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      Development of Therapeutic-Grade Small Interfering RNAs by Chemical Engineering

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          Abstract

          Recent successes in clinical trials have provided important proof of concept that small interfering RNAs (siRNAs) indeed constitute a new promising class of therapeutics. Although great efforts are still needed to ensure efficient means of delivery in vivo, the siRNA molecule itself has been successfully engineered by chemical modification to meet initial challenges regarding specificity, stability, and immunogenicity. To date, a great wealth of siRNA architectures and types of chemical modification are available for promoting safe siRNA-mediated gene silencing in vivo and, consequently, the choice of design and modification types can be challenging to individual experimenters. Here we review the literature and devise how to improve siRNA performance by structural design and specific chemical modification to ensure potent and specific gene silencing without unwarranted side-effects and hereby complement the ongoing efforts to improve cell targeting and delivery by other carrier molecules.

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          Most cited references220

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          Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs.

          MicroRNAs (miRNAs) are a class of noncoding RNAs that post-transcriptionally regulate gene expression in plants and animals. To investigate the influence of miRNAs on transcript levels, we transfected miRNAs into human cells and used microarrays to examine changes in the messenger RNA profile. Here we show that delivering miR-124 causes the expression profile to shift towards that of brain, the organ in which miR-124 is preferentially expressed, whereas delivering miR-1 shifts the profile towards that of muscle, where miR-1 is preferentially expressed. In each case, about 100 messages were downregulated after 12 h. The 3' untranslated regions of these messages had a significant propensity to pair to the 5' region of the miRNA, as expected if many of these messages are the direct targets of the miRNAs. Our results suggest that metazoan miRNAs can reduce the levels of many of their target transcripts, not just the amount of protein deriving from these transcripts. Moreover, miR-1 and miR-124, and presumably other tissue-specific miRNAs, seem to downregulate a far greater number of targets than previously appreciated, thereby helping to define tissue-specific gene expression in humans.
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            Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.

            Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-alpha in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.
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              Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.

              Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.
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                Author and article information

                Journal
                Front Genet
                Front Genet
                Front. Gene.
                Frontiers in Genetics
                Frontiers Research Foundation
                1664-8021
                20 August 2012
                2012
                : 3
                : 154
                Affiliations
                [1] 1simpleInterdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Aarhus University Aarhus C, Denmark
                Author notes

                Edited by: Kumiko Ui-Tei, University of Tokyo, Japan

                Reviewed by: Terrence Chi-Kong Lau, City University of Hong Kong, Hong Kong; Heh-In Im, Korea Institute of Science and Technology, South Korea

                *Correspondence: Jesper B. Bramsen, Interdisciplinary Nanoscience Center, Department of Molecular Biology and Genetics, Aarhus University, C.F. Mollersalle Building 1130, 8000 Aarhus C, Denmark. e-mail: jebb@ 123456mb.au.dk

                This article was submitted to Frontiers in Non-Coding RNA, a specialty of Frontiers in Genetics.

                Article
                10.3389/fgene.2012.00154
                3422727
                22934103
                923cdaac-0696-4977-996f-838ca468a360
                Copyright © 2012 Bramsen and Kjems.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

                History
                : 10 May 2012
                : 31 July 2012
                Page count
                Figures: 4, Tables: 0, Equations: 0, References: 255, Pages: 22, Words: 21017
                Categories
                Genetics
                Review Article

                Genetics
                off-target effect,sirna therapeutic,lna,rnai,chemical modification,sirna,ome,immunogenicity
                Genetics
                off-target effect, sirna therapeutic, lna, rnai, chemical modification, sirna, ome, immunogenicity

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