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      A Novel In Vivo siRNA Delivery System Specifically Targeting Liver Cells for Protection of ConA-Induced Fulminant Hepatitis

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          Abstract

          Background

          Fulminant hepatitis progresses to acute liver failure (ALF) when the extent of hepatocyte death exceeds the liver's regenerative capacity. Although small interfering RNA (siRNA) appears promising in animal models of hepatitis, the approach is limited by drawbacks associated with systemic administration of siRNA. The aim of this study is to develop a hepatocyte-specific delivery system of siRNA for treatment of fulminant hepatitis.

          Methodology/Principal Findings

          Galactose-conjugated liposome nano-particles (Gal-LipoNP) bearing siRNA was prepared, and the particle size and zeta potential of Gal-LipoNP/siRNA complexes were measured. The distribution, cytotoxicity and gene silence efficiency were studied in vivo in a concanavalin A (ConA)-induced hepatitis model. C57BL/6 mice were treated with Gal-LipoNP Fas siRNA by i.v. injection 72 h before ConA challenge, and hepatocyte injury was evaluated using serum alanine transferase (ALT) and aspartate transaminase (AST) levels, as well as liver histopathology and TUNEL-positive hepatocytes. The galactose-ligated liposomes were capable of encapsulating >96% siRNA and exhibited a higher stability than naked siRNA in plasma. Hepatocyte-specific targeting was confirmed by in vivo delivery experiment, in which the majority of Gal-LipoNP-siRNA evaded nuclease digestion and accumulated in the liver as soon as 6 h after administration. In vivo gene silencing was significant in the liver after treatment of Gal-Lipo-siRNA. In the ConA-induced hepatitis model, serum levels of ALT and AST were significantly reduced in mice treated with Gal-lipoNP-siRNA as compared with control mice. Additionally, tissue histopathology and apoptosis showed an overall reduction of injury in the Gal-LipoNP siRNA-treated mice.

          Conclusions/Significance

          This study is the first to our knowledge to demonstrate reduction of hepatic injury by liver-specific induction of RNA interference using Gal-LipoNP Fas siRNA, highlighting a novel RNAi-based therapeutic potential in many liver diseases.

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          Most cited references36

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          Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy.

          The results obtained in this study establish that liposome formulations incorporating a synthetic polyethylene glycol-derivatized phospholipid have a pronounced effect on liposome tissue distribution and can produce a large increase in the pharmacological efficacy of encapsulated antitumor drugs. This effect is substantially greater than that observed previously with conventional liposomes and is associated with a more than 5-fold prolongation of liposome circulation time in blood, a marked decrease in uptake by tissues such as liver and spleen, and a corresponding increased accumulation in implanted tumors. These and other properties described here have expanded considerably the prospects of liposomes as an effective carrier system for a variety of pharmacologically active macromolecules.
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            RNA interference targeting Fas protects mice from fulminant hepatitis.

            RNA interference (RNAi) is a powerful tool to silence gene expression post-transcriptionally. However, its potential to treat or prevent disease remains unproven. Fas-mediated apoptosis is implicated in a broad spectrum of liver diseases, where inhibiting hepatocyte death is life-saving. We investigated the in vivo silencing effect of small interfering RNA (siRNA) duplexes targeting the gene Fas (also known as Tnfrsf6), encoding the Fas receptor, to protect mice from liver failure and fibrosis in two models of autoimmune hepatitis. Intravenous injection of Fas siRNA specifically reduced Fas mRNA levels and expression of Fas protein in mouse hepatocytes, and the effects persisted without diminution for 10 days. Hepatocytes isolated from mice treated with Fas siRNA were resistant to apoptosis when exposed to Fas-specific antibody or co-cultured with concanavalin A (ConA)-stimulated hepatic mononuclear cells. Treatment with Fas siRNA 2 days before ConA challenge abrogated hepatocyte necrosis and inflammatory infiltration and markedly reduced serum concentrations of transaminases. Administering Fas siRNA beginning one week after initiating weekly ConA injections protected mice from liver fibrosis. In a more fulminant hepatitis induced by injecting agonistic Fas-specific antibody, 82% of mice treated with siRNA that effectively silenced Fas survived for 10 days of observation, whereas all control mice died within 3 days. Silencing Fas expression with RNAi holds therapeutic promise to prevent liver injury by protecting hepatocytes from cytotoxicity.
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              High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.

              We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this technique required injection directly into the liver vessels (portal vein, hepatic vein, or bile duct) and occlusion of outflow. The present study now demonstrates that high levels of plasmid DNA expression in hepatocytes can be easily obtained by tail vein injections. The highest levels of expression are achieved by rapidly injecting the plasmid DNA in large volumes, approximately 2.5 ml. This technique has great potential for a wide variety of laboratory studies.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                6 September 2012
                : 7
                : 9
                : e44138
                Affiliations
                [1 ]Multi-Organ Transplant Program, London Health Sciences Centre, London, Ontario, Canada
                [2 ]Department of Surgery, Pathology, and Oncology, University of Western Ontario, London, Ontario, Canada
                [3 ]Liver Transplant Center, The Third Affiliated Hospital of Sun Yat-sen University, Transplantation Research Institute of Sun Yat-sen University, Guangzhou, China
                [4 ]Jiangxi Provincial Institute of Medical Science, and Medical School of Nanchang University, Nanchang, China
                [5 ]Medistem Laboratories, San Diego, USA
                Duke University, United States of America
                Author notes

                Competing Interests: The authors have the following interests. Thomas E Ichim is employed by Medistem Laboratories. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

                Conceived and designed the experiments: NJ X. Zhang HW DQ VM GC WM. Performed the experiments: NJ X. Zhang Z. Zheng DC KS. Wrote the paper: NJ X. Zhang TI DQ GC WM.

                * These authors contribute to this work equally.

                ** Address correspondence and reprint requests to: Dr. Wei-Ping Min ( weiping.min@ 123456uwo.ca ), 339 Windermere Road, University Hospital C9-136, London, Ontario, N6A 5A5, Canada. Tel: 519-663-2985, Fax: 519-663-3295.

                Article
                PONE-D-12-07078
                10.1371/journal.pone.0044138
                3435394
                22970170
                3791ba55-d1db-4867-b16e-e41a8ff8412e
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 March 2012
                : 1 August 2012
                Page count
                Pages: 10
                Funding
                This study was supported by the Canadian Institute of Health Research, the Heart and Stroke Foundation of Canada, and a grant from National Nature Science Foundation of China (No. 81160286). DC was supported by the CIHR Strategic Training Program in Cancer Research. WM is a recipient of Department of Surgery Institute Scientist, University of Western Ontario. NJ was supported by the grants from the Major State Basic Research Development Program (973 Program) of China (No. 2009CB522404), National 12th Five-Year Science and Technology Plan major projects of China (No. 2012ZX10002017-005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Epigenetics
                RNA interference
                Gene Expression
                RNA interference
                Molecular Cell Biology
                Signal Transduction
                Signaling Cascades
                Apoptotic Signaling Cascade
                Signaling in Cellular Processes
                Apoptotic Signaling
                Materials Science
                Biomaterials
                Medicine
                Gastroenterology and Hepatology
                Liver Diseases
                Acute Liver Failure
                Infectious Diseases
                Viral Diseases
                Hepatitis

                Uncategorized
                Uncategorized

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