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1
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Shrilall C, Arbuthnot P, Ely A. In Vitro Transcribed Artificial Primary MicroRNA for the Inhibition of Hepatitis B Virus Gene Expression in Cultured Cells. Microorganisms 2025; 13:604. [PMID: 40142497 PMCID: PMC11946339 DOI: 10.3390/microorganisms13030604] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2024] [Revised: 02/07/2025] [Accepted: 02/26/2025] [Indexed: 03/28/2025] Open
Abstract
Available interventions for the management of chronic hepatitis B (hepB) exhibit limited efficacy and barriers to vaccination against the hepatitis B virus (HBV) have hampered prophylaxis programmes. Development of potent therapeutics capable of functional cure of chronic hepB thus remains a relevant medical objective. RNA interference (RNAi) can be exploited to effect potent and specific silencing of target genes through the introduction of RNA sequences that mimic the natural activators of the pathway. To achieve a therapeutic effect, artificial primary microRNAs (pri-miRNAs) have been used extensively to target various viruses, including HBV. To date artificial pri-miRNAs have exclusively been produced from DNA expression cassettes. Although this achieves impressive silencing, eventual translation of this platform to the clinic is complicated by the requirement for viral vectors to deliver DNA. Consequently, clinical translation has been slow. Recently, the use of in vitro transcribed RNA, specifically to produce mRNA vaccines at industrial scale, has gained significant interest. We therefore sought to evaluate the feasibility of using in vitro transcribed artificial pri-miRNAs for the inhibition of HBV gene expression. Artificial HBV-targeting pri-miR-31 sequences, which are highly effective when expressed in cells from a DNA template, demonstrated modest silencing of viral replication when incorporated into mRNA that was transcribed in vitro. Off-target effects were also observed. Characterisation revealed that intracellular processing of the artificial pri-miRNAs was inefficient and non-specific effects were caused by stimulation of the interferon response. Nevertheless, optimised nuclear delivery of the artificial pri-miRNAs should improve their processing and achieve better anti-hepB efficacy.
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Affiliation(s)
| | | | - Abdullah Ely
- Wits/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2050, South Africa; (C.S.); (P.A.)
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2
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Thangamani L, Balasubramanian B, Easwaran M, Natarajan J, Pushparaj K, Meyyazhagan A, Piramanayagam S. GalNAc-siRNA conjugates: Prospective tools on the frontier of anti-viral therapeutics. Pharmacol Res 2021; 173:105864. [PMID: 34474100 PMCID: PMC8405237 DOI: 10.1016/j.phrs.2021.105864] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 08/27/2021] [Accepted: 08/27/2021] [Indexed: 12/19/2022]
Abstract
The growing use of short-interfering RNA (siRNA)-based therapeutics for viral diseases reflects the most recent innovations in anti-viral vaccines and drugs. These drugs play crucial roles in the fight against many hitherto incurable diseases, the causes, pathophysiologies, and molecular processes of which remain unknown. Targeted liver drug delivery systems are in clinical trials. The receptor-mediated endocytosis approach involving the abundant asialoglycoprotein receptors (ASGPRs) on the surfaces of liver cells show great promise. We here review N-acetylgalactosamine (GalNAc)-siRNA conjugates that treat viral diseases such as hepatitis B infection, but we also mention that novel, native conjugate-based, targeted siRNA anti-viral drugs may also cure several life-threatening diseases such as hemorrhagic cystitis, multifocal leukoencephalopathy, and severe acute respiratory syndrome caused by coronaviruses and human herpes virus.
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Affiliation(s)
- Lokesh Thangamani
- Computational Biology Lab, Department of Bioinformatics, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
| | | | - Murugesh Easwaran
- International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India
| | - Jeyakumar Natarajan
- Data Mining and Text Mining Laboratory, Department of Bioinformatics, Bharathiar University, Coimbatore 641046, Tamil Nadu, India
| | - Karthika Pushparaj
- Department of Zoology, School of Biosciences, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641043, Tamil Nadu, India
| | - Arun Meyyazhagan
- Department of Life Sciences, CHRIST (Deemed to be University), Bengaluru 560029, Karnataka, India.
| | - Shanmughavel Piramanayagam
- Computational Biology Lab, Department of Bioinformatics, Bharathiar University, Coimbatore 641046, Tamil Nadu, India.
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3
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Hu W, Zheng H, Li Q, Wang Y, Liu X, Hu X, Liu W, Liu S, Chen Z, Feng W, Cai X, Li N. shRNA transgenic swine display resistance to infection with the foot-and-mouth disease virus. Sci Rep 2021; 11:16377. [PMID: 34385528 PMCID: PMC8361160 DOI: 10.1038/s41598-021-95853-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 07/29/2021] [Indexed: 12/15/2022] Open
Abstract
Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3+, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.
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Affiliation(s)
- Wenping Hu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.,Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Haixue Zheng
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
| | - Qiuyan Li
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.,Beijing Genprotein Biotechnology Company, Beijing, China
| | - Yuhang Wang
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Xiangtao Liu
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
| | - Xiaoxiang Hu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Wenjie Liu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Shen Liu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Zhisheng Chen
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Wenhai Feng
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Xuepeng Cai
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
| | - Ning Li
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.
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Rohani N, Ahmadi Moughari F, Eslahchi C. DisCoVering potential candidates of RNAi-based therapy for COVID-19 using computational methods. PeerJ 2021; 9:e10505. [PMID: 33680575 PMCID: PMC7919535 DOI: 10.7717/peerj.10505] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2020] [Accepted: 11/15/2020] [Indexed: 01/04/2023] Open
Abstract
The ongoing pandemic of a novel coronavirus (SARS-CoV-2) leads to international concern; thus, emergency interventions need to be taken. Due to the time-consuming experimental methods for proposing useful treatments, computational approaches facilitate investigating thousands of alternatives simultaneously and narrow down the cases for experimental validation. Herein, we conducted four independent analyses for RNA interference (RNAi)-based therapy with computational and bioinformatic methods. The aim is to target the evolutionarily conserved regions in the SARS-CoV-2 genome in order to down-regulate or silence its RNA. miRNAs are denoted to play an important role in the resistance of some species to viral infections. A comprehensive analysis of the miRNAs available in the body of humans, as well as the miRNAs in bats and many other species, were done to find efficient candidates with low side effects in the human body. Moreover, the evolutionarily conserved regions in the SARS-CoV-2 genome were considered for designing novel significant siRNA that are target-specific. A small set of miRNAs and five siRNAs were suggested as the possible efficient candidates with a high affinity to the SARS-CoV-2 genome and low side effects. The suggested candidates are promising therapeutics for the experimental evaluations and may speed up the procedure of treatment design. Materials and implementations are available at: https://github.com/nrohani/SARS-CoV-2.
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Affiliation(s)
- Narjes Rohani
- Department of Computer and Data Sciences, Faculty of Mathematical Sciences, Shahid Beheshti University, Tehran, Iran
- School of Biological Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran
| | - Fatemeh Ahmadi Moughari
- Department of Computer and Data Sciences, Faculty of Mathematical Sciences, Shahid Beheshti University, Tehran, Iran
| | - Changiz Eslahchi
- Department of Computer and Data Sciences, Faculty of Mathematical Sciences, Shahid Beheshti University, Tehran, Iran
- School of Biological Sciences, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran
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Földes F, Madai M, Papp H, Kemenesi G, Zana B, Geiger L, Gombos K, Somogyi B, Bock-Marquette I, Jakab F. Small Interfering RNAs Are Highly Effective Inhibitors of Crimean-Congo Hemorrhagic Fever Virus Replication In Vitro. Molecules 2020; 25:molecules25235771. [PMID: 33297527 PMCID: PMC7731286 DOI: 10.3390/molecules25235771] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 11/26/2020] [Accepted: 12/04/2020] [Indexed: 11/24/2022] Open
Abstract
Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the prioritized diseases of the World Health Organization, considering its potential to create a public health emergency and, more importantly, the absence of efficacious drugs and/or vaccines for treatment. The highly pathogenic characteristic of CCHFV restricts research to BSL-4 laboratories, which complicates effective research and developmental strategies. In consideration of antiviral therapies, RNA interference can be used to suppress viral replication by targeting viral genes. RNA interference uses small interfering RNAs (siRNAs) to silence genes. The aim of our study was to design and test siRNAs in vitro that inhibit CCHFV replication and can serve as a basis for further antiviral therapies. A549 cells were infected with CCHFV after transfection with the siRNAs. Following 72 h, nucleic acid from the supernatant was extracted for RT Droplet Digital PCR analysis. Among the investigated siRNAs we identified effective candidates against all three segments of the CCHF genome. Consequently, blocking any segment of CCHFV leads to changes in the virus copy number that indicates an antiviral effect of the siRNAs. In summary, we demonstrated the ability of specific siRNAs to inhibit CCHFV replication in vitro. This promising result can be integrated into future anti-CCHFV therapy developments.
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Affiliation(s)
- Fanni Földes
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Mónika Madai
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Henrietta Papp
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Gábor Kemenesi
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Brigitta Zana
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
| | - Lili Geiger
- Department of Laboratory Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary; (L.G.); (K.G.)
| | - Katalin Gombos
- Department of Laboratory Medicine, Medical School, University of Pécs, 7624 Pécs, Hungary; (L.G.); (K.G.)
| | - Balázs Somogyi
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
| | - Ildikó Bock-Marquette
- Regenerative Science, Sport and Medicina Research Group, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary;
| | - Ferenc Jakab
- National Laboratory of Virology, BSL-4 Laboratory, Szentágothai Research Centre, University of Pécs, 7624 Pécs, Hungary; (F.F.); (M.M.); (H.P.); (G.K.); (B.Z.); (B.S.)
- Institute of Biology, Faculty of Sciences, University of Pécs, 7622 Pécs, Hungary
- Correspondence: ; Tel.: +36-72-501-668 (ext. 29044)
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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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7
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González C, Tabernero D, Cortese MF, Gregori J, Casillas R, Riveiro-Barciela M, Godoy C, Sopena S, Rando A, Yll M, Lopez-Martinez R, Quer J, Esteban R, Buti M, Rodríguez-Frías F. Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy. World J Gastroenterol 2018; 24:2095-2107. [PMID: 29785078 PMCID: PMC5960815 DOI: 10.3748/wjg.v24.i19.2095] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Revised: 03/26/2018] [Accepted: 05/06/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5’ region that could be candidates for gene therapy.
METHODS The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5’ end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed.
RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5’ end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain.
CONCLUSION Two hyper-conserved regions detected in the HBX 5’ end may be of value for targeted gene therapy, regardless of the patients’ clinical stage or HBV genotype.
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Affiliation(s)
- Carolina González
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - David Tabernero
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Maria Francesca Cortese
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Josep Gregori
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
- Roche Diagnostics SL, Sant Cugat del Vallès 08174, Spain
| | - Rosario Casillas
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Mar Riveiro-Barciela
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
- Rafael Esteban and Maria Buti, Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Cristina Godoy
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Sara Sopena
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Ariadna Rando
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Marçal Yll
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Rosa Lopez-Martinez
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Josep Quer
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Rafael Esteban
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Maria Buti
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Francisco Rodríguez-Frías
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
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Ward H, Tang L, Poonia B, Kottilil S. Treatment of hepatitis B virus: an update. Future Microbiol 2016; 11:1581-1597. [PMID: 27855500 DOI: 10.2217/fmb-2016-0128] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Chronic hepatitis B virus infection is a global health concern as it affects over 240 million people worldwide and an estimated 686,000 people die annually as a result of complications of the disease. With the development of newer antiviral drugs, viral suppression of HBV is achievable, however elimination of HBV from infected individuals (functional cure) remains an issue. Due to persistence of HBV DNA (cccDNA) in infected cells, chronically infected patients who discontinue therapy prior to HBsAg loss or seroconversion are likely to relapse. Several novel therapeutic strategies are being researched and studied in clinical trials. Here we review these novel strategies to achieve sustained cure or elimination of HBV. These strategies include the targeting of the host or viral factors required for viral persistence as well as therapeutic vaccines.
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Affiliation(s)
- Haley Ward
- Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
| | - Lydia Tang
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Bhawna Poonia
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Shyam Kottilil
- Division of Clinical Care & Research, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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9
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Zhou B, Wang J, Zheng G, Qiu Z. Methylated urolithin A, the modified ellagitannin-derived metabolite, suppresses cell viability of DU145 human prostate cancer cells via targeting miR-21. Food Chem Toxicol 2016; 97:375-384. [PMID: 27725205 DOI: 10.1016/j.fct.2016.10.005] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2015] [Revised: 07/13/2016] [Accepted: 10/06/2016] [Indexed: 01/20/2023]
Abstract
Urolithins are bioactive ellagic acid-derived metabolites produced by human colonic microflora. Although previous studies have demonstrated the cytotoxicity of urolithins, the effect of urolithins on miRNAs is still unclear. In this study, the suppressing effects of methylated urolithin A (mUA) on cell viability in human prostate cancer DU145 cells was investigated. mUA induced caspase-dependent cell apoptosis, mitochondrial depolarization and down-regulation of Bcl-2/Bax ratio. The results showed that upon exposure to mUA, miR-21 expression was decreased and the expression of PTEN and Pdcd4 protein was elevated. mUA could further suppress Akt phosphorylation and increase protein expression of FOXO3a, and the effects of mUA on Akt phosphorylation and protein expression of FOXO3a were blocked by PTEN silence. Moreover, mUA suppressed the Wnt/β-catenin-mediated transcriptional activation of MMP-7 and c-Myc, and this function of mUA on MMP-7 and c-Myc was attenuated by over-expression of miR-21. In conclusion, our data suggest that mUA can suppress cell viability in DU145 cells through modulating miR-21 and its downstream series-wound targets, including PTEN, Akt and Wnt/β-catenin signaling.
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Affiliation(s)
- Benhong Zhou
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060, People's Republic of China
| | - Jing Wang
- Department of Pharmacy, Renmin Hospital of Wuhan University, Wuhan 430060, People's Republic of China
| | - Guohua Zheng
- Key Laboratory of Chinese Medicine Resource and Compound Prescription (Ministry of Education), Hubei University of Chinese Medicine, Wuhan 430065, People's Republic of China
| | - Zhenpeng Qiu
- College of Pharmacy, Hubei University of Chinese Medicine, No. 1, West Huangjiahu Road, Wuhan 430065, People's Republic of China.
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10
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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: 66] [Impact Index Per Article: 7.3] [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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11
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Chitosan and Sodium Alginate Combinations Are Alternative, Efficient, and Safe Natural Adjuvant Systems for Hepatitis B Vaccine in Mouse Model. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2016; 2016:7659684. [PMID: 27493674 PMCID: PMC4963576 DOI: 10.1155/2016/7659684] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Accepted: 06/09/2016] [Indexed: 02/06/2023]
Abstract
Hepatitis B viral (HBV) infections represent major public health problem and are an occupational hazard for healthcare workers. Current alum-adjuvanted HBV vaccine is the most effective measure to prevent HBV infection. However, the vaccine has some limitations including poor response in some vaccinee and being a frost-sensitive suspension. The goal of our study was to use an alternative natural adjuvant system strongly immunogenic allowing for a reduction in dose and cost. We tested HBV surface antigen (HBsAg) adjuvanted with chitosan (Ch) and sodium alginate (S), both natural adjuvants, either alone or combined with alum in mouse model. Mice groups were immunized subcutaneously with HBsAg adjuvanted with Ch or S, or triple adjuvant formula with alum (Al), Ch, and S, or double formulations with AlCh or AlS. These were compared to control groups immunized with current vaccine formula or unadjuvanted HBsAg. We evaluated the rate of seroconversion, serum HBsAg antibody, IL-4, and IFN-γ levels. The results showed that the solution formula with Ch or S exhibited comparable immunogenic responses to Al-adjuvanted suspension. The AlChS gave significantly higher immunogenic response compared to controls. Collectively, our results indicated that Ch and S are effective HBV adjuvants offering natural alternatives, potentially reducing dose.
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12
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Bitton Alaluf M, Shlomai A. New therapies for chronic hepatitis B. Liver Int 2016; 36:775-82. [PMID: 26854115 DOI: 10.1111/liv.13086] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/01/2016] [Accepted: 02/01/2016] [Indexed: 02/13/2023]
Abstract
Approximately 350 million people worldwide are chronically infected with hepatitis B virus (HBV), representing a significant public health challenge. Nucleos/tide analogues (NUCs) and interferon alpha (IFNα), the current standard of care for chronic infection, aim at preventing progression of the disease to cirrhosis, hepatocellular carcinoma (HCC) and death. However, in contrast to the case of hepatitis C virus infection, in which novel antiviral drugs cure the vast majority of treated patients, in regard to HBV, cure is rare due to the unusual persistence of viral DNA in the form of covalently closed circular DNA (cccDNA) within the nucleus of infected cells. Available therapies for HBV require lifelong treatment and surveillance, as reactivation frequently occurs following medication cessation and the occurrence of HCC is decreased but not eliminated, even after years of successful viral suppression. Progress has been made in the development of new therapeutics, and it is likely that only a combination of immune modulators, inhibitors of gene expression and replication and cccDNA-targeting drugs will eradicate chronic infection. This review aims to summarize the state of the art in HBV drug research highlighting those agents with the greatest potential for success based on in vitro as well as on data from clinical studies.
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Affiliation(s)
- Maya Bitton Alaluf
- Department of Medicine D, Beilinson Hospital Rabin Medical Center, Petah Tikva, Israel
| | - Amir Shlomai
- Department of Medicine D, Beilinson Hospital Rabin Medical Center, Petah Tikva, Israel.,The Liver Institute, Beilinson Hospital Rabin Medical Center, Petah Tikva, Israel.,The Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
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13
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14
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Louten J, Beach M, Palermino K, Weeks M, Holenstein G. MicroRNAs Expressed during Viral Infection: Biomarker Potential and Therapeutic Considerations. Biomark Insights 2016; 10:25-52. [PMID: 26819546 PMCID: PMC4718089 DOI: 10.4137/bmi.s29512] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 10/22/2015] [Accepted: 10/24/2015] [Indexed: 12/13/2022] Open
Abstract
MicroRNAs (miRNAs) are short sequences of noncoding single-stranded RNAs that exhibit inhibitory effects on complementary target mRNAs. Recently, it has been discovered that certain viruses express their own miRNAs, while other viruses activate the transcription of cellular miRNAs for their own benefit. This review summarizes the viral and/or cellular miRNAs that are transcribed during infection, with a focus on the biomarker and therapeutic potential of miRNAs (or their antagomirs). Several human viruses of clinical importance are discussed, namely, herpesviruses, polyomaviruses, hepatitis B virus, hepatitis C virus, human papillomavirus, and human immunodeficiency virus.
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Affiliation(s)
- Jennifer Louten
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Michael Beach
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Kristina Palermino
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Maria Weeks
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
| | - Gabrielle Holenstein
- Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA
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15
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Brahmania M, Feld J, Arif A, Janssen HLA. New therapeutic agents for chronic hepatitis B. THE LANCET. INFECTIOUS DISEASES 2016; 16:e10-21. [PMID: 26795693 DOI: 10.1016/s1473-3099(15)00436-3] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/07/2015] [Revised: 10/13/2015] [Accepted: 11/03/2015] [Indexed: 12/11/2022]
Abstract
The treatment goal for chronic hepatitis B is true eradication of the hepatitis B virus, but this is rarely achieved with first-line treatment regimens because of an inability to disrupt covalently closed circular DNA and an inadequate host immune response. Therefore, new antiviral agents are needed to target various stages of the hepatitis B virus lifecycle and modulation of the immune system. This Review provides a summary of available regimens with their strengths and limitations, and highlights future therapeutic strategies to target the virus and host immune response. These new agents can hopefully lead to a finite duration of treatment, and provide a functional and durable cure for chronic hepatitis B infection.
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Affiliation(s)
- Mayur Brahmania
- Toronto Centre for Liver Diseases, Toronto Western Hospital, University Health Network, Toronto, ON, Canada
| | - Jordan Feld
- Toronto Centre for Liver Diseases, Toronto Western Hospital, University Health Network, Toronto, ON, Canada
| | - Ambreen Arif
- Toronto Centre for Liver Diseases, Toronto Western Hospital, University Health Network, Toronto, ON, Canada
| | - Harry L A Janssen
- Toronto Centre for Liver Diseases, Toronto Western Hospital, University Health Network, Toronto, ON, Canada; Department of Gastroenterology and Hepatology, Erasmus Medical Center University Hospital, Rotterdam, Netherlands.
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16
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Ricardo-Lax I, Ramanan V, Michailidis E, Shamia T, Reuven N, Rice CM, Shlomai A, Shaul Y. Hepatitis B virus induces RNR-R2 expression via DNA damage response activation. J Hepatol 2015; 63:789-96. [PMID: 26026873 DOI: 10.1016/j.jhep.2015.05.017] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 04/21/2015] [Accepted: 05/19/2015] [Indexed: 12/16/2022]
Abstract
BACKGROUND & AIMS Hepatitis B virus (HBV) infects and replicates in quiescent hepatocytes, which are deficient in dNTPs, the critical precursors of HBV replication. Most tumor viruses promote dNTP production in host cells by inducing cell proliferation. Although HBV is known as a major cause of hepatocellular carcinoma, it does not lead to cellular proliferation. Instead, HBV acquires dNTPs by activating the expression of the R2 subunit of the Ribonucleotide Reductase (RNR) holoenzyme, the cell cycle gene that is rate-limiting for generation of dNTPs, without inducing the cell cycle. We wished to elucidate the molecular basis of HBV-dependent R2 expression in quiescent cells. METHODS Quiescent HepG2 cells were transduced with an HBV-containing lentiviral vector, and primary human hepatocytes were infected with HBV. DNA damage response and RNR-R2 gene expression were monitored under this condition. RESULTS We report here that HBV-induced R2 expression is mediated by the E2F1 transcription factor, and that HBV induces E2F1 accumulation, modification and binding to the R2 promoter. We found that Chk1, a known E2F1 kinase that functions in response to DNA damage, was activated by HBV. In cells where Chk1 was pharmacologically inhibited, or depleted by shRNA-mediated knockdown, HBV-mediated R2 expression was severely attenuated. Furthermore, we found that HBV attenuates DNA repair, thus reducing cellular dNTP consumption. CONCLUSIONS Our findings demonstrate that HBV exploits the Chk1-E2F1 axis of the DNA damage response pathway to induce R2 expression in a cell cycle-independent manner. This suggests that inhibition of this pathway may have a therapeutic value for HBV carriers.
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Affiliation(s)
- Inna Ricardo-Lax
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Vyas Ramanan
- Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States
| | - Eleftherios Michailidis
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States
| | - Tal Shamia
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Nina Reuven
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Charles M Rice
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States
| | - Amir Shlomai
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, United States.
| | - Yosef Shaul
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.
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17
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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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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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Gebbing M, Bergmann T, Schulz E, Ehrhardt A. Gene therapeutic approaches to inhibit hepatitis B virus replication. World J Hepatol 2015; 7:150-164. [PMID: 25729471 PMCID: PMC4342598 DOI: 10.4254/wjh.v7.i2.150] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2014] [Revised: 10/23/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
Acute and chronic hepatitis B virus (HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellular carcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.
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20
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Targeting the Achilles heel of the hepatitis B virus: a review of current treatments against covalently closed circular DNA. Drug Discov Today 2015; 20:548-61. [PMID: 25622780 DOI: 10.1016/j.drudis.2015.01.008] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Revised: 12/15/2014] [Accepted: 01/14/2015] [Indexed: 01/05/2023]
Abstract
Chronic infection with hepatitis B virus (HBV) often leads to the development of liver cancer and cirrhosis, creating immense sociological, clinical and economic burdens worldwide. Although current anti-HBV medications manage to control the disease progression and help restore normal liver functions, they often fail to eliminate the virus completely. A major reason for this failure is the presence of a stable viral genome in the hepatocyte nucleus: the covalently closed circular DNA (cccDNA). Targeting HBV cccDNA is a promising approach that could lead to a complete cure. Here, we review various research approaches that are directed toward eliminating HBV cccDNA. This is a brief, yet comprehensive, summary of current state-of-the-art developments in this emerging area of interest.
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21
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Hean J, Crowther C, Ely A, Ul Islam R, Barichievy S, Bloom K, Weinberg MS, van Otterlo WA, de Koning CB, Salazar F, Marion P, Roesch EB, Lemaitre M, Herdewijn P, Arbuthnot P. Inhibition of hepatitis B virus replication in vivo using lipoplexes containing altritol-modified antiviral siRNAs. ARTIFICIAL DNA, PNA & XNA 2014; 1:17-26. [PMID: 21687523 DOI: 10.4161/adna.1.1.11981] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/02/2010] [Revised: 04/06/2010] [Accepted: 04/07/2010] [Indexed: 01/15/2023]
Abstract
Chronic infection with the hepatitis B virus (HBV) occurs in approximately 6% of the world's population and carriers of the virus are at risk for complicating hepatocellular carcinoma. Current treatment options have limited efficacy and chronic HBV infection is likely to remain a significant global medical problem for many years to come. Silencing HBV gene expression by harnessing RNA interference (RNAi) presents an attractive option for development of novel and effective anti HBV agents. However, despite significant and rapid progress, further refinement of existing technologies is necessary before clinical application of RNAi-based HBV therapies is realized. Limiting off target effects, improvement of delivery efficiency, dose regulation and preventing reactivation of viral replication are some of the hurdles that need to be overcome. To address this, we assessed the usefulness of the recently described class of altritol-containing synthetic siRNAs (ANA siRNAs), which were administered as lipoplexes and tested in vivo in a stringent HBV transgenic mouse model. Our observations show that ANA siRNAs are capable of silencing of HBV replication in vivo. Importantly, non specific immunostimulation was observed with unmodified siRNAs and this undesirable effect was significantly attenuated by ANA modification. Inhibition of HBV replication of approximately 50% was achieved without evidence for induction of toxicity. These results augur well for future application of ANA siRNA therapeutic lipoplexes.
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Affiliation(s)
- Justin Hean
- Antiviral Gene Therapy Research Unit; School of Pathology; University of the Witwatersrand Medical School; South Africa
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Lipid nanoparticles as carriers for RNAi against viral infections: current status and future perspectives. BIOMED RESEARCH INTERNATIONAL 2014; 2014:161794. [PMID: 25184135 PMCID: PMC4145386 DOI: 10.1155/2014/161794] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Revised: 07/14/2014] [Accepted: 07/14/2014] [Indexed: 12/15/2022]
Abstract
The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.
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23
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Geng J, Wei H, Sun R, Tian Z. Construction and application of a novel hepatocyte-directed vector to simultaneous knockdown and overexpression of multiple genes. Liver Int 2014; 34:e246-56. [PMID: 24125589 DOI: 10.1111/liv.12336] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/17/2013] [Accepted: 09/15/2013] [Indexed: 02/13/2023]
Abstract
BACKGROUND & AIMS Liver disease, such as malignancy and hepatitis, often correlates with several genetic disorders. We aimed to construct a hepatocyte-specific vector that could manipulate multiple genes simultaneously. METHODS We selected a highly efficient hepatocyte-specific α-foetoprotein (AFP) enhancer/albumin promoter (an RNA polymerase II promoter) to express our gene of interest and transcribe microRNA-based shRNAs (shRNAmir). Multiple shRNAmirs were assembled together in tandem to enhance the gene-silencing effect. By employing the AFP enhancer/albumin promoter and inserting an internal ribosome entry site (IRES), a hepatocyte-specific, multi-reporter vector that overexpressed both β-galactosidase (LacZ) and DsRed2 while simultaneously knocking down both EGFP and luciferase expression was successfully constructed and functionally tested in vitro. RESULTS The reporter genes in the multireporter vector were easily replaced by immune-related genes to construct the Multi-Vector, which overexpressed human interleukin 10 and silenced both CCL5 and CX3CL1 (FKN) simultaneously in vivo; visualization of DsRed2 coexpressed to monitor vector function in vivo confirmed that the Multi-Vector was successfully introduced into the host. Simultaneous manipulation of these multiple genes by the Multi-Vector synergistically inhibited acute liver injury induced by Poly I:C/D-GalN injection in mice. The multifunctional cassette was also packaged in and successfully delivered by an adenoviral vector. CONCLUSIONS We successfully engineered a vector that can simultaneously regulate multiple genes from a single multigene-containing vector in a hepatocyte-specific manner, suggesting the possibility that this method could be extensively and practically utilized in liver gene therapy.
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Affiliation(s)
- Jianlin Geng
- Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China; Hefei National Laboratory for Physical Sciences at Microscale, Hefei, Anhui, China
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24
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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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25
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Zhang W, Lu Z, Kong G, Gao Y, Wang T, Wang Q, Cai N, Wang H, Liu F, Ye L, Zhang X. Hepatitis B virus X protein accelerates hepatocarcinogenesis with partner survivin through modulating miR-520b and HBXIP. Mol Cancer 2014; 13:128. [PMID: 24886421 PMCID: PMC4046021 DOI: 10.1186/1476-4598-13-128] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2014] [Accepted: 05/23/2014] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx. METHODS The expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo. RESULTS In this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo. CONCLUSION HBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.
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Affiliation(s)
- Weiying Zhang
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Zhanping Lu
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Guangyao Kong
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Yuen Gao
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Tao Wang
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Qi Wang
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Na Cai
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
| | - Honghui Wang
- State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
| | - Fabao Liu
- State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
| | - Lihong Ye
- State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071, P.R. China
| | - Xiaodong Zhang
- State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, P.R. China
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Sun CP, Wu TH, Chen CC, Wu PY, Shih YM, Tsuneyama K, Tao MH. Studies of efficacy and liver toxicity related to adeno-associated virus-mediated RNA interference. Hum Gene Ther 2014; 24:739-50. [PMID: 23829557 DOI: 10.1089/hum.2012.239] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.
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Affiliation(s)
- Cheng-Pu Sun
- Molecular Medicine Program, Taiwan International Graduate Program, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
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Abstract
Chronic HBV infection is a major public health concern affecting over 240 million people worldwide. Although suppression of HBV replication is achieved in the majority of patients with currently available newer antivirals, discontinuation of therapy prior to hepatitis B surface antigen loss or seroconversion is associated with relapse of HBV in the majority of cases. Thus, new therapeutic modalities are needed to achieve eradication of the virus from chronically infected patients in the absence of therapy. The basis of HBV persistence includes viral and host factors. Here, we review novel strategies to achieve sustained cure or elimination of HBV. The novel approaches include targeting the viral and or host factors required for viral persistence, and novel immune-based therapies, including therapeutic vaccines.
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Affiliation(s)
- Rama Kapoor
- Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research Inc., (formerly SAIC-Frederick, Inc.) Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA
- Laboratory of Immunoregulation, National Institute of Allergy & Infectious Diseases, NIH, Department of Health & Human Services, Bethesda, MD 20892, USA
| | - Shyam Kottilil
- Laboratory of Immunoregulation, National Institute of Allergy & Infectious Diseases, NIH, Department of Health & Human Services, Bethesda, MD 20892, USA
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Wu GY, Zheng XJ, Yin CC, Jiang D, Zhu L, Liu Y, Wei L, Wang Y, Chen HS. Inhibition of Hepatitis B Virus Replication by Bay 41-4109 and Its Association with Nucleocapsid Disassembly. J Chemother 2013; 20:458-67. [DOI: 10.1179/joc.2008.20.4.458] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/31/2022]
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Abstract
Though the pharmaceutical industry's infatuation with the therapeutic potential of RNA interference (RNAi) technology has finally come down from its initial lofty levels,[1] hope is by no means lost for the once-burgeoning enterprise, as recent clinical trials are beginning to show efficacy in areas ranging from amyloidosis to hypercholesterolemia to muscular dystrophy. With such resurgence comes a more informed perspective on the needs of such therapeutics: a renewed focus on true RNA drug development, and a desire for enhanced site-specific delivery.[2] In this review, we will discuss the latter with regard to hepatic targeting by synthetic vectors, covering the implications of organ and cellular physiology on conjugate structure, particle morphology, and active targeting. In presenting efficacy in a variety of disease models, we emphasize as well the extraordinary degree to which synthetic formulation improves upon and coordinates efforts with oligonucleotide development. Such advances in the understanding of and the technology behind RNAi have the potential to finally stabilize the long-term prospects RNA therapeutic development.
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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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Wang W, Peng H, Li J, Zhao X, Zhao F, Hu K. Controllable inhibition of hepatitis B virus replication by a DR1-targeting short hairpin RNA (shRNA) expressed from a DOX-inducible lentiviral vector. Virus Genes 2013; 46:393-403. [PMID: 23397077 PMCID: PMC7089079 DOI: 10.1007/s11262-013-0886-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2012] [Accepted: 01/22/2013] [Indexed: 12/14/2022]
Abstract
As a highly efficient delivery system, lentiviral vectors (LVs) have become a powerful tool to assess the antiviral efficacy of RNA drugs such as short hairpin RNA (shRNA) and decoys. Furthermore, recent advanced systems allow controlled expression of the effector RNA via coexpression of a tetracycline/doxycycline (DOX) responsive repressor (tTR-KRAB). Herein, this system was utilized to assess the antiviral effects of LV-encoded shRNAs targeting three conserved regions on the pregenomic RNA of hepatitis B virus (HBV), namely the region coding for the reverse transcriptase (RT) domain of the viral polymerase (LV-HBV-shRNA1), the core promoter (CP; LV-HBV-shRNA2), and the direct repeat 1 (DR1; LV-HBV-shRNA3). Transduction of just the LV-HBV-shRNA vectors into the stably HBV expressing HepG2.2.15 cell line showed significant reductions in secreted HBsAg and HBeAg, intracellular HBcAg as well as HBV RNA and DNA replicative intermediates for all vectors, however, most pronouncedly for the DR1-targeting shRNA3. The corresponding vector was therefore applied in the DOX-controlled system. Notably, strong interference with HBV replication was found in the presence of the inducer DOX whereas the antiviral effect was essentially ablated in its absence; hence, the silencing effect of the shRNA and consequently HBV replication could be strictly regulated by DOX. This newly established system may therefore provide a valuable platform to study the antiviral efficacy of RNA drugs against HBV in a regulated manner, and even be applicable in vivo.
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Affiliation(s)
- Weiwei Wang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China
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Mouler Rechtman M, Burdelova EO, Bar-Yishay I, Ben-Yehoyada M, Fishman S, Halpern Z, Shlomai A. The metabolic regulator PGC-1α links anti-cancer cytotoxic chemotherapy to reactivation of hepatitis B virus. J Viral Hepat 2013; 20:34-41. [PMID: 23231082 DOI: 10.1111/j.1365-2893.2012.01622.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Patients with chronic hepatitis B virus (HBV) infection are at an increased risk for a severe and a potentially fatal viral reactivation following anti-cancer therapy. The molecular mechanism for this induction of HBV expression is still unclear. Here, we show that treating hepatoma cell line expressing HBV with various anti-cancer cytotoxic agents results in a significant up-regulation of HBV expression. This HBV induction is at the transcriptional level and is time dependent. Interestingly, treating hepatoma cells with anti-cancer cytotoxic agents results in a robust induction of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), a metabolic and energy regulator that is normally induced in the liver under starvation conditions and that has been previously shown to strongly coactivate HBV transcription. Most importantly, HBV up-regulation following anti-cancer therapy depends on PGC-1α induction, because PGC-1α knock-down abolishes HBV induction. Finally, pretreatment of HBV-expressing cells with the antioxidant agent N-acetylcysteine attenuates the induction of both PGC-1α and HBV in response to anti-cancer treatment, suggesting that chemotherapy-associated PGC-1α induction is mediated by cellular oxidative stress that ultimately leads to HBV up-regulation. We conclude that cytotoxic anti-cancer chemotherapy has a direct and an immune system-independent effect on HBV gene expression, which is mediated by PGC-1α. Our results attribute to this metabolic regulator an unexpected role in linking anti-cancer treatment to HBV reactivation and make PGC-1α a potential target for future anti-HBV therapy, especially under conditions in which it is robustly induced, such as following anti-cancer treatment.
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Affiliation(s)
- M Mouler Rechtman
- The Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
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Qiu LP, Chen KP. Anti-HBV agents derived from botanical origin. Fitoterapia 2012; 84:140-57. [PMID: 23164603 DOI: 10.1016/j.fitote.2012.11.003] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2012] [Revised: 10/29/2012] [Accepted: 11/04/2012] [Indexed: 01/16/2023]
Abstract
There are 350,000 hepatitis B virus (HBV) carriers all over the world. Chronic HBV infection is at a high risk of developing liver cirrhosis and hepatocelluar carcinoma (HCC), and heavily threatened people's health. Two kinds of drugs approved by FDA for anti-HBV therapy are immunomodulators (interferon α, pegylated-interferon α) and nucleos(t)ide analogues (lamivudine, adefovir dipivoxil, entecavir, telbivudine, and tenofovir disoproxil fumarate). These drugs have been proved to be far from being satisfactory due to their low specificity, side effects, and high rate of drug resistance. There is an urgent need to discover and develop novel effective anti-HBV drugs. With vast resources, various structures, diverse biological activities and action mechanisms, as well as abundant clinical experiences, botanical agents become a promising source of finding new anti-HBV drugs. This review summarizes the recent research and development of anti-HBV agents derived from botanical origin on their sources and active components, inhibitory effects and possible toxicities, as well as action targets and mechanisms, and also addresses the advantages and the existing shortcomings in the development of botanical inhibitors. This information may not only broaden the knowledge of anti-HBV therapy, and offer possible alternative or substitutive drugs for CHB patients, but also provides considerable information for developing new safe and effective anti-HBV drugs.
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Affiliation(s)
- Li-Peng Qiu
- Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, China
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Bian Z, Xiao A, Cao M, Liu M, Liu S, Jiao Y, Yan W, Qi Z, Zheng Z. Anti-HBV efficacy of combined siRNAs targeting viral gene and heat shock cognate 70. Virol J 2012; 9:275. [PMID: 23158906 PMCID: PMC3534549 DOI: 10.1186/1743-422x-9-275] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2012] [Accepted: 11/12/2012] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) infection is a major health concern with more than two billion individuals currently infected worldwide. Because of the limited effectiveness of existing vaccines and drugs, development of novel antiviral strategies is urgently needed. Heat stress cognate 70 (Hsc70) is an ATP-binding protein of the heat stress protein 70 family. Hsc70 has been found to be required for HBV DNA replication. Here we report, for the first time, that combined siRNAs targeting viral gene and siHsc70 are highly effective in suppressing ongoing HBV expression and replication. METHODS We constructed two plasmids (S1 and S2) expressing short hairpin RNAs (shRNAs) targeting surface open reading frame of HBV(HBVS) and one plasmid expressing shRNA targeting Hsc70 (siHsc70), and we used the EGFP-specific siRNA plasmid (siEGFP) as we had previously described. First, we evaluated the gene-silencing efficacy of both shRNAs using an enhanced green fluorescent protein (EGFP) reporter system and flow cytometry in HEK293 and T98G cells. Then, the antiviral potencies of HBV-specific siRNA (siHBV) in combination with siHsc70 in HepG2.2.15 cells were investigated. Moreover, type I IFN and TNF-α induction were measured by quantitative real-time PCR and ELISA. RESULTS Cotransfection of either S1 or S2 with an EGFP plasmid produced an 80%-90% reduction in EGFP signal relative to the control. This combinational RNAi effectively and specifically inhibited HBV protein, mRNA and HBV DNA, resulting in up to a 3.36 log10 reduction in HBV load in the HepG2.2.15 cell culture supernatants. The combined siRNAs were more potent than siHBV or siHsc70 used separately, and this approach can enhance potency in suppressing ongoing viral gene expression and replication in HepG2.2.15 cells while forestalling escape by mutant HBV. The antiviral synergy of siHBV used in combination with siHsc70 produced no cytotoxicity and induced no production of IFN-α, IFN-β and TNF-α in transfected cells. CONCLUSIONS Our combinational RNAi was sequence-specific, effective against wild-type and mutant drug-resistant HBV strains, without triggering interferon response or producing any side effects. These findings indicate that combinational RNAi has tremendous promise for developing innovative therapy against viral infection.
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Affiliation(s)
- Zhongqi Bian
- Center for Infectious Diseases, Kunming General Hospital, PLA, 212 Daguan Rd, Kunming 650032, PR China.
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RNA interference targeting E637K mutation rescues hERG channel currents and restores its kinetic properties. Heart Rhythm 2012; 10:128-36. [PMID: 23022675 DOI: 10.1016/j.hrthm.2012.09.124] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/23/2012] [Indexed: 11/22/2022]
Abstract
BACKGROUND Long QT syndrome (LQTS) is a monogenic proarrhythmic disorder that predisposes affected individuals to sudden death from tachyarrhythmia. As an inherited disease, LQTS cannot be completely cured by conventional treatment modalities. Individualized gene therapy is a promising therapeutic approach. OBJECTIVE The purpose of this study was to investigate the role of small interference RNA (siRNA) on expression of E637K-hERG (human ether-a-go-go-related gene) mutant and whether it can be used to rescue the mutant's dominant-negative suppressive effects on hERG protein channel function. METHODS Western blot was performed to select the most sensitive siRNAs to target E637K-hERG mutant knockdown. Confocal laser scanning microscope was performed to monitor cellular localization of wild-type (WT)-hERG and E637K-hERG with or without siRNA. Patch-clamp technique was used to assess the effect of siRNA on the electrophysiologic characteristics of the rapidly activating delayed rectifier K(+) current I(Kr) of the hERG protein channel. RESULTS siRNA led to a significant decrease in the level of E637K-hERG protein but did not affect the level of WT-hERG protein. WT-hERG localization in cells coexpressing E637K-hERG mutant was restored to the membrane by siRNA. The siRNA-mediated inhibition of E637K-hERG mutant restored the maximum current and tail current amplitudes. Furthermore, siRNA treatment rescued the kinetic properties of WT/E637K-hERG protein channel to a level comparable to that of WT-hERG protein channel. CONCLUSION Our findings illustrated that siRNA can effectively inhibit E637K-hERG protein expression and rescue the dominant-negative effect of this mutation by restoring the kinetic properties of hERG protein channel. It has potential clinical implications with regard to the possibility of using siRNA in the treatment of LQTS.
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Li W, Zhao C, Hong Y, Li J, Zhang C, Xiong H. Regulation of Growth of Gastric Cancer Cells and Sensitivity to Cisplatin by Modulation of DNA Binding Inhibitor or Differentiation Protein Expression. Lab Med 2012. [DOI: 10.1309/lm2a3urfem0tsymc] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022] Open
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RNA Interference: A Promising Approach for the Treatment of Viral Hepatitis. HEPATITIS MONTHLY 2012. [DOI: 10.5812/hepatmon.4294] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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RNA Interference: A Promising Approach for the Treatment of Viral Hepatitis. HEPATITIS MONTHLY 2012. [DOI: 10.5812/kowsar.1735143x.4294] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
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Kumar M, Follenzi A, Garforth S, Gupta S. Control of HBV replication by antiviral microRNAs transferred by lentiviral vectors for potential cell and gene therapy approaches. Antivir Ther 2011; 17:519-28. [PMID: 22300804 DOI: 10.3851/imp2014] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/02/2011] [Indexed: 12/17/2022]
Abstract
BACKGROUND Because molecular mechanisms regulating host cell and virus interactions are not fully understood, we further defined roles of antiviral microRNAs (miRNAs) in HBV replication. METHODS We studied small interfering RNA sequences inserted into the miR-30 backbone in cell systems. Antiviral sequences were cloned into lentiviral vectors upstream of a green fluorescent protein reporter. Transduced cells included HepG2 or HepG2 2.2.15 cell lines and hTERT-FH-B fetal human liver cells. HBV replication was analysed by several assays. RESULTS In 2.2.15 cells treated with constructs primarily targeting HBV polymerase and surface antigen or HBV polymerase and X open reading frames, HBV core protein, HBV DNA and HBV RNA expression decreased. This antiviral effect was more pronounced when the two constructs were expressed together. Similarly, antiviral constructs decreased HBV replication in HepG2 cells transduced with adenoviral vector to express HBV. Although antiviral sequences were expressed in hTERT-FH-B cells, these cells were non-permissive for HBV, possibly owing to expression of miRNAs reported to inhibit HBV replication, whereas these miRNAs were absent in HepG2 cells. Expression of antiviral miRNAs did not affect cell viability or proliferation and no deleterious changes were observed in expression of native cellular miRNAs. Moreover, expression of antiviral miRNA did not affect engraftment and survival of transplanted cells in mice. CONCLUSIONS Identification of effective antiviral miRNAs and transfer of suitable constructs by lentiviral vectors will be helpful for pathophysiological studies of host cell-virus interactions. Simultaneously, this will advance potential mechanisms for cell/gene therapy in those afflicted with chronic hepatitis and refractory liver disease.
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Affiliation(s)
- Mukesh Kumar
- Department of Medicine, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA
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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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41
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Shah PS, Schaffer DV. Antiviral RNAi: translating science towards therapeutic success. Pharm Res 2011; 28:2966-82. [PMID: 21826573 PMCID: PMC5012899 DOI: 10.1007/s11095-011-0549-8] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2011] [Accepted: 07/25/2011] [Indexed: 01/07/2023]
Abstract
Viruses continuously evolve to contend with an ever-changing environment that involves transmission between hosts and sometimes species, immune responses, and in some cases therapeutic interventions. Given the high mutation rate of viruses relative to the timescales of host evolution and drug development, novel drug classes that are readily screened and translated to the clinic are needed. RNA interference (RNAi)-a natural mechanism for specific degradation of target RNAs that is conserved from plants to invertebrates and vertebrates-can potentially be harnessed to yield therapies with extensive specificity, ease of design, and broad application. In this review, we discuss basic mechanisms of action and therapeutic applications of RNAi, including design considerations and areas for future development in the field.
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Affiliation(s)
- Priya S. Shah
- Department of Chemical and Biolmolecular Engineering, University of California, Berkeley, California 94720 USA
| | - David V. Schaffer
- Department of Chemical and Biolmolecular Engineering, University of California, Berkeley, California 94720 USA
- Department of Bioengineering, University of California, Berkeley, California 94720 USA
- Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720 USA
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Blazquez L, Gonzalez-Rojas SJ, Abad A, Razquin N, Abad X, Fortes P. Increased in vivo inhibition of gene expression by combining RNA interference and U1 inhibition. Nucleic Acids Res 2011; 40:e8. [PMID: 22086952 PMCID: PMC3245954 DOI: 10.1093/nar/gkr956] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA—snRNA—interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.
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Affiliation(s)
- Lorea Blazquez
- Department of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), University of Navarra, Pio XII 55. 31008 Pamplona, Spain
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Han Q, Zhang C, Zhang J, Tian Z. Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I-dependent manner. Hepatology 2011; 54:1179-89. [PMID: 21721030 DOI: 10.1002/hep.24505] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/10/2011] [Accepted: 06/05/2011] [Indexed: 12/24/2022]
Abstract
UNLABELLED It is extensively accepted that hepatitis B virus (HBV) escapes from innate immunity by inhibiting type I interferon (IFN) production, but efficient intervention to reverse the immune tolerance is still not achieved. Here, we report that 5'-end triphosphate hepatitis B virus X gene (HBx)-RNAs (3p-HBx-short interfering [si]RNAs) exerted significantly stronger inhibitory effects on HBV replication than regular HBx-siRNAs in stably HBV-expressing hepatoplastoma HepG2.2.15 cells through extremely higher expression of type I IFNs, IFN-induced genes and proinflammatory cytokines, and retinoic acid inducible gene I (RIG-I) activation. Also, 3p-HBx-siRNA were more efficient to stimulate type I IFN response than HBx sequence-unrelated 3p-scramble-siRNA in HepG2.2.15 cells, indicating that a stronger immune-stimulating effect may partly result from the reversal of immune tolerance through decreasing HBV load. In RIG-I-overexpressed HepG2.2.15 cells, 3p-HBx-siRNAs exerted stronger inhibitory effects on HBV replication with greater production of type I IFNs; on the contrary, in RIG-I-silenced HepG2.2.15 cells or after blockade of IFN receptor by monoclonal antibody, inhibitory effect of 3p-HBx-siRNAs on HBV replication was largely attenuated, indicating that immunostimulatory function of 3p-HBx-siRNAs was RIG-I and type I IFN dependent. Moreover, in HBV-carrier mice, 3p-HBx-siRNA more strongly inhibited HBV replication and promoted IFN production than HBx-siRNA in primary HBV(+) hepatocytes and, therefore, significantly decreased serum hepatitis B surface antigen and increased serum IFN-β. CONCLUSION 3p-HBx-siRNAs may not only directly inhibit HBV replication, but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV-induced immune tolerance.
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Affiliation(s)
- Qiuju Han
- Institute of Immunopharmacology & Immunotherapy, School of Pharmaceutical Sciences, Shandong University, Jinan, China
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Ivacik D, Ely A, Arbuthnot P. Countering hepatitis B virus infection using RNAi: how far are we from the clinic? Rev Med Virol 2011; 21:383-96. [PMID: 21913277 DOI: 10.1002/rmv.705] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2011] [Revised: 07/04/2011] [Accepted: 07/06/2011] [Indexed: 12/14/2022]
Abstract
Globally, persistent HBV infection is a significant cause of public health problems. Currently available HBV therapies have variable efficacy and there is a need to develop improved treatment to prevent cirrhosis and hepatocellular carcinoma. Although RNA interference (RNAi)-based approaches have shown promise, accomplishing safe and sustained silencing by RNAi activators, as well as their efficient delivery to hepatocytes have hampered clinical translation of this very promising technology. Expressed silencers may be produced in a sustained manner from stable DNA templates, which makes them suited to treatment of chronic HBV infection. DNA expression cassettes can be incorporated into both viral and non-viral vectors, but in vivo delivery of these cassettes with non-viral vectors is currently inefficient. Synthetic short interfering RNAs (siRNAs), which may be chemically modified to improve stability, specificity and efficacy, are more conveniently delivered to their cytoplasmic sites of action with synthetic non-viral vectors. However, the short duration of action of this class of RNAi activator is a drawback for treatment of chronic HBV infection. Despite the impressive progress that has been made in developing highly effective HBV gene silencers, challenges continue to face implementation of RNAi-based HBV therapy. This review will discuss the current status of the topic and consider the developments that are required to advance RNAi-based HBV therapy to clinical application.
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Affiliation(s)
- Dejana Ivacik
- Antiviral Gene Therapy Research Unit, School of Pathology, Health Sciences Faculty, University of the Witwatersrand, Johannesburg, South Africa
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Ebert G, Poeck H, Lucifora J, Baschuk N, Esser K, Esposito I, Hartmann G, Protzer U. 5' Triphosphorylated small interfering RNAs control replication of hepatitis B virus and induce an interferon response in human liver cells and mice. Gastroenterology 2011; 141:696-706, 706.e1-3. [PMID: 21684282 DOI: 10.1053/j.gastro.2011.05.001] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2010] [Revised: 04/26/2011] [Accepted: 05/02/2011] [Indexed: 12/25/2022]
Abstract
BACKGROUND & AIMS Approved therapies for chronic hepatitis B include systemic administration of interferon (IFN)-alfa and inhibitors of hepatitis B virus (HBV) reverse-transcription. Systemic application of IFN-alfa is limited by side effects. Reverse-transcriptase inhibitors effectively control HBV replication, but rarely eliminate the virus and can select drug-resistant variants. We aimed to develop an alternative therapeutic approach that combines gene silencing with induction of IFN in the liver. METHODS To stimulate an immune response while inhibiting HBV activity, we designed 3 small interfering (si)RNAs that target highly conserved sequences and multiple HBV transcripts of all genotypes. A 5'-triphosphate (3p) was added to the siRNAs, turning them into a ligand for the cytosolic helicase retinoic acid-inducible protein I, which becomes activated and induces expression of type-I IFNs. Antiviral activity was investigated in cell lines that replicate HBV, in HBV-infected primary human hepatocytes, and in HBV transgenic mice. RESULTS 3p-double-stranded RNA (3p-RNA) activated retinoic acid-inducible protein I, induced a strong type I IFN response (expression of IFN-β) in liver cells and showed transient but strong antiviral activity. Bifunctional, HBV-specific, 3p-siRNAs controlled replication of HBV more efficiently and for longer periods of time than 3p-RNAs without silencing capacity or siRNAs that targeted identical sequences but did not contain 3p. CONCLUSIONS HBV-specific 3p-siRNAs are bifunctional antiviral molecules that induce production of type I IFNs in the liver and target HBV RNAs to inhibit viral replication.
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Affiliation(s)
- Gregor Ebert
- Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
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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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van der Laan LJW, Wang Y, Tilanus HW, Janssen HLA, Pan Q. AAV-mediated gene therapy for liver diseases: the prime candidate for clinical application? Expert Opin Biol Ther 2011; 11:315-27. [PMID: 21204741 DOI: 10.1517/14712598.2011.548799] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
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Teng X, Xu WZ, Hao ML, Fang Y, Zhao YX, Chen SJ, Li D, Gu HX. Differential inhibition of lamivudine-resistant hepatitis B virus by allele-specific RNAi. J Virol Methods 2010; 168:6-12. [DOI: 10.1016/j.jviromet.2010.04.007] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2009] [Revised: 02/25/2010] [Accepted: 04/13/2010] [Indexed: 10/19/2022]
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