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Zhu J, Ruan X, Li C, Yuan J, Yang Y, Zhang W, Zhang H, Zhuo Z, Yan FF, Huang CB, Hou F. Psychophysical Reverse Correlation Revealed Broader Orientation Tuning and Prolonged Reaction Time in Amblyopia. Invest Ophthalmol Vis Sci 2022; 63:3. [PMID: 35503229 PMCID: PMC9078079 DOI: 10.1167/iovs.63.5.3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Purpose Neural selectivity of orientation is a fundamental property of visual system. We aim to investigate whether and how the orientation selectivity changes in amblyopia. Methods Seventeen patients with amblyopia (27.1 ± 7.1 years) and 18 healthy participants (25.1 ± 2.7 years) took part in this study. They were asked to continuously detect vertical gratings embedded in a stream of randomly oriented gratings. Using a technique of subspace reverse correlation, the orientation-time perceptive field (PF) for the atypical grating detection task was derived for each participant. Detailed comparisons were made between the PFs measured with the amblyopic and healthy eyes. Results The PF of the amblyopic eyes showed significant differences in orientation and time domain compared with that of the normal eyes (cluster-based permutation test, ps < 0.05), with broader bandwidth of orientation tuning (31.41 ± 10.59 degrees [mean ± SD] vs. 24.76 ± 6.85 degrees, P = 0.039) and delayed temporal dynamics (483 ± 68 ms vs. 425 ± 58 ms, P = 0.015). None of the altered PF properties correlated with the contrast sensitivity at 1 cycle per degree (c/deg) in amblyopia. No difference in PFs between the dominant and non-dominant eyes in the healthy group was found. Conclusions The altered orientation-time PF to the low spatial frequency and high contrast stimuli suggests amblyopes had coarser orientation selectivity and prolonged reaction time. The broader orientation tuning probably reflects the abnormal lateral interaction in the primary visual cortex, whereas the temporal delay might indicate a high level deficit.
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Affiliation(s)
- Jinli Zhu
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Xiaowei Ruan
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Cheng Li
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Junli Yuan
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Yan Yang
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Wenhua Zhang
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Hanyi Zhang
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Zuopao Zhuo
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
| | - Fang-Fang Yan
- Key Laboratory of Behavioral Science, Institute of Psychology, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,Department of Psychology, University of Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Chang-Bing Huang
- Key Laboratory of Behavioral Science, Institute of Psychology, Chinese Academy of Sciences, Chaoyang District, Beijing, China.,Department of Psychology, University of Chinese Academy of Sciences, Shijingshan District, Beijing, China
| | - Fang Hou
- School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China
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Panda S, Banik U, Adhikary AK. Bioinformatics analysis reveals four major hexon variants of human adenovirus type-3 (HAdV-3) as the potential strains for development of vaccine and siRNA-based therapeutics against HAdV-3 respiratory infections. INFECTION, GENETICS AND EVOLUTION : JOURNAL OF MOLECULAR EPIDEMIOLOGY AND EVOLUTIONARY GENETICS IN INFECTIOUS DISEASES 2020; 85:104439. [PMID: 32585339 PMCID: PMC7308778 DOI: 10.1016/j.meegid.2020.104439] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 04/24/2020] [Accepted: 06/20/2020] [Indexed: 11/25/2022]
Abstract
Human adenovirus type 3 (HAdV-3) encompasses 15-87% of all adenoviral respiratory infections. The significant morbidity and mortality, especially among the neonates and immunosuppressed patients, demand the need for a vaccine or a targeted antiviral against this type. However, due to the existence of multiple hexon variants (3Hv-1 to 3Hv-25), the selection of vaccine strains of HAdV-3 is challenging. This study was designed to evaluate HAdV-3 hexon variants for the selection of potential vaccine candidates and the use of hexon gene as a target for designing siRNA that can be used as a therapy. Based on the data of worldwide distribution, duration of circulation, co-circulation and their percentage among all the variants, 3Hv-1 to 3Hv-4 were categorized as the major hexon variants. Phylogenetic analysis and the percentage of homology in the hypervariable regions followed by multi-sequence alignment, zPicture analysis and restriction enzyme analysis were carried out. In the phylogram, the variants were arranged in different clusters. The HVR encoding regions of hexon of 3Hv-1 to 3Hv-4 showed 16 point mutations resulting in 12 amino acids substitutions. The homology in HVRs was 81.81-100%. Therefore, the major hexon variants are substantially different from each other which justifies their inclusion as the potential vaccine candidates. Interestingly, despite the significant differences in the DNA sequence, there were many conserved areas in the HVRs, and we have designed functional siRNAs form those locations. We have also designed immunogenic vaccine peptide epitopes from the hexon protein using bioinformatics prediction tool. We hope that our developed siRNAs and immunogenic vaccine peptide epitopes could be used in the future development of siRNA-based therapy and designing a vaccine against HAdV-3.
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Affiliation(s)
- Somnath Panda
- Unit of Microbiology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia.
| | - Urmila Banik
- Unit of Pathology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia
| | - Arun K. Adhikary
- Unit of Microbiology, AIMST University, Faculty of Medicine, Jalan Bedong Semeling, 08100 Bedong, Kedah, Malaysia
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Levingstone TJ, Herbaj S, Redmond J, McCarthy HO, Dunne NJ. Calcium Phosphate Nanoparticles-Based Systems for RNAi Delivery: Applications in Bone Tissue Regeneration. NANOMATERIALS (BASEL, SWITZERLAND) 2020; 10:E146. [PMID: 31947548 PMCID: PMC7023416 DOI: 10.3390/nano10010146] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/04/2019] [Revised: 12/16/2019] [Accepted: 12/21/2019] [Indexed: 12/11/2022]
Abstract
Bone-related injury and disease constitute a significant global burden both socially and economically. Current treatments have many limitations and thus the development of new approaches for bone-related conditions is imperative. Gene therapy is an emerging approach for effective bone repair and regeneration, with notable interest in the use of RNA interference (RNAi) systems to regulate gene expression in the bone microenvironment. Calcium phosphate nanoparticles represent promising materials for use as non-viral vectors for gene therapy in bone tissue engineering applications due to their many favorable properties, including biocompatibility, osteoinductivity, osteoconductivity, and strong affinity for binding to nucleic acids. However, low transfection rates present a significant barrier to their clinical use. This article reviews the benefits of calcium phosphate nanoparticles for RNAi delivery and highlights the role of surface functionalization in increasing calcium phosphate nanoparticles stability, improving cellular uptake and increasing transfection efficiency. Currently, the underlying mechanistic principles relating to these systems and their interplay during in vivo bone formation is not wholly understood. Furthermore, the optimal microRNA targets for particular bone tissue regeneration applications are still unclear. Therefore, further research is required in order to achieve the optimal calcium phosphate nanoparticles-based systems for RNAi delivery for bone tissue regeneration.
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Affiliation(s)
- Tanya J. Levingstone
- School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland; (T.J.L.); (S.H.); (J.R.)
- Centre for Medical Engineering Research, School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland
- Advanced Processing Technology Research Centre, Dublin City University, 9 Dublin, Ireland
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, 2 Dublin, Ireland
| | - Simona Herbaj
- School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland; (T.J.L.); (S.H.); (J.R.)
- Centre for Medical Engineering Research, School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland
| | - John Redmond
- School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland; (T.J.L.); (S.H.); (J.R.)
- Centre for Medical Engineering Research, School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland
| | - Helen O. McCarthy
- School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, UK;
| | - Nicholas J. Dunne
- School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland; (T.J.L.); (S.H.); (J.R.)
- Centre for Medical Engineering Research, School of Mechanical and Manufacturing Engineering, Dublin City University, 9 Dublin, Ireland
- Advanced Processing Technology Research Centre, Dublin City University, 9 Dublin, Ireland
- Trinity Centre for Biomedical Engineering, Trinity Biomedical Sciences Institute, Trinity College Dublin, 2 Dublin, Ireland
- School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, UK;
- Department of Mechanical and Manufacturing Engineering, School of Engineering, Trinity College Dublin, 2 Dublin, Ireland
- Advanced Materials and Bioengineering Research Centre (AMBER), Royal College of Surgeons in Ireland and Trinity College Dublin, 2 Dublin, Ireland
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Abstract
Persistent hepatitis B virus (HBV) infection of hepatocytes is associated with a covalently closed circular DNA (cccDNA) episome. Although serologic hepatitis B surface antigen tests are negative, the presence of cccDNA is obviously increased in HBeAg-positive patients compared with that in HBeAg-negative patients, inactive carriers and patients. Moreover, trace cccDNA levels can also be found in the liver cells of patients with resolved hepatitis B infections. Therefore, clearance of cccDNA in hepatocytes could be an effective cure for HBV. In this review, we summarize the strategies that have been employed to eliminate cccDNA in recent years and discuss the future development of treatments for chronic hepatitis B.
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5
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Enhanced antiviral and antifibrotic effects of short hairpin RNAs targeting HBV and TGF-β in HBV-persistent mice. Sci Rep 2017. [PMID: 28634402 PMCID: PMC5478661 DOI: 10.1038/s41598-017-04170-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
The hepatitis B virus (HBV) causes acute and chronic liver infection, which may lead to liver cirrhosis and hepatocellular carcinoma. Current treatments including interferons and nucleotide analogs, have limited therapeutic effects, underscoring the need to identify effective therapeutic options to inhibit HBV replication and prevent complications. Previous animal models mimicking chronic HBV infection do not faithfully reflect disease progression in humans. Here, we used our established HBV-persistent mouse line with liver fibrosis to evaluate the efficacy of novel therapies. The combination of two short hairpin RNAs (dual-shRNA) against different coding regions of HBV delivered by a self-complementary AAV vector showed better antiviral effects than single shRNA both in vitro and in HBV-persistent mice. The dual-shRNA also exhibited stronger antifibrotic activity in vivo. Vector carrying shRNA against TGF-β, though did not inhibit HBV replication alone, enhanced the antiviral and antifibrotic activities of single and dual HBV shRNAs. Co-administration of TGF-β shRNA and HBV dual-shRNA decreased HBV DNA, HBV RNA, HBsAg, HBeAg, and liver fibrosis markers in serum and tissues, and improved liver morphology more effectively than single treatments. Our results suggest that the combination of shRNAs against HBV and TGF-β could be developed into a viable treatment for human HBV infection.
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6
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Li G, Jiang G, Lu J, Chen S, Cui L, Jiao J, Wang Y. Inhibition of hepatitis B virus cccDNA by siRNA in transgenic mice. Cell Biochem Biophys 2015; 69:649-54. [PMID: 24569930 DOI: 10.1007/s12013-014-9847-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The elimination of viral covalently closed circular DNA (cccDNA) from the nucleus of infected hepatocytes is an obstacle to achieving sustained viral clearance during antiviral therapy of chronic hepatitis B virus (HBV) infection. The aim of our study was to determine whether treatment with siRNA is able to suppress viral cccDNA amplification using a HBV-transgenic mice model. The experimental results revealed that siRNAs can serve as efficient alternative anti-HBV agents, because they showed better inhibitory effect on viral replication and antigen expression in transgenic mice. More importantly, the siRNA markedly inhibited HBV cccDNA amplification.
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MESH Headings
- Animals
- DNA, Circular/biosynthesis
- DNA, Circular/genetics
- DNA, Circular/metabolism
- DNA, Viral/biosynthesis
- DNA, Viral/genetics
- DNA, Viral/metabolism
- Gene Expression Regulation
- Hepatitis B Surface Antigens/metabolism
- Hepatitis B e Antigens/metabolism
- Hepatitis B virus/genetics
- Hepatitis B virus/immunology
- Hepatitis B virus/physiology
- Mice
- Mice, Transgenic
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Small Interfering/genetics
- Virus Replication/genetics
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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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7
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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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8
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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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9
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Zacharoulis D, Rountas C, Katsimpoulas M, Morianos J, Chatziandreou I, Vassilopoulos G. Efficient liver gene transfer with foamy virus vectors. Med Sci Monit Basic Res 2013; 19:214-20. [PMID: 23941977 PMCID: PMC3747017 DOI: 10.12659/msmbr.883996] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Background Liver gene transfer offers hope for the correction of genetic and acquired disorders. Efficient gene transfer in large animals can be obtained with hydrodynamic gene transfer (HGT), a method that can achieve sufficient levels of gene delivery. Material/Methods To test the relative efficiency between plasmid versus foamy virus (FV) vector-based liver gene transfer efficiency, we applied HGT in 4 juvenile pigs, using the same plasmid backbone, either naked or coated as a FV vector particle. Gene transfer efficiency and persistence of expression was assayed by PCR and real-time PCR, respectively, at 1 week and at 1 month after the infusions. Results HGT was tolerated well and no adverse reactions were observed. Plasmid injections resulted in no detectable DNA sequences at 1 week. At the 1 month time point, 2/15 liver sections analyzed were positive for the presence of plasmid DNA. When FV vectors were infused under identical conditions, 18/28 (64.3%) of the liver samples were positive for the presence of vector sequences, and the expression levels reached 29.7 and 15.6% of the endogenous GAPDH levels in the injected and the adjacent liver lobes. Conclusions Our results indicate that medium-term therapeutic levels of gene expression can be obtained with FV vectors, an effect that can be attributed to the potential of the HGT procedure and to the natural affinity of FV vectors for hepatocytes.
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10
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Bai J, Jiang K, Zhang L, Wang X, Wang X, Li Y, Jiang P. Protective efficacy of adenovirus-mediated small interfering RNAs against encephalomyocarditis virus challenge in mice. J Virol Methods 2012; 185:204-12. [DOI: 10.1016/j.jviromet.2012.07.004] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2012] [Revised: 06/26/2012] [Accepted: 07/03/2012] [Indexed: 10/28/2022]
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Hu HM, Chen Y, Liu L, Zhang CG, Wang W, Gong K, Huang Z, Guo MX, Li WX, Li W. C1orf61 acts as a tumor activator in human hepatocellular carcinoma and is associated with tumorigenesis and metastasis. FASEB J 2012; 27:163-73. [PMID: 23012322 DOI: 10.1096/fj.12-216622] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The genomic amplification of chromosome 1q long arm, the chromosomal region containing C1orf61, is a common event in human cancers. However, the expression pattern of chromosome 1 open reading frame 61 (C1orf61) in hepatocellular carcinoma (HCC) and its effects on HCC progression remain unclear. We have previously reported that C1orf61 is highly up-regulated during human embryogenesis. In this study, we report that C1orf61 expression is associated with the progression of liver disease. We found that C1orf61 is up-regulated in hepatic cirrhosis tissues and is further up-regulated in primary HCC tumors. Moreover, hepatitis B virus (HBV)-positive patients exhibited significantly higher levels of C1orf61 expression than HBV-negative patients. The evaluation of highly malignant HCC cell lines revealed high protein expression levels of C1orf61. Furthermore, the C1orf61 protein was found to be predominantly distributed within the cytoplasm. The ectopic expression of C1orf61 in the nonmalignant L02 cell line promoted cellular proliferation and colony formation in vitro, as well as cell cycle progression via the regulation of the expression of specific cell cycle-related proteins. In addition, the overexpression of C1orf61 in L02 cells facilitated cellular invasion and metastasis. The down-regulation of epithelial markers (E-cadherin and occludin) and the up-regulation of mesenchymal markers (N-cadherin, vimentin, and snail) suggested that the overexpression of C1orf61 induced the epithelial-mesenchymal transition (EMT) that is linked to metastasis. Taken together, our findings demonstrate, for the first time, the roles of C1orf61 in HCC tumorigenesis and metastasis.
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Affiliation(s)
- Hai-Ming Hu
- College of Life Sciences, Wuhan University, Wuhan, China
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12
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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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13
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Papadaki M, Siapati EK, Vassilopoulos G. A foamy virus vector system for stable and efficient RNAi expression in mammalian cells. Hum Gene Ther 2011; 22:1293-303. [PMID: 21456885 DOI: 10.1089/hum.2010.223] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The promise of the RNA interference (RNAi) technology is equally dependent on the efficiency and stability of gene silencing. The aim of the present study was the development of foamy virus (FV) vectors for stable RNAi, utilizing two potent RNA polymerase III (Pol III) promoters. Using green fluorescent protein as a target gene, we examined the efficiency of mouse U6 (mU6) and human H1 Pol III promoters in different human cell lines and mouse hematopoietic stem cells (HSCs) ex vivo and in vivo, following bone marrow transplantation. Both our mU6 and H1 FV vectors mediated very efficient gene silencing with as low as one vector copy per cell. However, transduction of human cell lines with FV vectors expressing short hairpin RNA from mU6 led to the gradual elimination of cells in culture, as opposed to H1-harboring cells, underscoring the importance of the expression system or cellular context in the evaluation of the overall RNAi effects. The efficiency and stability of the H1 vectors were further shown by the successful silencing of BCR-ABL in K562 cells. Accordingly, mU6 vectors induced efficient and stable gene silencing in mouse HSCs following bone marrow transplantation. Our work is the first in vivo study on the efficiency and stability of RNAi gene silencing in HSCs with FV vectors, currently a safe alternative for viral gene transfer.
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Affiliation(s)
- Magdalini Papadaki
- Division of Genetics and Gene Therapy, Center for Basic Research II, Biomedical Research Foundation of the Academy of Athens, Athens, Greece
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14
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Eekels JJM, Geerts D, Jeeninga RE, Berkhout B. Long-term inhibition of HIV-1 replication with RNA interference against cellular co-factors. Antiviral Res 2010; 89:43-53. [PMID: 21093490 DOI: 10.1016/j.antiviral.2010.11.005] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2010] [Revised: 11/04/2010] [Accepted: 11/08/2010] [Indexed: 12/12/2022]
Abstract
In this study we tested whether HIV-1 replication could be inhibited by stable RNAi-mediated knockdown of cellular co-factors. Cell lines capable of expressing shRNAs against 30 candidate co-factors implicated at different steps of the viral replication cycle were generated and analyzed for effects on cell viability and inhibition of HIV-1 replication. For half of these candidate co-factors we obtained knockdown cell lines that are less susceptible to virus replication. For three co-factors (ALIX, ATG16 and TRBP) the cell lines were resistant to HIV-1 replication for up to 2 months. With these cells we could test the hypothesis that HIV-1 is not able to escape from RNAi-mediated suppression of cellular co-factors, which was indeed not detected.
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Affiliation(s)
- Julia J M Eekels
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
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15
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Zhang YL, Cheng T, Cai YJ, Yuan Q, Liu C, Zhang T, Xia DZ, Li RY, Yang LW, Wang YB, Yeo AET, Shih JWK, Zhang J, Xia NS. RNA Interference inhibits hepatitis B virus of different genotypes in vitro and in vivo. BMC Microbiol 2010; 10:214. [PMID: 20696079 PMCID: PMC2927532 DOI: 10.1186/1471-2180-10-214] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2010] [Accepted: 08/10/2010] [Indexed: 12/19/2022] Open
Abstract
Background Hepatitis B virus (HBV) infection increases the risk of liver disease and hepatocellular carcinoma. Small interfering RNA (siRNA) can be a potential new tool for HBV therapy. Given the high heterogeneity of HBV strains and the sensitivity towards sequences changes of siRNA, finding a potent siRNA inhibitor against the conservative site on the HBV genome is essential to ensure a therapeutic application. Results Forty short hairpin RNA (shRNA) expression plasmids were constructed to target conserved regions among nine HBV genotypes. HBV 1.3-fold genome plasmids carrying various genotypes were co-transfected with shRNA plasmids into either Huh7 cells or mice. The levels of various viral markers were examined to assess the anti-HBV efficacy of siRNA. Four (B245, B376, B1581 and B1789) were found with the ability to potently inhibit HBV RNA, DNA, surface antigen (HBsAg), e antigen (HBeAg) and core antigen (HBcAg) expression in HBV genotypes A, B, C, D and I (a newly identified genotype) in Huh7 cells and in mice. No unusual cytotoxicity or off-target effects were noted. Conclusions Such siRNA suggests an alternate way of inhibiting various HBV genotypes in vitro and in vivo, promising advances in the treatment of HBV.
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Affiliation(s)
- Ya-Li Zhang
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science, Xiamen University, Xiamen, Fujian Province, China
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16
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Sun D, Rösler C, Kidd-Ljunggren K, Nassal M. Quantitative assessment of the antiviral potencies of 21 shRNA vectors targeting conserved, including structured, hepatitis B virus sites. J Hepatol 2010; 52:817-26. [PMID: 20400195 DOI: 10.1016/j.jhep.2009.10.038] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2009] [Revised: 10/16/2009] [Accepted: 10/19/2009] [Indexed: 12/12/2022]
Abstract
BACKGROUND & AIMS RNA interference (RNAi) may offer new treatment options for chronic hepatitis B. Replicating via an RNA intermediate, hepatitis B virus (HBV) is known to be principally vulnerable to RNAi. However, beyond delivery, the relevant issues of potential off-target effects, target site conservation in circulating HBV strains, and efficacy of RNAi itself have not systematically been addressed, nor can the different existing data be quantitatively compared. The aim of this study was to provide such information. METHODS To focus on the intracellular RNAi process itself and minimise other variables affecting overall RNAi efficacy, we used a robust co-transfection system to quantitatively assess the relative potencies of 21 small-hairpin (sh) RNA vectors, targeting conserved sites throughout the HBV genome, against viral RNAs, proteins, nucleocapsids, and secreted virions under standardised conditions. RESULTS The approach enabled a distinct efficacy ranking, with the six most potent shRNAs achieving 95% reductions in virion formation, sequence-specifically and without detectable interferon induction, yet by differentially affecting different steps. Efficacy correlated poorly with predictions and was not principally abolished by target structure. Sequence comparisons suggest that truly conserved, RNAi-targetable sequences comprise less than 500 nucleotides of the circulating HBV genomes. CONCLUSIONS The HBV genome can harbour only a finite number of optimal target sites, but current predictions are poorly suited to constrain the number of possible candidates. However, the small size of the highly conserved sequence space suggests experimental identification as a viable option.
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Affiliation(s)
- Dianxing Sun
- Bethune International Peace Hospital, Departmrnt of Liver Disease, 398 West Zhongshan Road, 050082 Shijiazhuang, PR China
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17
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Yang CJ, Liu YK, Liu CL, Shen CN, Kuo ML, Su CC, Tseng CP, Yen TC, Shen CR. Inhibition of acidic mammalian chitinase by RNA interference suppresses ovalbumin-sensitized allergic asthma. Hum Gene Ther 2010; 20:1597-606. [PMID: 19548841 DOI: 10.1089/hum.2008.092] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Asthma, a chronic helper T cell type 2-mediated inflammatory disease, is characterized by airway hyperresponsiveness and inflammation. Growing evidence suggests that increased expression of acidic mammalian chitinase (AMCase) may play a role in the pathogenesis of asthma. In the present study, we sought to develop an RNA interference approach to suppress allergic asthma in mice through silencing of AMCase expression. Mice sensitized with ovalbumin (OVA) were intratracheally administered a recombinant adeno-associated virus expressing short hairpin RNA (rAAV-shRNA) against AMCase. In OVA-sensitized mice, the development of allergic symptoms was significantly associated with elevated AMCase expression. After administration of rAAV-shRNA, there was a significant reduction of AMCase expression in the lung and in bronchoalveolar lavage fluid (BALF) cells of sensitized mice. Sensitized mice receiving rAAV-shRNA showed a significant improvement in allergic symptoms, including airway hyperresponsiveness (AHR), eosinophil infiltration, eotaxin, interleukin-13 secretion in BALF, and serum OVA-specific IgE level. Our data suggest the hyperexpression of AMCase in asthma can be suppressed by rAAV-mediated shRNA. Silencing AMCase expression by shRNA may be a promising therapeutic strategy in asthma.
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Affiliation(s)
- Ching-Jen Yang
- Department of Chemical and Materials Engineering, Chang Gung University, Taoyuan 333, Taiwan
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18
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Chattopadhyay S, Ely A, Bloom K, Weinberg MS, Arbuthnot P. Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles. Biochem Biophys Res Commun 2009; 389:484-9. [PMID: 19733548 DOI: 10.1016/j.bbrc.2009.09.004] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2009] [Accepted: 09/01/2009] [Indexed: 12/29/2022]
Abstract
RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.
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Affiliation(s)
- Saket Chattopadhyay
- Antiviral Gene Therapy Research Unit, University of the Witwatersrand, Johannesburg, South Africa
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19
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von Eije KJ, ter Brake O, Berkhout B. Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAs. J Gene Med 2009; 11:459-67. [PMID: 19384894 DOI: 10.1002/jgm.1329] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
BACKGROUND RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. METHODS In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. RESULTS Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. CONCLUSIONS These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.
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Affiliation(s)
- Karin J von Eije
- Laboratory of Experimental Virology, Department of Medical Microbiology and Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
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20
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Kim JW, Lee SH, Park YS, Jeong SH, Kim N, Lee DH. [Inhibition of in vitro hepatitis B virus replication by lentivirus-mediated short-hairpin RNA against HBx]. THE KOREAN JOURNAL OF HEPATOLOGY 2009; 15:15-24. [PMID: 19346782 DOI: 10.3350/kjhep.2009.15.1.15] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUNDS/AIMS Hepatitis B virus (HBV) replicates via RNA intermediates, which could serve as targets for RNA interference (RNAi). Vector-mediated short-hairpin RNA (shRNA) can induce sustained RNAi in comparison to small interfering RNA. Lentiviral vector is known to induce prolonged RNAi with high transduction efficiency. In this study, we sought to test the in vitro efficacy of shRNA delivered by a lentiviral vector in suppressing the replication of HBV. METHODS Two shRNA sequences against the hepatitis B viral protein HBx (sh1580 and sh1685) were cloned downstream of the U6 promoter in an HIV-based plasmid to generate third-generation lentiviral vectors. HepAD38 cells were transduced with anti-HBx lentiviral vectors, and HBV replication was induced for 5 days. HBV DNA was isolated and quantified using real-time PCR. RESULTS Lentiviral vectors encoding the shRNA against HBV transduced HepAD38 cells with high efficacy. The total intracellular HBV DNA content was significantly reduced by both sh1580 and sh1685 (2.9% and 12.0%, respectively; P<0.05). HBV covalently closed circular DNA (cccDNA) was also suppressed significantly (19.7% and 25.5%, respectively; P<0.05). CONCLUSIONS Lentivirus-mediated delivery of shRNA against HBx can effectively suppress the replication of HBV and reduce HBV cccDNA in cell culture systems.
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Affiliation(s)
- Jin-Wook Kim
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Seoul National University Bungdang Hospital, Seongnam, Korea.
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21
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Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication. PLoS One 2009; 4:e5671. [PMID: 19479060 PMCID: PMC2682565 DOI: 10.1371/journal.pone.0005671] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2009] [Accepted: 05/03/2009] [Indexed: 01/12/2023] Open
Abstract
RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection.
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22
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Starkey JL, Chiari EF, Isom HC. Hepatitis B virus (HBV)-specific short hairpin RNA is capable of reducing the formation of HBV covalently closed circular (CCC) DNA but has no effect on established CCC DNA in vitro. J Gen Virol 2009; 90:115-26. [PMID: 19088280 DOI: 10.1099/vir.0.004408-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the source of HBV transcripts and persistence in chronically infected patients. The novel aspect of this study was to determine the effect of RNA interference (RNAi) on HBV CCC DNA when administered prior to establishment of HBV replication or during chronic HBV infection. HBV replication was initiated in HepG2 cells by transduction with HBV baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA, thus simulating chronic HBV infection. HepG2 cells were transduced with short hairpin RNA (shRNA)-expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication, and the levels of HBV RNA, HBV surface antigens (HBsAg) and replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced until day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. We conclude that RNAi may have a therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. Our data support previous findings demonstrating the stability of HBV CCC DNA following antiviral therapy. This study also reports the development of a novel HBV baculovirus subculture system that can be used to evaluate antiviral effects on chronic HBV replication.
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Affiliation(s)
- Jason L Starkey
- Department of Microbiology and Immunology, Milton S. Hershey Medical Center, The Penn State University College of Medicine, Hershey, PA 17033, USA
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23
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Carmona S, Jorgensen MR, Kolli S, Crowther C, Salazar FH, Marion PL, Fujino M, Natori Y, Thanou M, Arbuthnot P, Miller AD. Controlling HBV Replication in Vivo by Intravenous Administration of Triggered PEGylated siRNA-Nanoparticles. Mol Pharm 2009; 6:706-17. [DOI: 10.1021/mp800157x] [Citation(s) in RCA: 95] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- Sergio Carmona
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Michael R. Jorgensen
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Soumia Kolli
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Carol Crowther
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Felix H. Salazar
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Patricia L. Marion
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Masato Fujino
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Yukikazu Natori
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Maya Thanou
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Patrick Arbuthnot
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
| | - Andrew D. Miller
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Private Bag 3, WITS 2050, South Africa, Imperial College Genetic Therapies Centre, Department of Chemistry, Flowers Building, Armstrong Road, Imperial College London, London SW7 2AZ, U.K., Stanford University, Stanford, California, Hepadnavirus Testing, Inc., Mountain View, California, RNAi Co., Cosmos Hongo Bldg. 10F, 4-1-4, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan, and
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24
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Crowther C, Ely A, Hornby J, Mufamadi S, Salazar F, Marion P, Arbuthnot P. Efficient Inhibition of Hepatitis B Virus Replication In Vivo, Using Polyethylene Glycol-Modified Adenovirus Vectors. Hum Gene Ther 2008; 19:1325-31. [DOI: 10.1089/hum.2008.066] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
- Carol Crowther
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa
| | - Abdullah Ely
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa
| | - Judith Hornby
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa
| | - Steven Mufamadi
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa
| | | | | | - Patrick Arbuthnot
- Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Hematology, University of the Witwatersrand, Johannesburg, South Africa
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25
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Rauschhuber C, Xu H, Salazar FH, Marion PL, Ehrhardt A. Exploring gene-deleted adenoviral vectors for delivery of short hairpin RNAs and reduction of hepatitis B virus infection in mice. J Gene Med 2008; 10:878-89. [PMID: 18470951 DOI: 10.1002/jgm.1207] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND RNA interference based therapeutic approaches hold promise for the treatment of patients chronically infected with hepatitis B virus (HBV). To conquer HBV infection, long-term suppression of target transcripts in all hepatocytes without toxic effects may be required. The present study explored gene-deleted adenoviral vectors (GD-AdV) lacking all viral coding sequences for delivery of the previously described short hairpin RNA (shRNA) HBVU6no.2, which was demonstrated to result in post-transcriptional knock-down of HBV transcripts. METHODS We established conditions for shRNA delivery expressed from GD-AdV in vitro and in vivo and observed up to 96% shRNA-mediated knockdown of luciferase expressed in mouse liver. To investigate in vivo efficacy of HBVU6no.2 expressed from a GD-AdV, we explored a transient and a transgenic mouse model for HBV infection. RESULTS We observed an up to 68% drop in serum HBV surface antigen (HBsAg) levels in the transient and the transgenic mouse model for HBV infection, respectively. Interestingly, we detected an up to 86% drop in HBsAg levels in both animal models after administration of a control GD-AdV encoding beta-galactosidase. In concordance with reduced serum HBsAg levels, we observed reduced HBV replication as demonstrated by Southern blot analysis of HBV genomes. CONCLUSIONS The present study demonstrates that GD-AdV can be used against HBV infection but the design of DNA sequences including shRNAs contained in the vector and virus-host interactions during superinfection needs to be carefully considered.
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Affiliation(s)
- Christina Rauschhuber
- Max von Pettenkofer-Institute, Department of Virology, Ludwig-Maximilians-Universität München, Munich, Germany
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26
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Vector design for liver-specific expression of multiple interfering RNAs that target hepatitis B virus transcripts. Antiviral Res 2008; 80:36-44. [PMID: 18499277 DOI: 10.1016/j.antiviral.2008.04.001] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2008] [Revised: 04/08/2008] [Accepted: 04/10/2008] [Indexed: 12/11/2022]
Abstract
RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence-specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue-specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes.
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27
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Feng Z, Jiang P, Wang X, Li Y, Jiang W. Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo. Antiviral Res 2007; 77:186-94. [PMID: 18199493 DOI: 10.1016/j.antiviral.2007.11.005] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2007] [Revised: 10/31/2007] [Accepted: 11/21/2007] [Indexed: 10/22/2022]
Abstract
Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS), which is responsible for the heavy economic losses in stockbreeding. There are no specific antiviral drugs for treatment of the virus infection. We have now constructed two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against either ORF1 (rAdS1) or ORF2 (rAdS2) of PCV2 and measured the inhibition of PCV2 replication. The results showed that delivery of these shRNAs by recombinant adenovirus into PK15 cells could induce a significant inhibition of viral RNA and DNA replication and protein synthesis level in cells subsequently infected with PCV2. The antiviral effect was dose-dependent and could sustain at least for 120h and the inhibition of virus replication could be significantly strengthened by combination of rAdS1 with rAdS2. Mice injected with shRNA before PCV2 infection showed substantial and low level of PCV2 DNA replication in the spleen during the period of 21-28 days post-PCV2 infection. These results indicated that shRNAs generated by adenovirus could sufficiently and continuously inhibit PCV2 infection in vitro as well as in vivo. The adenovirus based shRNA targeting ORF1 and ORF2 of PCV2 might be a new potential alternative strategy for controlling PCV2 infection.
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Affiliation(s)
- Zhixin Feng
- Key Laboratory of Animal Diseases Diagnostic and Immunology, College of Veterinary Medicine, Nanjing Agricultural University, Ministry of Agriculture, Nanjing 210095, China
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28
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Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference. J Virol 2007; 82:2895-903. [PMID: 18077712 DOI: 10.1128/jvi.02035-07] [Citation(s) in RCA: 102] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but-in contrast to what is observed with nef-no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.
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29
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Chen Y, Cheng G, Mahato RI. RNAi for treating hepatitis B viral infection. Pharm Res 2007; 25:72-86. [PMID: 18074201 PMCID: PMC2217617 DOI: 10.1007/s11095-007-9504-0] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2007] [Accepted: 11/14/2007] [Indexed: 12/18/2022]
Abstract
Chronic hepatitis B virus (HBV) infection is one of the leading causes of liver cirrhosis and hepatocellular carcinoma (HCC). Current treatment strategies of HBV infection including the use of interferon (IFN)-α and nucleotide analogues such as lamivudine and adefovir have met with only partial success. Therefore, it is necessary to develop more effective antiviral therapies that can clear HBV infection with fewer side effects. RNA interference (RNAi), by which a small interfering RNA (siRNA) induces the gene silence at a post-transcriptional level, has the potential of treating HBV infection. The successful use of chemically synthesized siRNA, endogenous expression of small hairpin RNA (shRNA) or microRNA (miRNA) to silence the target gene make this technology towards a potentially rational therapeutics for HBV infection. However, several challenges including poor siRNA stability, inefficient cellular uptake, widespread biodistribution and non-specific effects need to be overcome. In this review, we discuss several strategies for improving the anti-HBV therapeutic efficacy of siRNAs, while avoiding their off-target effects and immunostimulation. There is an in-depth discussion on the (1) mechanisms of RNAi, (2) methods for siRNA/shRNA production, (3) barriers to RNAi-based therapies, and (4) delivery strategies of siRNA for treating HBV infection.
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Affiliation(s)
- Yong Chen
- Huai-An 4th People’s Hospital, Jiangsu, China
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
| | - Guofeng Cheng
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
| | - Ram I. Mahato
- Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, 19 S Manassas Street, Memphis, Tennessee 38103 USA
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Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells. Chin Med J (Engl) 2007. [DOI: 10.1097/00029330-200712020-00004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
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Abstract
RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.
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Affiliation(s)
- Yan Ma
- Stanley Ho Centre for Emerging Infectious Diseases, and Li Ka Shing Institute of Health Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
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Arbuthnot P, Longshaw V, Naidoo T, Weinberg MS. Opportunities for treating chronic hepatitis B and C virus infection using RNA interference. J Viral Hepat 2007; 14:447-59. [PMID: 17576386 DOI: 10.1111/j.1365-2893.2006.00818.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Activating the RNA interference (RNAi) pathway to achieve silencing of specific genes is one of the most exciting new developments of molecular biology. A particularly interesting use of this technology is inhibition of defined viral gene expression. In this review, we discuss the potential application of RNAi to treatment of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Globally, these hepatotropic viruses are the most important causes of cirrhosis and liver cancer. Available treatments have their limitations, which makes development of novel effective RNAi-based therapies for HBV and HCV especially significant. Several investigations carried out in vitro and in vivo are summarized, which demonstrate proof of principle that HBV and HCV can be inhibited by RNAi activators. Challenges facing further development of this technology to a stage of clinical application are discussed.
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Affiliation(s)
- P Arbuthnot
- Hepatitis B Virus Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.
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Wang XY, Zhang JM, Yin YK, Xie Y, Huang YX, Wu XH, Weng XH. Inhibition of hepatitis B virus expression and replication by RNA interference. Shijie Huaren Xiaohua Zazhi 2007; 15:1688-1694. [DOI: 10.11569/wcjd.v15.i15.1688] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the RNA interference on hepatitis B virus (HBV) replication by a reverse transcription virus vector which can express short hairpin RNA inside cells.
METHODS: pSIREN vectors with inserted oligonucleotides targeting on reverse transcriptase (RT) regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells. Viral antigens were measured by enzyme-linked immunosorbent assay (ELISA). HBV core particle DNA was measured and quantified by real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) and Southern blot. Viral RNA was analyzed by Northern blot.
RESULTS: Three RNA interfering targets were identified, and three corresponding retrovirus vectors, named 154i, 312i and 734i, were obtained. It was found that 312i markedly inhibited the expression of pHBV3.8, and the levels of HBsAg and HBeAg were 39% and 41% of those in the negative control group (P = 0.001, P = 0.000). RFQ-PCR showed that the level of HBV core particle DNA was significantly lower in 312i group than that in the negative control group (21.3% ± 1.1% vs 100.0% ± 10.6%, P = 0.0046). Southern and Northern blot demonstrated a lowest replication and transcription level of HBV in 312i group (10.5%, 12.0%).
CONCLUSION: A new RNAi system is identified in the RT regions of HBV genome, and the corresponding retrovirus vectors, which can remarkably inhibit the replication and expression of HBV, are also constructed.
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Kayhan H, Karatayli E, Turkyilmaz AR, Sahin F, Yurdaydin C, Bozdayi AM. Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2 2.2.15 cell lines. Arch Virol 2007; 152:871-9. [PMID: 17245534 DOI: 10.1007/s00705-006-0918-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2006] [Accepted: 12/06/2006] [Indexed: 12/18/2022]
Abstract
In this study, the effect of RNAi on HBV replication was observed in a cell culture model, HepG2 2.2.15 cell line, which supports human HBV ayw replication and expression. Aim of the study was to investigate effects of shRNAs (small hairpin RNAs) targeting hepatitis B virus mRNAs on the viral replication in HepG2 2.2.15 cells. We selected three target HBV mRNA regions with different putative secondary structures to test whether the secondary structure of RNA may affect the inhibition efficacy on the target HBV RNA. Three HBV-specific siRNAs (small interfering RNA) were designed targeting X (1689-1708), Core (2229-2248) and S (765-784 nt) transcripts. HepG2 2.2.15 cells were transfected with shRNA expressing plasmids, P765, P2229 and P1689 targeting S, core and X region, respectively or a mock plasmid targeting lacZ gene. The culture media was collected throughout six days after transfection and analyzed by real-time PCR. Viral DNA production was suppressed for 7 days. The HBV DNA levels were decreased by 73, 72 and 79% with P765, P2229 and P1689 vectors, respectively. In conclusion, the shRNAs designed for X, core and S regions, specifically and significantly suppressed HBV DNA. siRNAs potentially may be used in treatment of hepatitis B.
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Affiliation(s)
- H Kayhan
- Institute of Hepatology, Ankara University, Ankara, Turkey
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35
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Bastone P, Romen F, Liu W, Wirtz R, Koch U, Josephson N, Langbein S, Löchelt M. Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors. Gene Ther 2007; 14:613-20. [PMID: 17203107 DOI: 10.1038/sj.gt.3302890] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
As serious side effects affected recent virus-mediated gene transfer studies, novel vectors with improved safety profiles are urgently needed. In the present study, replication-deficient retroviral vectors based on feline foamy virus (FFV) were constructed and analyzed. The novel FFV vectors are devoid of almost the complete env gene plus the internal promoter - accessory bel gene cassette including the gene for the viral transcriptional transactivator Bel1/Tas. In these Bel1/Tas-independent vectors, expression of the lacZ (beta-galactosidase) marker gene is directed by the heterologous, constitutively active human ubiquitin C promoter (ubi). Env-transcomplemented vectors have un-concentrated titers of more than 10(5) transducing units/ml. The vectors allow efficient transduction of a broad array of diverse target cells, which can be increased by repeated vector exposure. However, the number of lacZ marker gene expressing cells decreased slightly upon serial passages of the transduced cells. Vectors carrying a self-inactivating (SIN) deletion of the TATA box and most parts of the viral promoter were not rescued by wt FFV whereas those with the intact or a partially deleted promoter were readily reactivated. This finding indicates that the viral promoters are in fact non-functional, pointing to a highly advantageous safety profile of these new FFV-ubi-lacZ-SIN vectors.
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Affiliation(s)
- P Bastone
- Abt. Genomveränderungen und Karzinogenese, Forschungsschwerpunkt Infektion und Krebs, Deutsches Krebsforschungszentrum, Heidelberg, Germany
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36
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Fichou Y, Férec C. The potential of oligonucleotides for therapeutic applications. Trends Biotechnol 2006; 24:563-70. [PMID: 17045686 DOI: 10.1016/j.tibtech.2006.10.003] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2006] [Revised: 08/21/2006] [Accepted: 10/03/2006] [Indexed: 12/15/2022]
Abstract
Viral-derived particles have been widely used and described in gene therapy clinical trials. Although substantial results have been achieved, major safety issues have also arisen. For more than a decade, oligonucleotides have been seen as an alternative to gene complementation by viral vectors or DNA plasmids, either to correct the genetic defect or to silence gene expression. The development of RNA interference has strengthened the potential of this approach. Recent clinical trials have also tested the ability of aptamer molecules and decoy oligonucleotides to sequestrate pathogenic proteins. Here, we review the potential of oligonucleotides in gene therapy, outline what has already been accomplished, and consider what remains to be done.
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Affiliation(s)
- Yann Fichou
- Inserm U613, Université de Bretagne Occidentale, 46 rue Félix Le Dantec, 29275 Brest Cedex, France
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37
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Xin KQ, Mizukami H, Urabe M, Toda Y, Shinoda K, Yoshida A, Oomura K, Kojima Y, Ichino M, Klinman D, Ozawa K, Okuda K. Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells. J Virol 2006; 80:11899-910. [PMID: 17005662 PMCID: PMC1676308 DOI: 10.1128/jvi.00890-06] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.
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Affiliation(s)
- Ke-Qin Xin
- Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan
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38
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Jia F, Zhang YZ, Liu CM. Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery. J Biotechnol 2006; 128:32-40. [PMID: 17049658 DOI: 10.1016/j.jbiotec.2006.09.007] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2006] [Revised: 08/25/2006] [Accepted: 09/14/2006] [Indexed: 12/11/2022]
Abstract
RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.
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Affiliation(s)
- Fang Jia
- Molecular Virology Research Center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China
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39
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Alam AKMS, Florey O, Weber M, Pillai RG, Chan C, Tan PH, Lechler RI, McClure MO, Haskard DO, George AJT. Knockdown of mouse VCAM-1 by vector-based siRNA. Transpl Immunol 2006; 16:185-93. [PMID: 17138052 DOI: 10.1016/j.trim.2006.08.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2006] [Accepted: 08/01/2006] [Indexed: 11/26/2022]
Abstract
Graft rejection is critically dependent on the recruitment of leukocytes via adhesion molecules on the endothelium, and inhibition of these interactions can prolong graft survival. We have therefore developed an approach using siRNA to inhibit the expression of VCAM-1 in endothelial cells. We transfected siRNA constructs into murine corneal and vascular endothelium and looked at expression of VCAM-1 and other surface molecules by flow cytometry. Adhesion assays (both static and under flow) were used to determine the effect of VCAM-1 inhibition. The activation of cellular stress responses was assessed by RT-PCR. Constructs encoding siRNA can block expression of VCAM-1 in both corneal and vascular endothelial cells (in the latter case after cytokine stimulation). Inhibition of VCAM-1 expression reduced the ability of T cells to adhere to endothelium. However, there were non-specific effects of siRNA expression, including upregulation of (Programmed Death Ligand 1) PDL1 and decreased cell growth. Analysis of stress pathways showed that the endothelial cells transfected with siRNA had upregulated molecules associated with cell stress. While these data are supportive of a potential therapeutic role for siRNA constructs in blocking the expression of adhesion molecules, they also highlight potential non-specific effects of siRNA that must be carefully considered in any application of this technology.
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Affiliation(s)
- A K M Shamsul Alam
- Department of Immunology, Division of Medicine, Imperial College London, Hammersmith Campus, London W12 0NN, UK
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40
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Grimm D, Kay MA. Therapeutic short hairpin RNA expression in the liver: viral targets and vectors. Gene Ther 2006; 13:563-75. [PMID: 16453009 DOI: 10.1038/sj.gt.3302727] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Over 500 million people worldwide are infected with one or more different and unrelated types of human hepatitis virus. Such individuals are at a high risk of developing acute or chronic hepatic disease, and ultimately dying from sequelae. Although a vaccine is available for hepatitis A and B virus, treatment options for chronically infected patients are limited, and particularly ineffective in case of hepatitis C virus (HCV) infection. A promising new avenue currently being explored is to harness the power of RNA interference for development of an antiviral therapy. The timing to pursue this particular approach is excellent, with the first in vivo animal models for HCV infection becoming available, and the technology for liver-specific expression of short hairpin RNAs advancing at a rapid pace. Here, we critically review these important current developments, and discuss the next steps to bring this novel approach into the clinics.
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Affiliation(s)
- D Grimm
- Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA 94305, USA
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41
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Guo Y, Guo H, Zhang L, Xie H, Zhao X, Wang F, Li Z, Wang Y, Ma S, Tao J, Wang W, Zhou Y, Yang W, Cheng J. Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. J Virol 2006; 79:14392-403. [PMID: 16254373 PMCID: PMC1280207 DOI: 10.1128/jvi.79.22.14392-14403.2005] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNA's side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.
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Affiliation(s)
- Yong Guo
- Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
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42
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Taxman DJ, Livingstone LR, Zhang J, Conti BJ, Iocca HA, Williams KL, Lich JD, Ting JPY, Reed W. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol 2006; 6:7. [PMID: 16433925 PMCID: PMC1409772 DOI: 10.1186/1472-6750-6-7] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2005] [Accepted: 01/24/2006] [Indexed: 11/18/2022] Open
Abstract
Background RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. Results To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. Conclusion The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.
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Affiliation(s)
- Debra J Taxman
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - Laura R Livingstone
- Program of Molecular Biology and Biotechnology; University of North Carolina, Chapel Hill, NC 27599, USA
| | - Jinghua Zhang
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - Brian J Conti
- Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
| | - Heather A Iocca
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - Kristi L Williams
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - John D Lich
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - Jenny P-Y Ting
- Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center; University of North Carolina, Chapel Hill, NC 27599, USA
| | - William Reed
- Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina, Chapel Hill, NC 27599, USA
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43
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Xuan B, Qian Z, Hong J, Huang W. EsiRNAs inhibit Hepatitis B virus replication in mice model more efficiently than synthesized siRNAs. Virus Res 2006; 118:150-5. [PMID: 16423421 DOI: 10.1016/j.virusres.2005.12.005] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2005] [Revised: 12/12/2005] [Accepted: 12/12/2005] [Indexed: 12/20/2022]
Abstract
RNA interference (RNAi) has been proved to be a promising strategy to combat Hepatitis B virus (HBV) infection by way of cell culture and animal model studies. In this work, esiRNAs (endoribonuclease-prepared siRNAs) targeting all of the four open reading frames (ORFs) of HBV genome were prepared. In vitro experiment showed that esiHBVP suppressed HBsAg expression most effectively. Its capacity to suppress HBV replication in vivo was then tested. A single dose of 1 microg esiHBVP was able to reduce HBsAg and HBeAg level in the mouse serum by 90 and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33 and 45%. Immunostaining of HBcAg showed that esiHBVP inhibited HBcAg expression more potently than chemically synthesized siRNA. Quantification of HBV DNA in the mouse serum showed 1 microg eiHBVP treatment reduced serum HBV DNA copy number to 18% that of the untreated control, while 1 microg siRNA treatment only reduced that to 63%. In conclusion, the data presented here proved that esiRNA is much more efficient in suppressing HBV replication than chemically synthesized siRNA, and it might be a better therapeutic agent to fight against HBV infection.
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Affiliation(s)
- Baoqin Xuan
- Department of Biochemistry, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai 200433, China
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44
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Raoul C, Barker SD, Aebischer P. Viral-based modelling and correction of neurodegenerative diseases by RNA interference. Gene Ther 2005; 13:487-95. [PMID: 16319945 DOI: 10.1038/sj.gt.3302690] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Experimental recapitulation of recessive human genetic neurodegenerative disease in rodents can be classically addressed through genetic disruption of the related gene. Although very informative, this specific gene targeting is restricted to mice and precludes a species scale-up towards non-human primates. Concomitantly, this requirement to silence a specific gene in a broad range of animal models is important in the design of therapeutic approaches to dominantly inherited neurodegenerative diseases. The emergence of RNA interference (RNAi), a highly specific mechanism of post-translational gene silencing, has opened a plethora of biological application ranging from reverse genetic analysis to therapeutic schemes. Recombinant viral vectors, by promoting a long-lasting delivery of genetic instructions in a broad range of cellular types of different species origins, represent potential platforms mandating silencing of specific gene products through RNAi. This review aims at providing an overview of the different viral systems engineered so far for efficient in vitro and in vivo delivery of RNAi instructions. Additionally, the potential of RNAi for functional analysis and therapy for polyglutamine disorders or amyotrophic lateral sclerosis is discussed.
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Affiliation(s)
- C Raoul
- Institute of Neurosciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
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45
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Ren GL, Bai XF, Zhang Y, Chen HM, Huang CX, Wang PZ, Li GY, Zhang Y, Lian JQ. Stable inhibition of hepatitis B virus expression and replication by expressed siRNA. Biochem Biophys Res Commun 2005; 335:1051-9. [PMID: 16111658 DOI: 10.1016/j.bbrc.2005.07.170] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2005] [Accepted: 07/26/2005] [Indexed: 12/14/2022]
Abstract
RNA interference might be an efficient antiviral therapy for some obstinate illness. Here, we studied the effects of hepatitis B virus (HBV)-specific 21-nt small interfering RNAs (siRNA) on HBV gene expression and replication in 2.2.15 cells. Seven vectors expressing specific hairpin siRNA driven by the RNA polymerase II-promoter were constructed and transfected into 2.2.15 cells. In the cell strain that can stably express functional siRNA, the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg) secretion into culture media was inhibited by 86% and 91%, respectively, as shown by an enzyme-linked immunosorbent assay. Immunofluorescence and Western blot indicated similar results. HBV DNA was markedly restrained by 3.28-fold, as assessed by the fluorescent quantitation PCR. Moreover, the HBV mRNA was significantly reduced by 80% based on semiquantitative RT-PCR. In conclusion, the specific siRNA can knock down the HBV gene expression and replication in vitro, and the silence effects have no relationship with interferon response.
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Affiliation(s)
- Guang-Li Ren
- The State Key Discipline and Diagnosis and Treatment Center of Infectious Diseases of Chinese People Liberation Army, Tang Du Hospital, Fourth Military Medical University, Xin Yi Road, Fang Zhi District, Xi'an 710038, China.
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46
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N/A, 马 瑾. N/A. Shijie Huaren Xiaohua Zazhi 2005; 13:2179-2182. [DOI: 10.11569/wcjd.v13.i18.2179] [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] [Indexed: 02/06/2023] Open
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