/j.jconrel.2012.12.023] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2012] [Revised: 12/14/2012] [Accepted: 12/17/2012] [Indexed: 12/17/2022]
Abstract
The vaginal tract is a suitable site for the administration of both local and systemic acting drugs. There are numerous vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. Despite the potential in drug delivery, the vagina is a complex and dynamic organ that requires greater understanding. The recent discovery that injections of double stranded RNA (dsRNA) in Caenorhabditis elegans (C. elegans) results in potent gene specific silencing, was a major scientific revolution. This phenomenon known as RNA interference (RNAi), is believed to protect host genome against invasion by mobile genetic elements such as transposons and viruses. Gene silencing or RNAi has opened new potential opportunities to study the function of a gene in an organism. Furthermore, its therapeutic potential is being investigated in the field of sexually transmitted infections such as human immunodeficiency virus (HIV) and other diseases such as age-related macular degeneration (AMD), diabetes, hypercholesterolemia, respiratory disease, and cancer. This review will focus on the therapeutic potential of siRNA for the treatment and/or prevention of infectious diseases such as HIV, HPV, and HSV within the vaginal tract. Specifically, formulation design parameters to improve siRNA stability and therapeutic efficacy in the vaginal tract will be discussed along with challenges, advancements, and future directions of the field.
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Affiliation(s)
- Sidi Yang
- Faculty of Pharmacy, University of Manitoba, 750 McDermot Ave, Winnipeg, Manitoba, Canada
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Hinton TM, Monaghan P, Green D, Kooijmans SA, Shi S, Breheney K, Tizard M, Nicolazzo JA, Zelikin AN, Wark K. Biodistribution of polymer hydrogel capsules for the delivery of therapeutics. Acta Biomater 2012; 8:3251-60. [PMID: 22659177 DOI: 10.1016/j.actbio.2012.05.028] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2011] [Revised: 05/18/2012] [Accepted: 05/23/2012] [Indexed: 10/28/2022]
Abstract
A key phase in the development of intelligently designed nanoparticle delivery vehicles for new therapeutic agents is to gain an understanding of their interaction with tissues and cells. We report a series of in vitro and in vivo experiments aimed at tracking a potential delivery vehicle for therapeutic agents, including vaccine peptides and drugs derived from poly(methacrylic acid) hydrogel capsules in certain organs and cell types. For the in vitro studies, two immortal liver-derived cell lines (Huh7 and Hepa1-6) and primary cultures of mouse hepatocytes were incubated with Alexa 647 labelled fluorescent capsules to track their internalization and intracellular distribution by confocal microscopy. Capsules, 500nm in diameter, were taken up into the cells in a time-dependent manner in all three cell lines. Capsules were observed in plasma membrane-derived vesicles within the cells. After 24h a significant proportion of the capsules was observed in lysosomes. To understand the behaviour of the capsules in vivo, Alexa 488 labelled fluorescent capsules were intravenously injected into Sprague-Dawley rats and after 24h the fate of the capsules in a number of organs was determined by flow cytometry and confocal microscopy. By flow cytometry, the majority of the capsules were detected in the spleen whilst similar numbers were found in the lung and liver. By confocal microscopy, the majority of the capsules were found in the liver and spleen with significantly less capsules in the lung, heart and kidney. Colocalization of capsules with cell-type specific markers indicated that in lung, heart and kidney, the majority of the capsules were located in endothelial cells. In the spleen ~50% of the capsules were found in CD163-positive cells, whereas in the liver, almost all capsules were located in CD163-positive cells, indicating uptake by Kupffer cells. Electron microscopy confirmed the presence of capsules within Kupffer cells.
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22
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Thakur N, Qureshi A, Kumar M. VIRsiRNAdb: a curated database of experimentally validated viral siRNA/shRNA. Nucleic Acids Res 2012; 40:D230-6. [PMID: 22139916 PMCID: PMC3245049 DOI: 10.1093/nar/gkr1147] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2011] [Revised: 10/04/2011] [Accepted: 11/09/2011] [Indexed: 12/22/2022] Open
Abstract
RNAi technology has been emerging as a potential modality to inhibit viruses during past decade. In literature a few siRNA databases have been reported that focus on targeting human and mammalian genes but experimentally validated viral siRNA databases are lacking. We have developed VIRsiRNAdb, a manually curated database having comprehensive details of 1358 siRNA/shRNA targeting viral genome regions. Further, wherever available, information regarding alternative efficacies of above 300 siRNAs derived from different assays has also been incorporated. Important fields included in the database are siRNA sequence, virus subtype, target genome region, cell type, target object, experimental assay, efficacy, off-target and siRNA matching with reference viral sequences. Database also provides the users with facilities of advance search, browsing, data submission, linking to external databases and useful siRNA analysis tools especially siTarAlign which align the siRNA with reference viral genomes or user defined sequences. VIRsiRNAdb contains extensive details of siRNA/shRNA targeting 42 important human viruses including influenza virus, hepatitis B virus, HPV and SARS Corona virus. VIRsiRNAdb would prove useful for researchers in picking up the best viral siRNA for antiviral therapeutics development and also for developing better viral siRNA design tools. The database is freely available at http://crdd.osdd.net/servers/virsirnadb.
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Affiliation(s)
| | | | - Manoj Kumar
- Bioinformatics Centre, Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh-160036, India
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23
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Han Q, Zhang C, Zhang J, Tian Z. Involvement of activation of PKR in HBx-siRNA-mediated innate immune effects on HBV inhibition. PLoS One 2011; 6:e27931. [PMID: 22174754 PMCID: PMC3234243 DOI: 10.1371/journal.pone.0027931] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2011] [Accepted: 10/28/2011] [Indexed: 12/25/2022] Open
Abstract
RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-hepatitis B virus (anti-HBV) therapy. Recent studies have revealed that siRNAs can induce an innate immune response in vitro and in vivo. Here, HBVx (HBx) mRNA expression and HBV replication were significantly inhibited, followed by the enhancement of expression of type I interferons (IFNs), IFN-stimulated genes (ISG15 and ISG56) and proinflammatory cytokines after HepG2.2.15 cells were transfected with chemically synthesized HBx-siRNAs. Transfection with HBx-siRNAs also significantly increased expression of dsRNA-dependent protein kinase R (PKR) in HepG2.2.15 cells, followed by activation of downstream signaling events such as eukaryotic initiation factor 2α (eIF2-α). In PKR-over-expressing HepG2.2.15 cells, HBx-siRNAs exerted more potent inhibitory effects on HBV replication and greater production of type I IFNs. By contrast, the inhibitory effect of HBx-siRNAs on HBV replication was attenuated when PKR was inhibited or silenced, demonstrating that HBx-siRNAs greatly promoted PKR activation, leading to the higher production of type I IFN. Therefore, we concluded that PKR is involved in the innate immune effects mediated by HBx-siRNAs and further contributes to HBV inhibition. The bifunctional siRNAs with both gene silencing and innate immune activation properties may represent a new potential strategy for treatment of HBV.
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Affiliation(s)
- Qiuju Han
- Institute of Immunopharmacology and Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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Arbuthnot P. MicroRNA-like antivirals. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2011; 1809:746-55. [PMID: 21616187 DOI: 10.1016/j.bbagrm.2011.05.006] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2011] [Revised: 04/28/2011] [Accepted: 05/09/2011] [Indexed: 12/26/2022]
Abstract
Employing engineered DNA templates to express antiviral microRNA (miRNA) sequences has considerable therapeutic potential. The durable silencing that may be achieved with these RNAi activators is valuable to counter chronic viral infections, such as those caused by HIV-1, hepatitis B, hepatitis C and dengue viruses. Early use of expressed antiviral miRNAs entailed generation of cassettes containing Pol III promoters (e.g. U6 and H1) that transcribe virus-targeting short hairpin RNA mimics of precursor miRNAs. Virus escape from single gene silencing elements prompted later development of combinatorial antiviral miRNA expression cassettes that form multitargeting siRNAs from transcribed long hairpin RNA and polycistronic primary miRNA sequences. Weaker Pol III and Pol II promoters have also been employed to control production of antiviral miRNA mimics, improve dose regulation and address concerns about toxicity caused by saturation of the endogenous miRNA pathway. Efficient delivery of expressed antiviral sequences remains challenging and utilizing viral vectors, which include recombinant adenoviruses, adeno-associated viruses and lentiviruses, has been favored. Investigations using recombinant lentiviruses to transduce CD34+ hematological precursor cells with expressed HIV-1 gene silencers are at advanced stages and show promise in preclinical and clinical trials. Although the use of expressed antiviral miRNA sequences to treat viral infections is encouraging, eventual therapeutic application will be dependent on rigorously proving their safety, efficient delivery to target tissues and uncomplicated large scale preparation of vector formulations. This article is part of a special issue entitled: MicroRNAs in viral gene regulation.
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Abstract
RNA interference (RNAi) is an ancient defensive mechanism in eukaryotes to control gene expressing and defend their genomes from foreign invaders. It refers to the phenomenon that double-stranded RNA results in the sequence-specific silencing of target gene expression. Although it was documented in a relatively short time ago, intensive research has facilitated making its mechanism clear. Researchers have found that it was a powerful tool for analyzing the functions of genes and treating tumors, infectious diseases and genetic abnormalities that are associated with a dominant gene defect. However, delivery in vivo, low blood stability and poor intracellular uptake present significant challenges for the development of RNAi reagents in clinical use. Thus, long-term inducible RNAi was designed. There are hundreds of millions of hepatitis B virus (HBV) carriers in the world at present, a portion of whom will lose their lives after several years due to chronic complications such as cirrhosis, hepatocellular carcinomas or both. Although a preventive vaccine is now available, the present therapeutic options for chronically infected patients are limited and of low efficiency. Admittedly, to date most RNAi experiments have been done in vitro, but it is hoped that they may be developed into a therapeutic strategy for HBV in the near future. In this article the principles and construction of long-term RNA are discussed. Its therapeutic potentiality and attention to the potential hazards will also outlined. We conclude that this ancient defensive mechanism can be recruited as a powerful weapon in the fight against HBV.
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Affiliation(s)
- Jin Shui Pan
- Department of Gastroenterology, Zhongshan Hospital Xiamen University, Xiamen, China
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Kim JW, Lee SH, Park YS, Jeong SH, Kim N, Lee DH. [Inhibition of in vitro hepatitis B virus replication by lentivirus-mediated short-hairpin RNA against HBx]. THE KOREAN JOURNAL OF HEPATOLOGY 2009; 15:15-24. [PMID: 19346782 DOI: 10.3350/kjhep.2009.15.1.15] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUNDS/AIMS Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. METHODS Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. RESULTS Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). CONCLUSIONS Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.
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Affiliation(s)
- Jin-Wook Kim
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Seoul National University Bungdang Hospital, Seongnam, Korea.
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Park YG. [Inhibition of hepatitis B virus replication by RNA interference]. THE KOREAN JOURNAL OF HEPATOLOGY 2009; 15:1-6. [PMID: 19346780 DOI: 10.3350/kjhep.2009.15.1.1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
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Comparative study of anti-hepatitis B virus RNA interference by double-stranded adeno-associated virus serotypes 7, 8, and 9. Mol Ther 2008; 17:352-9. [PMID: 19066602 DOI: 10.1038/mt.2008.245] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.Molecular Therapy (2009) 17 2, 352-359 doi:10.1038/mt.2008.245.
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Sakamoto N, Tanabe Y, Yokota T, Satoh K, Sekine-Osajima Y, Nakagawa M, Itsui Y, Tasaka M, Sakurai Y, Cheng-Hsin C, Yano M, Ohkoshi S, Aoyagi Y, Maekawa S, Enomoto N, Kohara M, Watanabe M. Inhibition of hepatitis C virus infection and expression in vitro and in vivo by recombinant adenovirus expressing short hairpin RNA. J Gastroenterol Hepatol 2008; 23:1437-1447. [PMID: 17683479 PMCID: PMC7166320 DOI: 10.1111/j.1440-1746.2007.05076.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 04/12/2007] [Indexed: 12/15/2022]
Abstract
BACKGROUND AND AIM We have reported previously that synthetic small interfering RNA (siRNA) and DNA-based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. In this study, we investigated the effects of the siRNA targeting HCV-RNA in vivo. METHODS We constructed recombinant retrovirus and adenovirus expressing short hairpin RNA (shRNA), and transfected into replicon-expressing cells in vitro and transgenic mice in vivo. RESULTS Retroviral transduction of Huh7 cells to express shRNA and subsequent transfection of an HCV replicon into the cells showed that the cells had acquired resistance to HCV replication. Infection of cells expressing the HCV replicon with an adenovirus expressing shRNA resulted in efficient vector delivery and expression of shRNA, leading to suppression of the replicon in the cells by approximately 10(-3). Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/loxP switching system resulted in specific suppression of virus protein synthesis in the liver. CONCLUSION Taken together, our results support the feasibility of utilizing gene targeting therapy based on siRNA and/or shRNA expression to counteract HCV replication, which might prove valuable in the treatment of hepatitis C.
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Affiliation(s)
- Naoya Sakamoto
- Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.
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Inhibition of hepatitis B virus gene expression and replication by endoribonuclease-prepared siRNA. J Virol Methods 2008; 150:27-33. [PMID: 18378325 PMCID: PMC7112819 DOI: 10.1016/j.jviromet.2008.02.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2007] [Revised: 02/11/2008] [Accepted: 02/13/2008] [Indexed: 02/07/2023]
Abstract
Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.
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32
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Arbuthnot P, Thompson LJ. Harnessing the RNA interference pathway to advance treatment and prevention of hepatocellular carcinoma. World J Gastroenterol 2008; 14:1670-81. [PMID: 18350598 PMCID: PMC2695907 DOI: 10.3748/wjg.14.1670] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2007] [Revised: 01/27/2008] [Indexed: 02/06/2023] Open
Abstract
Primary liver cancer is the fifth most common malignancy in the world and is a leading cause of cancer-related mortality. Available treatment for hepatocellular carcinoma (HCC), the commonest primary liver cancer, is rarely curative and there is a need to develop therapy that is more effective. Specific and powerful gene silencing that can be achieved by activating RNA interference (RNAi) has generated enthusiasm for exploiting this pathway for HCC therapy. Many studies have been carried out with the aim of silencing HCC-related cellular oncogenes or the hepatocarcinogenic hepatitis B virus (HBV) and hepatitis C virus (HCV). Proof of principle studies have demonstrated promising results, and an early clinical trial assessing RNAi-based HBV therapy is currently in progress. Although the data augur well, there are several significant hurdles that need to be overcome before the goal of RNAi-based therapy for HCC is realized. Particularly important are the efficient and safe delivery of RNAi effecters to target malignant tissue and the limitation of unintended harmful non-specific effects.
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Abstract
Molecular analyses have become an integral part of biomedical research as well as clinical medicine. The definition of the molecular and genetic basis of many human diseases has led to a better understanding of their pathogenesis and has in addition offered new perspectives for their diagnosis, therapy and prevention. Genetically, liver diseases can be classified as hereditary monogenic, acquired monogenic, complex genetic and diseases. Based on this classification, gene therapy is based on six concepts: gene repair, gene substitution, cell therapy, block of gene expression or function, DNA vaccination as well as gene augmentation. While recent developments are promising, various delivery, targeting and safety issues need to be addressed before gene therapy will enter clinical practice. In the future, molecular diagnosis and therapy liver diseases will be part of our patient management and complement existing diagnostic, therapeutic and preventive strategies.
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Affiliation(s)
- H E Blum
- Department of Medicine II, University Hospital, D-79106 Freiburg, Germany.
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34
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Chen Y, Mahato RI. siRNA pool targeting different sites of human hepatitis B surface antigen efficiently inhibits HBV infection. J Drug Target 2008; 16:140-8. [PMID: 18274934 PMCID: PMC2778861 DOI: 10.1080/10611860701878750] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The main objective was to determine whether a pool of small interfering RNAs (siRNAs) targeting different regions of hepatitis B virus surface antigen (HBsAg) efficiently inhibits hepatitis B virus (HBV) infection. siRNAs targeting different regions of HBsAg were transfected into HBV-producing HepG2.2.15 cells and at 72 h post-transfection, the culture medium was collected for ELISA to determine HBsAg, while total RNA was isolated from the cells for real-time PCR. Three siRNA sequences that efficiently inhibited HBV infection were converted into small hairpin RNAs (shRNAs) and then cloned into a single plasmid psiSTRIKE driven by a single U6 promoter. These shRNA expressing plasmids were tested for HBsAg gene silencing in HepG2.2.15 cells. A pool of siRNAs targeting HBsAg efficiently inhibited HBV replication and antigen expression when transfected into HepG2.2.15 cells, compared with the use of single siRNA. Similarly, the plasmid encoding three different shRNAs driven by a single U6 promoter was more effective in silencing HBsAg at DNA, mRNA and protein levels compared with the plasmid encoding single shRNA. No apoptotic change was observed in the cells when the plasmid was transfected at a dose of 0.5-2 microg/1 x 10(6) cells after complex formation with Lipofectamine LTX. Furthermore, transfection with siRNA or shRNA did not increase interferon-gamma (IFNs-gamma) release, suggesting no induction of IFN response. In conclusion, a pool of chemically synthesised siRNAs as well as the shRNA expression plasmid encoding multiple shRNAs targeting different regions of HBsAg showed high gene silencing in HepG2.2.15 cells.
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Affiliation(s)
- Yong Chen
- Huaian 4th People's Hospital, Jiangsu, China
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN TN38103-3308
| | - Ram I Mahato
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN TN38103-3308
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35
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Chen Y, Cheng G, Mahato RI. RNAi for treating hepatitis B viral infection. Pharm Res 2007; 25:72-86. [PMID: 18074201 PMCID: PMC2217617 DOI: 10.1007/s11095-007-9504-0] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2007] [Accepted: 11/14/2007] [Indexed: 12/18/2022]
Abstract
Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection.
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Affiliation(s)
- Yong Chen
- Huai-An 4th People’s Hospital, Jiangsu, China
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
| | - Guofeng Cheng
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
| | - Ram I. Mahato
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
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36
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Pardo M, Bartolomé J, Carreño V. Current therapy of chronic hepatitis B. Arch Med Res 2007; 38:661-77. [PMID: 17613358 DOI: 10.1016/j.arcmed.2006.12.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2006] [Accepted: 12/04/2006] [Indexed: 12/24/2022]
Affiliation(s)
- Margarita Pardo
- Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain
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37
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Chou YC, Chen ML, Hu CP, Chen YL, Chong CL, Tsai YL, Liu TL, Jeng KS, Chang C. Transforming growth factor-beta1 suppresses hepatitis B virus replication primarily through transcriptional inhibition of pregenomic RNA. Hepatology 2007; 46:672-81. [PMID: 17580335 DOI: 10.1002/hep.21726] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
UNLABELLED Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with pivotal roles in the regulation of cellular functions and immune responses. In this study, we found that TGF-beta1 was able to effectively suppress hepatitis B virus (HBV) replication. In the presence of TGF-beta1, the level of viral replicative intermediates was dramatically decreased, both in actively dividing cells and in confluent cells. At the same time, the levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF-beta1 treatment. Interestingly, the inhibitory activity of TGF-beta1 was associated with preferential reduction of the level of pregenomic RNA compared with pre-C mRNA. Further analysis indicated that TGF-beta1 might exert its antiviral effect primarily through reducing expression of the HBV core protein by transcriptional regulation instead of posttranscriptional modification. CONCLUSION TGF-beta1 may play a dual role in HBV infection, in the suppression of immune responses against viral infection and in the direct inhibition of viral replication, resulting in minimization of liver damage in patients with chronic hepatitis.
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Affiliation(s)
- Yu-Chi Chou
- Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
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38
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Arbuthnot P, Longshaw V, Naidoo T, Weinberg MS. Opportunities for treating chronic hepatitis B and C virus infection using RNA interference. J Viral Hepat 2007; 14:447-59. [PMID: 17576386 DOI: 10.1111/j.1365-2893.2006.00818.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Activating the RNA interference (RNAi) pathway to achieve silencing of specific genes is one of the most exciting new developments of molecular biology. A particularly interesting use of this technology is inhibition of defined viral gene expression. In this review, we discuss the potential application of RNAi to treatment of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Globally, these hepatotropic viruses are the most important causes of cirrhosis and liver cancer. Available treatments have their limitations, which makes development of novel effective RNAi-based therapies for HBV and HCV especially significant. Several investigations carried out in vitro and in vivo are summarized, which demonstrate proof of principle that HBV and HCV can be inhibited by RNAi activators. Challenges facing further development of this technology to a stage of clinical application are discussed.
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Affiliation(s)
- P Arbuthnot
- Hepatitis B Virus Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.
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39
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Wen WH, Liu JY, Qin WJ, Zhao J, Wang T, Jia LT, Meng YL, Gao H, Xue CF, Jin BQ, Yao LB, Chen SY, Yang AG. Targeted inhibition of HBV gene expression by single-chain antibody mediated small interfering RNA delivery. Hepatology 2007; 46:84-94. [PMID: 17596868 DOI: 10.1002/hep.21663] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Abstract
UNLABELLED RNA interference is highly effective at inhibiting HBV gene expression and replication. However, before small interfering RNA (siRNA) can be used in the clinic, it is essential to develop a system to target their delivery. Antibody-mediated delivery is a novel approach for targeting siRNA to appropriate cells. In this report, we asked whether this siRNA delivery strategy would be effective against HBV. Of 5 candidates, a specific siRNA that effectively inhibited HBV gene expression and replication was determined. Two fusion proteins, s-tP and sCkappa-tP, were constructed to contain a single chain of the human variable fragment, scFv, against hepatitis B surface antigen (HBsAg), a truncated protamine (tP), and in the case of sCkappa-tP, a constant region of the kappa chain (Ckappa). S-tP and sCkappa-tP were developed to provide targeted delivery of the siRNA, siRNA expressing cassettes (SEC), and siRNA-producing plasmids. Fluorescein isothiocyanate-siRNA, fluorescein isothiocyanate-SEC, and plasmid DNA were specifically delivered into HBsAg-positive cells using the sCkappa-tP fusion protein, and effectively inhibited HBV gene expression and replication. HBV gene expression was also inhibited by siRNA or siRNA-producing plasmids in HBV transgenic mice. CONCLUSION Our results describe a potential method for the targeted delivery of siRNA or siRNA-producing plasmids against HBV, using anti-HBsAg fusion proteins.
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Affiliation(s)
- Wei-Hong Wen
- State Key Laboratory of Cancer Biology, Department of Immunology, Xijing Hospital, Fourth Military Medical University, Xi'an, China
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40
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Kayhan H, Karatayli E, Turkyilmaz AR, Sahin F, Yurdaydin C, Bozdayi AM. Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2 2.2.15 cell lines. Arch Virol 2007; 152:871-9. [PMID: 17245534 DOI: 10.1007/s00705-006-0918-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2006] [Accepted: 12/06/2006] [Indexed: 12/18/2022]
Abstract
In this study, the effect of RNAi on HBV replication was observed in a cell culture model, HepG2 2.2.15 cell line, which supports human HBV ayw replication and expression. Aim of the study was to investigate effects of shRNAs (small hairpin RNAs) targeting hepatitis B virus mRNAs on the viral replication in HepG2 2.2.15 cells. We selected three target HBV mRNA regions with different putative secondary structures to test whether the secondary structure of RNA may affect the inhibition efficacy on the target HBV RNA. Three HBV-specific siRNAs (small interfering RNA) were designed targeting X (1689-1708), Core (2229-2248) and S (765-784 nt) transcripts. HepG2 2.2.15 cells were transfected with shRNA expressing plasmids, P765, P2229 and P1689 targeting S, core and X region, respectively or a mock plasmid targeting lacZ gene. The culture media was collected throughout six days after transfection and analyzed by real-time PCR. Viral DNA production was suppressed for 7 days. The HBV DNA levels were decreased by 73, 72 and 79% with P765, P2229 and P1689 vectors, respectively. In conclusion, the shRNAs designed for X, core and S regions, specifically and significantly suppressed HBV DNA. siRNAs potentially may be used in treatment of hepatitis B.
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Affiliation(s)
- H Kayhan
- Institute of Hepatology, Ankara University, Ankara, Turkey
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41
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Konishi M, Wu CH, Kaito M, Hayashi K, Watanabe S, Adachi Y, Wu GY. siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations. J Viral Hepat 2006; 13:756-61. [PMID: 17052275 DOI: 10.1111/j.1365-2893.2006.00752.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.
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Affiliation(s)
- Masayoshi Konishi
- Division of Gastroenterology & Hepatology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA
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42
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Jia F, Zhang YZ, Liu CM. Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery. J Biotechnol 2006; 128:32-40. [PMID: 17049658 DOI: 10.1016/j.jbiotec.2006.09.007] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2006] [Revised: 08/25/2006] [Accepted: 09/14/2006] [Indexed: 12/11/2022]
Abstract
RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.
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Affiliation(s)
- Fang Jia
- Molecular Virology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
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43
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Abstract
Silencing gene expression through a process known as RNA interference (RNAi) has been known in the plant world for many years. In recent years, knowledge of the prevalence of RNAi and the mechanism of gene silencing through RNAi has started to unfold. It is now believed that RNAi serves in part as an innate response against invading viral pathogens and, indeed, counter silencing mechanisms aimed at neutralizing RNAi have been found in various viral pathogens. During the past few years, it has been demonstrated that RNAi, induced by specifically designed double‐stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Furthermore, it is now apparent that in in vitro and in some in vivo models, the prospects for this technology in developing therapeutic applications are robust. However, many key questions and obstacles in the translation of RNAi into a potential therapeutic platform still remain, including the specificity and longevity of the silencing effect, and, most importantly, the delivery of the dsRNA that induces the system. It is expected that for the specific examples in which the delivery issue could be circumvented or resolved, RNAi may hold promise for the development of gene‐specific therapeutics. Copyright © 2006 John Wiley & Sons, Ltd.
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Affiliation(s)
- Mali Ketzinel‐Gilad
- Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel
| | - Yosef Shaul
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Eithan Galun
- Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel
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44
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Kim YH, Lee JH, Paik NW, Rho HM. RNAi-Based Knockdown of HBx mRNA in HBx-Transformed and HBV-Producing Human Liver Cells. DNA Cell Biol 2006; 25:412-7. [PMID: 16848683 DOI: 10.1089/dna.2006.25.412] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
RNA interference (RNAi) is the process of sequence-specific gene silencing induced by 21-23-nt RNA of small interfering RNA (siRNA). The HBx of hepatitis B virus (HBV) causing human liver diseases has been known as a multifunctional protein which affects transcription, cell growth, and apoptotic cell death. Here, we demonstrate that the HBx-specific siRNA (siRNAx) and short hairpin RNA (shRNAx) effectively induce the degradation of HBx mRNA in HBx-transformed and HBV-producing human liver cells by up to 80-90%. Also, the HBx expression in HBx-transformed cells was continuously silenced by retransformation with the shRNAx expression vector. These results imply that HBx-driven RNAi, either delivery of siRNAx or expression of shRNAx, provides a promising anti-HBV approach to suppress the HBx expression in human hepatoma cells.
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Affiliation(s)
- Young Ha Kim
- Indang Institute of Molecular Biology, Inje University, Seoul, Korea
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45
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Abstract
RNA interference (RNAi) is a sequence-specific gene-silencing mechanism that has been proposed to function as a defence mechanism of eukaryotic cells against viruses and transposons. RNAi was first observed in plants in the form of a mysterious immune response to viral pathogens. But RNAi is more than just a response to exogenous genetic material. Small RNAs termed microRNA (miRNA) regulate cellular gene expression programs to control diverse steps in cell development and physiology. The discovery that exogenously delivered short interfering RNA (siRNA) can trigger RNAi in mammalian cells has made it into a powerful technique for generating genetic knock-outs. It also raises the possibility to use RNAi technology as a therapeutic tool against pathogenic viruses. Indeed, inhibition of virus replication has been reported for several human pathogens including human immunodeficiency virus, the hepatitis B and C viruses and influenza virus. We reviewed the field of antiviral RNAi research in 2003 (Haasnoot et al. 2003), but many new studies have recently been published. In this review, we present a complete listing of all antiviral strategies published up to and including December 2004. The latest developments in the RNAi field and their antiviral application are described.
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Affiliation(s)
- Volker Erdmann
- Institute of Chemistry/Biochemistry, Free University Berlin, Thielallee 63, 14195 Berlin, Germany
| | - Jan Barciszewski
- Institute of Bioorganic Chemistry, Polish Academy of Scienes, Noskowskiego 12/14, 61-704 Poznan, Poland
| | - Jürgen Brosius
- Institute of Experimental Pathology, Molecular Neurobiology (ZMBE), University of Münster, Von-Esmarch-Str. 56, 48149 Münster, Germany
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46
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Ren X, Luo G, Xie Z, Zhou L, Kong X, Xu A. Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells. J Hepatol 2006; 44:663-70. [PMID: 16466826 DOI: 10.1016/j.jhep.2005.10.029] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/21/2005] [Revised: 10/14/2005] [Accepted: 10/21/2005] [Indexed: 12/22/2022]
Abstract
BACKGROUND/AIMS Hepatitis B virus (HBV) infection is a world-wide health problem. Recent studies have demonstrated the efficacy of RNA interference (RNAi) against HBV replication at cell culture and animal levels using transient transfection. The present study was to determine whether the stable transfection of short hairpin RNA (shRNA)-producing vector could achieve potent and sustained inhibition of the HBV replication in 2.2.15 cells. METHODS shRNA-producing vector against HBV and the empty vector were stably transfected into the 2.2.15 cells respectively. A series of experiments were performed in the producing stable lines to determine the changes of viral protein expression and replication. RESULTS The HBV protein expression and viral replication were suppressed dramatically and stably by the integrated shRNA-producing vectors. Most importantly, this suppression effect persists after 30 passages. CONCLUSIONS Our data provided the possibility of continuous and stable inhibition of HBV protein expression and replication in patients using RNAi, suggesting a potential clinical application of this novel approach. Furthermore, the established stable transfected cell lines provided a good platform for understanding the mechanism of anti-HBV by RNAi.
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Affiliation(s)
- Xiangrong Ren
- State Key Laboratory of Biocontrol and Department of Biochemistry, College of Life Sciences, Sun Yat-sen Zhongshan University, Guangzhou 510275, People's Republic of China [corrected]
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47
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Guo Y, Guo H, Zhang L, Xie H, Zhao X, Wang F, Li Z, Wang Y, Ma S, Tao J, Wang W, Zhou Y, Yang W, Cheng J. Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. J Virol 2006; 79:14392-403. [PMID: 16254373 PMCID: PMC1280207 DOI: 10.1128/jvi.79.22.14392-14403.2005] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.
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Affiliation(s)
- Yong Guo
- Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
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48
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Ren JL, Pan JS, Cheng T, Dong J, Lu YP, Huang SJ, Shi HX, Wang L, Lian YM. RNA interference inhibits hepatitis B virus gene expression and replication in HepG2-N10 cells. CHINESE JOURNAL OF DIGESTIVE DISEASES 2006; 7:230-236. [PMID: 17054586 DOI: 10.1111/j.1443-9573.2006.00268.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
OBJECTIVE RNA interference (RNAi) refers to the phenomenon of sequence-specific degradation of homologous mRNA induced by double-stranded RNA. It has been successfully utilized to down-regulate endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, the effect of vector-based small interfering RNA (siRNA) promoted by pSilencer2.0-U6 inhibit hepatitis B virus (HBV) replication in cell culture was evaluated. METHODS Three fragments of short nucleic acids, respectively, targeting on S, X and C region of HBV genome were inserted into pSilencer vectors after they were annealed with their partly antisense strands. The recombination plasmids were pS, pX and pC. These expression plasmids were transfected into HepG2-N10 cells, a cell line which stably expresses hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg) and adw2 subtype Dane particles. The effect of RNAi was evaluated from the changes of DNA, RNA and protein levels. Viral antigens were measured by ELISA. Viral mRNA was analyzed by RT-PCR. The covalent closed circular DNA and genome DNA of HBV secreted into the culture media were measured by quantitative real-time PCR. Analysis of variance was performed for the results. RESULTS Vector-based RNA interference could potently reduce HBsAg (pS vs pN: 47%, pX vs pN: 30%, and pC vs pN: 25%, P < 0.001) and HBeAg (pX vs pN: 57% and pC vs pN: 66%, P < 0.001) expression in cell culture. Furthermore, RT-PCR analysis showed that viral mRNAs were effectively degraded, thus eliminating the messengers for protein expression as well as templates for reverse transcription (pS and pC vs pN, P < 0.001; pX vs pN, P = 0.003). Quantitative real-time PCR analysis of HBV DNA revealed that vector-based RNA interference can inhibit HBV replication efficiently (pS, pX and pC vs pN, P < 0.001). CONCLUSIONS Our results indicate that RNAi can inhibit HBV gene expression and replication, and it might have the potential to revolutionize the treatment of HBV.
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Affiliation(s)
- Jian Lin Ren
- Department of Gastroenterology, Zhongshan Hospital, Xiamen University, Xiamen, Fujian Province, China.
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Warmann SW, Armeanu S, Frank H, Buck H, Graepler F, Lemken ML, Heitmann H, Seitz G, Lauer UM, Bitzer M, Fuchs J. In vitro gene targeting in human hepatoblastoma. Pediatr Surg Int 2006; 22:16-23. [PMID: 16374644 DOI: 10.1007/s00383-005-1573-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
Poor treatment results in advanced hepatoblastoma (HB) made alternative treatment approaches desirable. Gene-directed tumor therapy is increasingly investigated in different malignancies. The aim of this study was to analyze possible alternatives of gene transfer into HB cells and to study therapeutic applications based on different strategies. Liposomal transfection of HB cells was assessed using liver-specific promoters, and adenovirus and Sendai virus transductions were performed in vitro. Transfer efficiencies were measured via flow cytometry determining expression of vector-encoded marker gene green fluorescent protein. Gene silencing of the anti-apoptotic bcl-2 gene in HUH6 cells was performed using lipofection of small interfering RNA (siRNA). Additionally, suicide gene therapy was carried out through a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer, leading to a potent intracellular prodrug transformation of 5-fluorocytosine into 5-fluorouracil. Treatment efficiencies were monitored via MTT viability assay. Highest gene transfer rates (86%) were observed using adenovirus transduction. We furthermore observed a significant therapeutic effect of adenovirus-mediated YCD::YUPRT suicide gene transfer. Liposomal-mediated anti-bcl-2 siRNA transfer led to a significant improvement of cisplatin treatment in HUH6 cells. Liver-specific promoters were found to be strongly active in HUH6 cells (mixed HB-derived), but less active in HepT1 cells (embryonal HB-derived). Liposomal transfection and viral transduction are effective approaches to genetically manipulate HB cells in vitro. For the first time, we demonstrate a positive effect of siRNA gene silencing in this malignancy. Additionally, we successfully investigated a model of adenovirus-based suicide gene therapy in HB cell cultures. Our data strongly encourage further studies assessing these alternative treatment approaches.
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Affiliation(s)
- Steven W Warmann
- Department of Pediatric Surgery, University of Tübingen, Hoppe-Seyler-Street 3, 72076 Tübingen, Germany.
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Chen Z, Xu ZF, Ye JJ, Yao HP, Zheng S, Ding JY. Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression. J Zhejiang Univ Sci B 2005; 6:236-41. [PMID: 15754419 PMCID: PMC1389730 DOI: 10.1631/jzus.2005.b0236] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
OBJECTIVES To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. METHODS (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. RESULTS Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. CONCLUSION Our results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.
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Affiliation(s)
- Zhe Chen
- Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
| | - Ze-feng Xu
- Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
| | - Jing-jia Ye
- Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
| | - Hang-ping Yao
- Immunology Institute, School of Medicine, Zhejiang University, Hangzhou 310031, China
| | - Shu Zheng
- Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
- †E-mail:;
| | - Jia-yi Ding
- Cancer Institute, Second affiliated hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China
- †E-mail:;
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