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Hu W, Zheng H, Li Q, Wang Y, Liu X, Hu X, Liu W, Liu S, Chen Z, Feng W, Cai X, Li N. shRNA transgenic swine display resistance to infection with the foot-and-mouth disease virus. Sci Rep 2021; 11:16377. [PMID: 34385528 PMCID: PMC8361160 DOI: 10.1038/s41598-021-95853-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2020] [Accepted: 07/29/2021] [Indexed: 12/15/2022] Open
Abstract
Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3+, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.
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Affiliation(s)
- Wenping Hu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.,Key Laboratory of Animal Genetics and Breeding and Reproduction of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China
| | - Haixue Zheng
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
| | - Qiuyan Li
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.,Beijing Genprotein Biotechnology Company, Beijing, China
| | - Yuhang Wang
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Xiangtao Liu
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China
| | - Xiaoxiang Hu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Wenjie Liu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Shen Liu
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Zhisheng Chen
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Wenhai Feng
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China
| | - Xuepeng Cai
- State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Diseases Reference Laboratory, Lanzhou Veterinarian Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China.
| | - Ning Li
- State Key Laboratory of AgroBiotechnology, China Agricultural University, Beijing, China.
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Khalil IA, Yamada Y, Harashima H. Optimization of siRNA delivery to target sites: issues and future directions. Expert Opin Drug Deliv 2018; 15:1053-1065. [PMID: 30198792 DOI: 10.1080/17425247.2018.1520836] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Affiliation(s)
- Ikramy A. Khalil
- Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
- Faculty of Pharmacy, Assiut University, Assiut, Egypt
| | - Yuma Yamada
- Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
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Chakraborty C, Sharma AR, Sharma G, Doss CGP, Lee SS. Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine. MOLECULAR THERAPY. NUCLEIC ACIDS 2017; 8:132-143. [PMID: 28918016 PMCID: PMC5496203 DOI: 10.1016/j.omtn.2017.06.005] [Citation(s) in RCA: 551] [Impact Index Per Article: 68.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/12/2017] [Revised: 06/07/2017] [Accepted: 06/09/2017] [Indexed: 12/21/2022]
Abstract
In the past few years, therapeutic microRNA (miRNA) and small interfering RNA (siRNA) are some of the most important biopharmaceuticals that are in commercial space as future medicines. This review summarizes the patents of miRNA- and siRNA-based new drugs, and also provides a snapshot about significant biopharmaceutical companies that are investing for the therapeutic development of miRNA and siRNA molecules. An insightful view about individual siRNA and miRNA drugs has been depicted with their present status, which is gaining attention in the therapeutic landscape. The efforts of the biopharmaceuticals are discussed with the status of their preclinical and/or clinical trials. Here, some of the setbacks have been highlighted during the biopharmaceutical development of miRNA and siRNA as individual therapeutics. Finally, a snapshot is illustrated about pharmacokinetics, pharmacodynamics with absorption, distribution, metabolism, and excretion (ADME), which is the fundamental development process of these therapeutics, as well as the delivery system for miRNA- and siRNA-based drugs.
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Affiliation(s)
- Chiranjib Chakraborty
- Department of Bioinformatics and Biochemistry, Galgotias University, Greater Noida 201306, Uttar Pradesh, India; Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon 24252, Republic of Korea.
| | - Ashish Ranjan Sharma
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon 24252, Republic of Korea
| | - Garima Sharma
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon 24252, Republic of Korea
| | - C George Priya Doss
- Department of Integrative Biology, VIT University, Vellore 632014, Tamil Nadu, India
| | - Sang-Soo Lee
- Institute for Skeletal Aging & Orthopedic Surgery, Hallym University-Chuncheon Sacred Heart Hospital, Chuncheon 24252, Republic of Korea.
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Polysaccharide-based Nanoparticles for Gene Delivery. Top Curr Chem (Cham) 2017; 375:31. [DOI: 10.1007/s41061-017-0114-y] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2016] [Accepted: 01/25/2017] [Indexed: 12/17/2022]
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Ezzat WM, Amr KS. Insights for hepatitis C virus related hepatocellular carcinoma genetic biomarkers: Early diagnosis and therapeutic intervention. World J Hepatol 2016; 8:1251-1261. [PMID: 27843535 PMCID: PMC5084054 DOI: 10.4254/wjh.v8.i30.1251] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2016] [Revised: 07/15/2016] [Accepted: 09/08/2016] [Indexed: 02/06/2023] Open
Abstract
The current review explores the role of emerging molecular contributing factors in liver carcinogenesis on top of hepatitis C virus (HCV). Here we will try to discuss the role genetic and epigenetic factors in pathogenesis of hepatocellular carcinoma. Understanding the role of these factors will help in discovering the mystery of liver carcinogenesis on top of chronic HCV infection. Moreover, use of the studied molecular factors will provide the hepatologists with tailored diagnostic promising biomarkers and flatten the way for establishment of emerging molecular treatment based on exploring the molecular subscription of this aggressive liver cancer.
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EMMPRIN reduction via scFv-M6-1B9 intrabody affects α3β1-integrin and MCT1 functions and results in suppression of progressive phenotype in the colorectal cancer cell line Caco-2. Cancer Gene Ther 2014; 21:246-55. [DOI: 10.1038/cgt.2014.24] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2014] [Revised: 04/30/2014] [Accepted: 04/30/2014] [Indexed: 12/15/2022]
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Abstract
siRNA therapeutics has developed rapidly and already there are clinical trials ongoing or planned; however, the delivery of siRNA into cells, tissues or organs remains to be a major obstacle. Lipid-based vectors hold the most promising position among non-viral vectors, as they have a similar structure to cell or organelle membranes. But when used in the form of liposomes, these vectors have shown some problems. Therefore, either the nature of lipids themselves or forms used should be improved. As a novel class of lipid like materials, lipidoids have the advantages of easy synthesis and the ability for delivering siRNA to obtain excellent silencing activity. However, the toxicities of lipidoids have not been thoroughly studied. pH responsive lipids have also gained great attention recently, though some of the amine-based lipids are not novel in terms of chemical structures. More complex self-assembly structures, such as LPD (LPH) and LCP, may provide a good solution to siRNA delivery. They have demonstrated controlled particle morphology and size and siRNA delivery activity for both in vitro and in vivo.
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Affiliation(s)
- Shubiao Zhang
- Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Sindhu A, Arora P, Chaudhury A. Illuminating the gateway of gene silencing: perspective of RNA interference technology in clinical therapeutics. Mol Biotechnol 2012; 51:289-302. [PMID: 21947958 PMCID: PMC7091241 DOI: 10.1007/s12033-011-9456-9] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
A novel laboratory revolution for disease therapy, the RNA interference (RNAi) technology, has adopted a new era of molecular research as the next generation "Gene-targeted prophylaxis." In this review, we have focused on the chief technological challenges associated with the efforts to develop RNAi-based therapeutics that may guide the biomedical researchers. Many non-curable maladies, like neurodegenerative diseases and cancers have effectively been cured using this technology. Rapid advances are still in progress for the development of RNAi-based technologies that will be having a major impact on medical research. We have highlighted the recent discoveries associated with the phenomenon of RNAi, expression of silencing molecules in mammals along with the vector systems used for disease therapeutics.
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Affiliation(s)
- Annu Sindhu
- Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, 125001 Haryana India
| | - Pooja Arora
- Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, 125001 Haryana India
| | - Ashok Chaudhury
- Department of Bio and Nano Technology, Guru Jambheshwar University of Science and Technology, Hisar, 125001 Haryana India
- Present Address: Crop Science Department, NC State University, Raleigh, NC 27606 USA
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Zhao ZX, Gao SY, Wang JC, Chen CJ, Zhao EY, Hou WJ, Feng Q, Gao LY, Liu XY, Zhang LR, Zhang Q. Self-assembly nanomicelles based on cationic mPEG-PLA-b-Polyarginine(R15) triblock copolymer for siRNA delivery. Biomaterials 2012; 33:6793-807. [PMID: 22721724 DOI: 10.1016/j.biomaterials.2012.05.067] [Citation(s) in RCA: 92] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2012] [Accepted: 05/29/2012] [Indexed: 12/25/2022]
Abstract
Due to the absence of safe and effective carriers for in vivo delivery, the applications of small interference RNA (siRNA) in clinic for therapeutic purposes have been limited. In this study, a biodegradable amphiphilic tri-block copolymer (mPEG(2000)-PLA(3000)-b-R(15)) composed of monomethoxy poly(ethylene glycol), poly(d,l-lactide) and polyarginine was synthesized and further self-assembled to cationic polymeric nanomicelles for in vivo siRNA delivery, with an average diameter of 54.30 ± 3.48 nm and a zeta potential of approximately 34.8 ± 1.77 mV. The chemical structures of the copolymers were well characterized by (1)H NMR spectroscopy and FT-IR spectra. In vitro cytotoxicity and hemolysis assays demonstrated that the polymeric nanomicelles showed greater cell viability and haemocompatibility than those of polyethyleneimine (PEI) or R(15) peptide. In vitro experiments demonstrated that EGFR targeted siRNA formulated in micelleplexes exhibited approximately 65% inhibition of EGFR expression on MCF-7 cells in a sequence-specific manner, which was comparable to Lipofectamine™ 2000. The results of intravenous administration showed Micelleplex/EGFR-siRNA significantly inhibited tumor growth in nude mice xenografted MCF-7 tumors, with a remarkable inhibition of EGFR expression. Furthermore, no positive activation of the innate immune responses and no significant body weight loss was observed during treatment suggested that this polymeric micelle delivery system is non-toxic. In conclusion, the present nanomicelles based on cationic mPEG(2000)-PLA(3000)-b-R(15) copolymer would be a safe and efficient nanocarrier for in vivo delivery of therapeutic siRNA.
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Affiliation(s)
- Zhi-Xia Zhao
- State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
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11
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Lan Y, Zhao K, He W, Wang G, Lu H, Song D, Gao F. Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line. J Virol Methods 2012; 179:414-8. [PMID: 22138683 PMCID: PMC7112858 DOI: 10.1016/j.jviromet.2011.11.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2011] [Revised: 10/26/2011] [Accepted: 11/09/2011] [Indexed: 12/26/2022]
Abstract
Porcine hemagglutinating encephalomyelitis virus (PHEV), which causes porcine encephalomyelitis and is widespread among swine worldwide. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, two siRNA expression plasmids (shN1 and shN2) were generated to target two different coding regions of the nucleocapsid protein (N) of PHEV. The shRNAs were transiently transfected into a porcine kidney cell line, PK-15, to determine whether these constructs inhibited PHEV production. Our results revealed that both shRNAs were highly capable of inhibiting viral RNA genome replication, especially shN2. Next, stable transfection of shN2 was used to produce two siRNA stably expressing PK-15 cell clones (shN2-1 and shN2-2), and these two lines were infected with PHEV. The analysis of cytopathic effects (CPE) demonstrated that shN2-1 and shN2-2 were capable of protecting cells against PHEV infection with high specificity and efficiency. Furthermore, effective inhibition of viral replication persisted for up to 120 h by a TCID(50) assay. These results indicated that RNAi targeting of the N gene could facilitate studies of the specific function of viral genes associated with PHEV replication and may have potential therapeutic applications.
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Affiliation(s)
- Yungang Lan
- College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China
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12
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Zhang S, Zhao Y, Zhi D, Zhang S. Non-viral vectors for the mediation of RNAi. Bioorg Chem 2012; 40:10-18. [DOI: 10.1016/j.bioorg.2011.07.005] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2011] [Revised: 07/20/2011] [Accepted: 07/22/2011] [Indexed: 12/01/2022]
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Abstract
Small interfering RNAs (siRNAs) are potent molecules capable of blocking gene expression after entering cell cytoplasm. Despite their strong efficacy, they need to be carried by nanoscale delivery systems that can protect them against degradation in biological fluids, increase their cellular uptake and favor their subcellular distribution. Several studies have highlighted the potential of local pulmonary delivery of siRNAs for the treatment of lung diseases. For this purpose, nanoscale delivery systems were addressed to target passively or actively the target cell. This review discusses the possibilities of approaching lung delivery of nanoscale particles carrying siRNAs.
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Nimesh S, Gupta N, Chandra R. Strategies and advances in nanomedicine for targeted siRNA delivery. Nanomedicine (Lond) 2011; 6:729-46. [PMID: 21718181 DOI: 10.2217/nnm.11.15] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
siRNA are a rapidly emerging class of new therapeutic molecules for the treatment of inherited and acquired diseases. However, poor cellular uptake and instability in physiological conditions limits its therapeutic potential, hence a need to develop a delivery system that can protect and efficiently transport siRNA to the target cells has arisen. Nanoparticles have been proposed as suitable delivery vectors with reduced cytotoxicity and enhanced efficacy. These delivery vectors form condensed complexes with siRNA which, in turn, provides protection to siRNA against enzymatic degradation and further leads to tissue and cellular targeting. Nanoparticles derived from polymers, such as chitosan and polyethylenimine have found numerous applications owing to ease of manipulation, high stability, low cost and high gene carrying capability. This article focuses on various aspects of nanomedicine based siRNA delivery with emphasis on targeted delivery to tumors.
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Affiliation(s)
- Surendra Nimesh
- Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
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Truong NP, Jia Z, Burgess M, Payne L, McMillan NAJ, Monteiro MJ. Self-catalyzed degradable cationic polymer for release of DNA. Biomacromolecules 2011; 12:3540-8. [PMID: 21838265 DOI: 10.1021/bm2007423] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.
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Affiliation(s)
- Nghia P Truong
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane QLD 4072, Australia
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The sense strand pre-cleaved RNA duplex mediates an efficient RNA interference with less off-target and immune response effects. Appl Microbiol Biotechnol 2010; 90:583-9. [DOI: 10.1007/s00253-010-3065-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2010] [Revised: 12/03/2010] [Accepted: 12/03/2010] [Indexed: 01/31/2023]
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Li J, Guo H, Shi Z, Tu C. In vitro inhibition of CSFV replication by retroviral vector-mediated RNA interference. J Virol Methods 2010; 169:316-21. [PMID: 20691206 PMCID: PMC7112837 DOI: 10.1016/j.jviromet.2010.07.036] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2010] [Revised: 07/28/2010] [Accepted: 07/29/2010] [Indexed: 11/30/2022]
Abstract
Classical swine fever (CSF) is a highly contagious viral disease of pigs which causes major economic losses worldwide. No specific drug is currently available for the effective treatment of CSFV infection. RNA interference (RNAi) technology depends on effective delivery systems, for which several effective vectors have recently been developed. Three retroviral plasmids containing siRNA genes targeting different regions of Npro and NS4A have been constructed, and 3 replication-incompetent retroviral vectors have been produced in the human embryo kidney cell line GP2-293 by retroviral plasmid transfection. PK-15 cells were then infected with these replication-incompetent retroviral vectors and screened for siRNA stably expressing PK-15 cell clones. Growth of CSFV in such siRNA stably expressing cell clones resulted in a 186-fold reduction in viral genome copies and, at 72 h post-infection, only a small % of cells showed infection by indirect immunofluorescence microscopy, and effective inhibition of virus replication persisted for up to 120 h. Retroviral vector-mediated RNAi can therefore be used to study the specific function of viral genes associated with CSFV replication and may have potential therapeutic application.
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Affiliation(s)
- Jiangnan Li
- College of Animal Science and Veterinary Medicine, Jilin University, 5333 XiAn Da Road, Changchun 130062, China
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Gupta AK, Eshraghi Y, Gliniak C, Gosain AK. Nonviral transfection of mouse calvarial organ in vitro using Accell-modified siRNA. Plast Reconstr Surg 2010; 125:494-501. [PMID: 19910849 DOI: 10.1097/prs.0b013e3181c82df1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Understanding the biology of cranial suture fusion and the precise role of involved molecules implicated in the process will help to identify key factors involved in regulation of suture fusion. Modulation of these key factors may serve as a tissue-engineering technique to replace the traditional surgical procedures for the correction of premature suture fusion. Modulation of gene expression by RNA interference is a widely used technique with high potential. Because there is no available report of calvarial organ transfection in vitro, the authors studied the development of a successful nonviral delivery technique of small inhibitory RNA (siRNA) to an in vitro calvarial organ culture system. METHODS In this study, 19-day-old male CD1 mice were euthanized and parallel craniotomies made through the parietal and frontal calvaria, 2 mm to either side of the sagittal suture, with care taken to preserve the underlying dura mater. Organs grown in vitro in a defined medium were transfected with transforming growth factor-beta1-specific Accell-modified siRNA followed by RNA isolation and quantitative polymerase chain reaction analysis. RESULTS Transfection of a calvarial organ with transforming growth factor-beta1-specific Accell-modified siRNA effectively knocks down the mRNA level. CONCLUSIONS Observations from this study indicate that in an in vitro calvarial organ culture system, a specific, efficient, and durable RNA interference activity can be achieved when Accell-modified siRNA is used. In addition to bypassing the need for toxic lipid carriers, the modifications introduced in Accell-modified siRNAs make it more stable and less off-target. This technique can potentially be used for in vivo studies once the initial effect of gene-specific siRNA on in vitro suture fusion has been determined.
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Affiliation(s)
- Ashim K Gupta
- Cleveland, Ohio From the Department of Plastic Surgery, Case Western Reserve School of Medicine
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Chen J, Tian H, Guo Z, Xia J, Kano A, Maruyama A, Jing X, Chen X. A Highly Efficient siRNA Carrier of PBLG Modified Hyperbranched PEI. Macromol Biosci 2009; 9:1247-53. [DOI: 10.1002/mabi.200900249] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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Abstract
RNA interference (RNAi) as a mechanism to selectively degrade mRNA (mRNA) expression has emerged as a potential novel approach for drug target validation and the study of functional genomics. Small interfering RNAs (siRNA) therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Although other challenges remain, delivery strategies for siRNA become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics. This review provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy.
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Affiliation(s)
- Kun Gao
- University of North Carolina, Chapel Hill, North Carolina 27599, USA
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Li G, Jiang P, Li Y, Wang X, Huang J, Bai J, Cao J, Wu B, Chen N, Zeshan B. Inhibition of porcine reproductive and respiratory syndrome virus replication by adenovirus-mediated RNA interference both in porcine alveolar macrophages and swine. Antiviral Res 2009; 82:157-65. [PMID: 19428607 DOI: 10.1016/j.antiviral.2009.02.202] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2008] [Revised: 02/02/2009] [Accepted: 02/26/2009] [Indexed: 01/18/2023]
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Consequently, there is a need to develop a new antiviral strategy. In this study, two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against ORF1b of PRRSV S1 strain were constructed and the inhibition of PRRSV replication was determined. The results showed that pretreatment with these shRNAs delivered by recombinant adenovirus could induce a significant inhibition of viral RNA and protein level in Marc-145 cells infected with PRRSV S1 strains. One recombinant adenovirus (rAd-P2) was found to be also effective in inhibiting the replication of highly virulent PRRSV SY0608 strain in Marc-145 cells and porcine alveolar macrophages at both the protein and ORF1b mRNA level. The antiviral effect was dose-dependent and sustained for at least 96h. Twenty 6-week old piglets were assigned to four groups each with five piglets. Groups 1 and 2 were inoculated intramuscularly with rAd-P2 and mock construct rAd-mP2 individually. After 24h, groups 1, 2 and 3 were challenged intramuscularly with the SY0608 strain. Group 4 remained unchallenged but with PBS as mock. The results showed that the viral load of PRRSV in serum and lung tissue of swine was suppressed effectively by rAd-P2. The clinical signs and pathological lesions in the pigs inoculated with rAd-P2 were milder than those in rAd-mP2 negative and PRRSV control. These results indicated that shRNAs mediated by the adenovirus could inhibit PRRSV infection sufficiently in vitro as well as in vivo. RNAi mediated by recombinant adenovirus might be a potential new tool for controlling PRRSV infection. Of course, the protective efficiency of rAd-P2 should be made by using a large number of pigs in future.
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Affiliation(s)
- Guangming Li
- Ministry of Agriculture, Nanjing Agriculture University, Jiangsu, China
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State of the art and perspectives for the delivery of antisense oligonucleotides and siRNA by polymeric nanocarriers. Int J Pharm 2008; 364:237-48. [PMID: 18619528 DOI: 10.1016/j.ijpharm.2008.06.011] [Citation(s) in RCA: 92] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2008] [Revised: 06/07/2008] [Accepted: 06/10/2008] [Indexed: 02/07/2023]
Abstract
Knocking down gene expression using either antisense oligonucleotides (AS-ODNs) or small interfering RNA (siRNAs) has raised a lot of interest in designing new pathways for therapeutics. Despite their potentialities, these negatively charged and hydrophilic molecules request chemical modifications or a carrier that allows cell recognition, cell internalization and moreover subcellular penetration. Although chemical modifications were brought to the basic AS-ODNs and siRNAs, their sensitivity to degradation and poor intracellular penetration is still hampering their clinical applications. We present here the potentialities of polymeric carriers or the use of alternative administration route such as oral, ocular and skin delivery to improve their delivery and to circumvent the hurdles for their clinical applications.
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White PJ. Barriers to successful delivery of short interfering RNA after systemic administration. Clin Exp Pharmacol Physiol 2008; 35:1371-6. [PMID: 18565190 DOI: 10.1111/j.1440-1681.2008.04992.x] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
1. RNA interference in vivo has tremendous potential, both with respect to the elucidation of protein function in animals and as a therapeutic platform in humans. In vitro, short interfering RNA (siRNA) has been shown to completely silence gene expression in mammalian cells at low picomolar concentrations. 2. Although many good publications have shown specific silencing to occur in vivo, there are few that have transferred the combination of maximal efficacy and high potency to this setting. The present review considers the biological barriers that limit the movement of siRNA from vascular lumen to target cell cytoplasm and the strategies that have been used to overcome them. 3. Intravenous administration of siRNA results in rapid, extensive removal of siRNA from the blood via renal excretion, tissue distribution and nuclease degradation. Movement across vascular capillaries appears to be a limiting factor in some cases; few examples of silencing have been reported in organs with a conventional capillary endothelium. 4. Cellular uptake and endosomal trapping are significant barriers, but can be overcome using strategies such as antibody mediated cellular uptake or polyethyleneimine-mediated endosomal escape.
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Affiliation(s)
- Paul J White
- Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.
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Li SD, Chono S, Huang L. Efficient oncogene silencing and metastasis inhibition via systemic delivery of siRNA. Mol Ther 2008; 16:942-6. [PMID: 18388916 DOI: 10.1038/mt.2008.51] [Citation(s) in RCA: 145] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
The selective delivery of small interfering RNA (siRNA) to metastatic tumors remains a challenging task. We have developed a nanoparticle (NP) formulation composed of siRNA, a carrier DNA, a polycationic peptide, and cationic liposomes. The NP was obtained by a self-assembling process, followed by surface modification with a polyethylene glycol (PEG)-conjugated ligand, anisamide. The NP was PEGylated and a ligand was presented to target sigma receptor-expressing murine melanoma cells, B16F10. The lung metastasis model was established by intravenous (i.v.) injection of the B16F10 cells into C57BL/6 mice. A mixture of siRNA against MDM2, c-myc, and vascular endothelial growth factor (VEGF) co-formulated in the targeted NP caused simultaneous silencing of each of the oncogenes in the metastatic nodules. Two consecutive i.v. injections of siRNA in the targeted NP significantly reduced the lung metastasis (approximately 70-80%) at a relatively low dose (0.45 mg/kg), whereas free siRNA and the nontargeted NP showed little effect. This targeted NP formulation significantly prolonged the mean survival time of the animals by 30% as compared to the untreated controls. At the therapeutic dose, the targeted NP showed little local and systemic immunotoxicity and did not decrease the body weight or damage the major organs.
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Affiliation(s)
- Shyh-Dar Li
- Division of Molecular Pharmaceutics, Department of Pharmaceutical Sciences, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, USA
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25
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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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Abstract
We have developed a self-assembled nanoparticle (NP) that efficiently delivers small interfering RNA (siRNA) to the tumor by intravenous (IV) administration. The NP was obtained by mixing carrier DNA, siRNA, protamine, and lipids, followed by post-modification with polyethylene glycol and a ligand, anisamide. Four hours after IV injection of the formulation into a xenograft model, 70-80% of injected siRNA/g accumulated in the tumor, approximately 10% was detected in the liver and approximately 20% recovered in the lung. Confocal microscopy showed that fluorescent-labeled siRNA was efficiently delivered into the cytoplasm of the sigma receptor expressing NCI-H460 xenograft tumor by the targeted NPs, whereas free siRNA and non-targeted NPs showed little uptake. Three daily injections (1.2 mg/kg) of siRNA formulated in the targeted NPs silenced the epidermal growth factor receptor (EGFR) in the tumor and induced approximately 15% tumor cell apoptosis. Forty percent tumor growth inhibition was achieved by treatment with targeted NPs, while complete inhibition lasted for 1 week when combined with cisplatin. The serum level of liver enzymes and body weight monitoring during the treatment indicated a low level of toxicity of the formulation. The carrier itself also showed little immunotoxicity (IMT).
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27
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Zhang S, Zhao B, Jiang H, Wang B, Ma B. Cationic lipids and polymers mediated vectors for delivery of siRNA. J Control Release 2007; 123:1-10. [PMID: 17716771 DOI: 10.1016/j.jconrel.2007.07.016] [Citation(s) in RCA: 299] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2007] [Accepted: 07/19/2007] [Indexed: 01/13/2023]
Abstract
RNA interference (RNAi) is one of the most importantly protective phenomena forming from the process combating against virus. Since its high efficiency for silencing the expression of proteins at the posttranscriptional level, RNAi shows great prospect in therapeutics for diseases. However, the delivery of siRNA into cells, tissues or organs remains to be a big obstacle for its applications. Many vectors for siRNA delivery have been developed including viral vectors and non-viral vectors, among them non-viral vectors have the advantages of low toxicity, ease of synthesis and low immune response over viral ones. Cationic liposomes and polymer particles, major varieties of non-viral vectors, used for gene delivery, have shown to be suitable for the delivery of siRNA. Based on the concise introduction of RNAi, this article reviews the non-viral delivery systems of siRNA, hoping to provide helpful information for the development of delivery systems of siRNA, and to summarize literatures about siRNA delivery published in recent years.
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Affiliation(s)
- Shubiao Zhang
- SEAC-ME Key Laboratory of Biotechnology and Bioresources Utilization, Dalian Nationalities University, Dalian 116600, Liaoning, China.
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28
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Li SD, Huang L. Targeted delivery of antisense oligodeoxynucleotide and small interference RNA into lung cancer cells. Mol Pharm 2007; 3:579-88. [PMID: 17009857 DOI: 10.1021/mp060039w] [Citation(s) in RCA: 206] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Selective gene inhibition by antisense oligodeoxynucleotide (AS-ODN) or by small interference RNA (siRNA) therapeutics promises the treatment of diseases that cannot be cured by conventional drugs. However, antisense therapy is hindered due to poor stability in physiological fluids and limited intracellular uptake. To address these problems, a ligand targeted and sterically stabilized nanoparticle formulation has been developed in our lab. Human lung cancer cells often overexpress the sigma receptor and, thus, can be targeted with a specific ligand such as anisamide. AS-ODN or siRNA against human survivin was mixed with a carrier DNA, calf thymus DNA, before complexing with protamine, a highly positively charged peptide. The resulting particles were coated with cationic liposomes consisting of DOTAP and cholesterol (1:1, molar ratio) to obtain LPD (liposome-polycation-DNA) nanoparticles. Ligand targeting and steric stabilization were then introduced by incubating preformed LPD nanoparticles with DSPE-PEG-anisamide, a PEGylated ligand lipid developed earlier in our lab, by the postinsertion method. Nontargeted nanoparticles coated with DSPE-PEG were also prepared as a control. Antisense activities of nanoparticles were determined by survivin mRNA down-regulation, survivin protein down-regulation, ability to trigger apoptosis in tumor cells, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to anticancer drugs. We found that tumor cell delivery and antisense activity of PEGylated nanoparticles were sequence dependent and rely on the presence of anisamide ligand. The uptake of oligonucleotide in targeted, PEGylated nanoparticles could be competed by excess free ligand. Our results suggest that the ligand targeted and sterically stabilized nanoparticles can provide a selective delivery of AS-ODN and siRNA into lung cancer cells for therapy.
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MESH Headings
- Cell Line, Tumor
- Cell Survival/drug effects
- DNA/chemistry
- Drug Delivery Systems/methods
- Enzyme-Linked Immunosorbent Assay
- Humans
- Inhibitor of Apoptosis Proteins
- Liposomes
- Lung Neoplasms/genetics
- Lung Neoplasms/pathology
- Lung Neoplasms/therapy
- Microtubule-Associated Proteins/genetics
- Microtubule-Associated Proteins/metabolism
- Molecular Structure
- Nanoparticles/chemistry
- Neoplasm Proteins/genetics
- Neoplasm Proteins/metabolism
- Oligodeoxyribonucleotides, Antisense/administration & dosage
- Oligodeoxyribonucleotides, Antisense/chemistry
- Oligodeoxyribonucleotides, Antisense/genetics
- Polyethylene Glycols/chemistry
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Small Interfering/administration & dosage
- RNA, Small Interfering/chemistry
- RNA, Small Interfering/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Survivin
- Technology, Pharmaceutical/methods
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Affiliation(s)
- Shyh-Dar Li
- Division of Molecular Pharmaceutics, School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599, USA
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29
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Abstract
The rate of biopharmaceutical approvals has leveled off, but some milestones bode well for the future.
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Affiliation(s)
- Gary Walsh
- Industrial Biochemistry Programme, University of Limerick, Limerick, Ireland.
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30
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Chen W, Liu M, Jiao Y, Yan W, Wei X, Chen J, Fei L, Liu Y, Zuo X, Yang F, Lu Y, Zheng Z. Adenovirus-mediated RNA interference against foot-and-mouth disease virus infection both in vitro and in vivo. J Virol 2006; 80:3559-66. [PMID: 16537624 PMCID: PMC1440392 DOI: 10.1128/jvi.80.7.3559-3566.2006] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Foot-and-mouth disease virus (FMDV) infection is responsible for the heavy economic losses in stockbreeding each year. Because of the limited effectiveness of existing vaccines and antiviral drugs, the development of new strategies is needed. RNA interference (RNAi) is an effective means of suppressing virus replication in vitro. Here we demonstrate that treatment with recombinant, replication-defective human adenovirus type 5 (Ad5) expressing short-hairpin RNAs (shRNAs) directed against either structural protein 1D (Ad5-NT21) or polymerase 3D (Ad5-POL) of FMDV totally protects swine IBRS-2 cells from homologous FMDV infection, whereas only Ad5-POL inhibits heterologous FMDV replication. Moreover, delivery of these shRNAs significantly reduces the susceptibility of guinea pigs and swine to FMDV infection. Three of five guinea pigs inoculated with 10(6) PFU of Ad5-POL and challenged 24 h later with 50 50% infectious doses (ID50) of homologous virus were protected from the major clinical manifestation of disease: the appearance of vesicles on the feet. Two of three swine inoculated with an Ad5-NT21-Ad5-POL mixture containing 2 x 10(9) PFU each and challenged 24 h later with 100 ID50 of homologous virus were protected from the major clinical disease, but treatment with a higher dose of adenovirus mixture cannot promote protection of animals. The inhibition was rapid and specific because treatment with a control adenovirus construct (Ad5-LacZ) expressing Escherichia coli galactosidase-specific shRNA showed no marked antiviral activity. Our data highlight the in vivo potential of RNAi technology in the case of FMD.
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Affiliation(s)
- Weizao Chen
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, People's Republic of China
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31
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Trepanier JB, Tanner JE, Alfieri C. Oligonucleotide-Based Therapeutic Options against Hepatitis C Virus Infection. Antivir Ther 2006. [DOI: 10.1177/135965350601100315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
The hepatitis C virus (HCV) is the cause of a silent pandemic that, due to the chronic nature of the disease and the absence of curative therapy, continues to claim an ever-increasing number of lives. Current antiviral regimens have proven largely unsatisfactory for patients with HCV drug-resistant genotypes. It is therefore important to explore alternative therapeutic stratagems whose mode of action allows them to bypass viral resistance. Antisense oligonucleotides, ribozymes, small interfering RNAs, aptamers and deoxyribozymes constitute classes of oligonucleotide-based compounds designed to target highly conserved or functionally crucial regions contained within the HCV genome. The therapeutic expectation for such compounds is the elimination of HCV from infected individuals. Progress in oligonucleotide-based HCV antivirals towards clinical application depends on development of nucleotide designs that bolster efficacy while minimizing toxicity, improvement in liver-targeting delivery systems, and refinement of small-animal models for preclinical testing.
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Affiliation(s)
- Janie B Trepanier
- Sainte-Justine Hospital Research Centre, and the Department of Microbiology and Immunology, Université de Montréal, Montréal, Québec, Canada
| | | | - Caroline Alfieri
- Sainte-Justine Hospital Research Centre, and the Department of Microbiology and Immunology, Université de Montréal, Montréal, Québec, Canada
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