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Berlin Brandenburg

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  • 1
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 July 2015, Vol.112(30), pp.E4007-16
    Description: Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.
    Keywords: Aav ; Adeno-Associated Viral Vector ; RNA Interference ; Off-Targeting ; Short Hairpin RNA ; Gene Transfer Techniques ; RNA Interference ; RNA, Small Interfering -- Metabolism
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 2
    Language: English
    In: Journal of medicinal chemistry, 13 March 2014, Vol.57(5), pp.1627-42
    Description: HCV infections are a major global health burden. After the identification of the virus in 1989, insights into viral replication and drug development have long been hampered by the lack of efficient cell culture models. Their establishment was an important prerequisite for the development of selective antivirals. This review describes the initial difficulties to achieve HCV replication in cell culture, finally leading to the establishment of subgenomic replicons and the infectious virus model (HCVcc). The review further summarizes the current state of HCV cell culture systems with respect to available virus isolates, engineered genomes, and cell types allowing efficient HCV propagation. Finally, we comment on how these cell culture models contributed to the development of directly acting antivirals.
    Keywords: Cell Culture Techniques -- History ; Hepacivirus -- Physiology
    ISSN: 00222623
    E-ISSN: 1520-4804
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  • 3
    Language: English
    In: Gastroenterology, January 2013, Vol.144(1), pp.13-15
    Keywords: Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
    Source: ScienceDirect Journals (Elsevier)
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  • 4
    Language: English
    In: JAMA, 27 September 2016, Vol.316(12), pp.1254-5
    Description: Ralf et al talk about hepatitis C virus (HCV). With the robust HCV replicon whole-cell system available for screening of small molecules, the search for inhibitors of HCV became a major focus of pharmaceutical and biotech companies. This was propelled by the desire to identify direct-acting antivirals with the ultimate goal of replacing the then current standard of care for treating HCV infection, the combination of injectable interferon plus ribavirin, which was limited by severe adverse effects, modest cure rates, and limited genotype coverage.
    Keywords: Hepacivirus -- Physiology ; Hepatitis C -- Virology ; Liver -- Virology
    ISSN: 00987484
    E-ISSN: 1538-3598
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  • 5
    Language: English
    In: Nature, September 2018, Vol.561(7722), pp.253-257
    Description: Zika virus (ZIKV) has recently emerged as a global health concern owing to its widespread diffusion and its association with severe neurological symptoms and microcephaly in newborns. However, the molecular mechanisms that are responsible for the pathogenicity of ZIKV remain largely unknown. Here we use human neural progenitor cells and the neuronal cell line SK-N-BE2 in an integrated proteomics approach to characterize the cellular responses to viral infection at the proteome and phosphoproteome level, and use affinity proteomics to identify cellular targets of ZIKV proteins. Using this approach, we identify 386 ZIKV-interacting proteins, ZIKV-specific and pan-flaviviral activities as well as host factors with known functions in neuronal development, retinal defects and infertility. Moreover, our analysis identified 1,216 phosphorylation sites that are specifically up- or downregulated after ZIKV infection, indicating profound modulation of fundamental signalling pathways such as AKT, MAPK-ERK and ATM-ATR and thereby providing mechanistic insights into the proliferation arrest elicited by ZIKV infection. Functionally, our integrative study identifies ZIKV host-dependency factors and provides a comprehensive framework for a system-level understanding of ZIKV-induced perturbations at the levels of proteins and cellular pathways.
    Keywords: Proteomics ; Host-Pathogen Interactions -- Physiology ; Proteome -- Analysis ; Zika Virus -- Pathogenicity
    ISSN: 00280836
    E-ISSN: 1476-4687
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  • 6
    Language: English
    In: Gastroenterology, August 2012, Vol.143(2), pp.429-438.e8
    Description: Hepatitis C virus (HCV) is a common cause of chronic liver disease. Many patients do not clear the viral infection; little is known about the mechanisms of HCV persistence or the frequent failure of interferon (IFN) to eliminate it. Better culture systems are needed to study viral replication in quiescent liver cells. We used human hepatoma (Huh7.5) cells and those that had undergone proliferation arrest and differentiation (Huh7.5 ) to study the persistence of HCV infection following exposure of the cells to IFN-α and to compare the antiviral effects of IFN-α and IFN-λ. We validated these results with primary human hepatocytes and Huh7 cells that expressed an IFN-inducible fluorophore. Following infection of Huh7.5 cells, HCV replicated persistently and released infectious particles. Long-term exposure of the cells to IFN-α reduced HCV replication ∼1000-fold but did not eliminate the virus; viral replication rebounded after withdrawal of IFN, as it does in patients with chronic HCV infection. HCV replicated at higher levels, but not exclusively, in cells that had a low level of response to IFN-α. Following incubation of cells with equipotent concentrations of IFN-α or IFN-λ, Huh7.5 cells expressed a wider pattern of IFN-stimulated genes than undifferentiated Huh7.5 cells or primary human hepatocytes, indicating that the antiviral response depends on the differentiation status of the cells. We developed a cell culture system using hepatoma cells to study persistent HCV infection during the type I or type III IFN-induced antiviral response. The level and range of the antiviral responses were associated with the differentiation status of the cells. We propose that HCV exploits the stochastic nature of the response of hepatocytes to IFN to sustain persistence.
    Keywords: Differentiated Hepatoma Cells ; Cell Culture System ; Model ; Bacterial Artificial Chromosome ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 7
    Language: English
    In: Gastroenterology
    Description: Dysfunctional CD8+ T cells are believed to contribute to the ability of hepatitis C virus (HCV) to evade the immune response. Most studies have focused on the effector functions of HCV-specific CD8+ T cells or their surface expression of inhibitory receptors. There is currently no information available about the ability of HCV-specific CD8+ T cells to inhibit viral replication (antiviral efficacy). To analyze the antiviral efficacy of virus-specific CD8+ T cells we utilized an immunological model based on a cell line that expresses HLA-A*02 and contains a stably replicating HCV reporter replicon. We isolated HCV-specific CD8+ T cells from 18 HLA-A*02–positive patients with chronic HCV infection and 15 subjects that resolved HCV infection (7 spontaneous, 8 after therapy). Replicon cells were labeled with virus-specific peptides; inhibition of HCV replication was determined by measuring luciferase activity after 72 hours co-culture with virus-specific CD8+ T cells. HCV-specific CD8+ T cells from patients with chronic HCV infection had a significantly lower antiviral efficacy than influenza-, Epstein-Barr virus-, and cytomegalovirus-specific CD8+ T cells. Antiviral efficacy was associated with the ability of virus-specific CD8+ T cells to secrete interferon (IFN)-γ. Antiviral efficacy of HCV-specific CD8+ T cells was linked to surface expression of CD127 and PD-1. The cytokines interleukin (IL)-2, IL-7, and IL-15 increased the antiviral efficacy of CD127-positive, but not of CD127-negative, HCV-specific CD8+ T cells. Spontaneous, but not antiviral therapy-induced, viral clearance was associated with increased antiviral efficacy. The ability of CD8+ T cells to inhibit HCV replication is associated with their ability to secrete IFN-γ and their surface expression of CD127 and PD-1.
    Keywords: Immune Response ; Liver Disease ; T Cell Exhaustion ; Antiviral Therapy ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 8
    Language: English
    In: Gastroenterology, 2011, Vol.141(3), pp.1057-1066
    Description: Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of and mouse support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.
    Keywords: Hcv Mouse Model ; Hcv Assembly ; Liver Disease ; Virology ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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  • 9
    Language: English
    In: Bioorganic & Medicinal Chemistry, 2011, Vol.19(13), pp.4067-4074
    Description: The development of small molecule inhibitors of the viral protease is of considerable interest for the treatment of emergent flaviviral diseases such as Dengue or West Nile fever. Until today little progress has been made in finding drug-like compounds that inhibit the protease and provide a starting point for lead optimization. We describe here the initial steps of a drug discovery effort that focused on the styryl pharmacophore, combined with a ketoamide function to serve as electrophilic trap for the catalytic serine. This resulted in a fragment-like lead compound with reasonable target affinity and good ligand efficiency, which was extensively modified to explore structure–activity relationships. Selected compounds were cross-tested against the West Nile virus protease and thrombin, indicating that selectivity for one or more flaviviral proteases can be achieved. Finally, the antiviral activity of several protease inhibitors was confirmed in a cell-culture model of Dengue virus replication. The SAR presented here may serve as starting point for further drug discovery efforts with the aim of targeting flaviviral proteases.
    Keywords: Ketoamides ; Ns2b–Ns3 Protease ; Protease Inhibitors ; Dengue Virus ; Flavivirus ; Medicine ; Chemistry ; Anatomy & Physiology
    ISSN: 0968-0896
    E-ISSN: 1464-3391
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  • 10
    Language: English
    In: Gastroenterology, July 2014, Vol.147(1), pp.48-64
    Description: Although there has been much research into the pathogenesis and treatment of hepatitis B virus (HBV) and hepatitis D virus (HDV) infections, we still do not completely understand how these pathogens enter hepatocytes. This is because in vitro infection studies have only been performed in primary human hepatocytes. Development of a polarizable, HBV-susceptible human hepatoma cell line and studies of primary hepatocytes from have provided important insights into the viral and cellular factors involved in virus binding and infection. The large envelope (L) protein on the surface of HBV and HDV particles has many different functions and is required for virus entry. The L protein mediates attachment of virions to heparan sulfate proteoglycans on the surface of hepatocytes. The myristoylated N-terminal preS1 domain of the L protein subsequently binds to the sodium taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1), the recently identified bona fide receptor for HBV and HDV. The receptor functions of NTCP and virus entry are blocked, in vitro and in vivo, by Myrcludex B, a synthetic -acylated preS1 lipopeptide. Currently, the only agents available to treat chronic HBV infection target the viral polymerase, and no selective therapies are available for HDV infection. It is therefore important to study the therapeutic potential of virus entry inhibitors, especially when combined with strategies to induce immune-mediated killing of infected hepatocytes.
    Keywords: Hepatitis B Virus Receptor ; Sodium Taurocholate Cotransporting Polypeptide ; Scl10a1 ; Medicine
    ISSN: 0016-5085
    E-ISSN: 1528-0012
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