Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translation

Eisenhardt,; Serwe,; Renz,; Engels,; Caselmann,

doi:10.1046/j.1365-2362.1999.00552.x

Cited by 33 publications

(13 citation statements)

References 53 publications

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“…Error bars represent the range of duplicate data points. Previous studies aiming to select ASOs targeting conserved IRES sequences have not identified the 25-to 40-nt region as a priority target region but instead have pointed to other IRES regions, namely, the region around stem-loop III and the region around the initiation ATG codon (1,2,16,27,41). In some of these studies, the authors have omitted the 25-to 40-nt region from the experimental system (1, 2, 16).…”

Section: Discussionmentioning

confidence: 99%

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Laxton

Brady

Moschos

et al. 2011

Antimicrob Agents Chemother

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We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC 50 ]) and cytotoxic (50% cytotoxic concentration [CC 50 ]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC 50 ‫؍‬ 4 nM) and low cytotoxicity (CC 50 >880 nM) targeting the 25-to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25-to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25-to 40-nt region have no antiviral effect, revealing important regulatory features of the 25-to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.Hepatitis C virus (HCV) infection is a major cause of liver disease, with Ͼ170 million infected people worldwide being at risk from liver failure and hepatocarcinoma. Current standard of care (SOC) using pegylated alpha 2a interferon (IFN-␣2a)-ribavirin is failing 40 to 50% of treated HCV patients, so new therapies are urgently required. New directly acting antiviral therapies for hepatitis C are becoming available, but these will eventually fail many HCV patients due to emerging drugresistant mutations in HCV strains (6, 7). HCV genomic RNA is an attractive antiviral target because it holds genetic information for viral proteins and contains regions of highly conserved sequence required for HCV replication/translation. Many groups have identified antisense oligonucleotides (ASOs) capable of inhibiting HCV RNA replication and viral polyprotein synthesis in vitro. Clinical trials on chronically HCV-infected patients show that modified ASOs targeting HCV can result in Ͼ2-log-unit decreases in viral loads, although the mechanism driving clinical antiviral activity has yet to be fully validated as antisense (14,28,33). Moreover, locked nucleic acid (LNA) represents a new generation of ASOs with improved affinity of binding to RNA targets, increased sequence specificity, greater biostability ag...

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Section: Discussionmentioning

confidence: 99%

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Laxton

Brady

Moschos

et al. 2011

Antimicrob Agents Chemother

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“…Such elements can be either viral proteins (NS2-NS3 protease, NS3-NS4A serine proteinase, NS3 RNA helicase, or RNA-dependent RNA polymerase [3,24,34,36]), viral cis-acting RNA elements (internal ribosomal entry site [IRES] antisense oligonucleotides or ribozymes [1,26]), or cellular elements (IFN-␣, ribavirin, and IMP dehydrogenase [EC 1.1.1.205] inhibitors [18,31,41,43]). Current knowledge of the human genome, combined with array technology and pathogen infection models, will likely lead to more defined host-pathogen-related targets for future drug design (17,23).…”

mentioning

confidence: 99%

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

Stuyver

Whitaker

McBrayer

et al. 2003

Antimicrob Agents Chemother

181

215

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Hepatitis C virus (HCV) is one of the most important Flaviviridae infections in humans and is responsible for the second most common cause of viral hepatitis. Presently, nearly 2% of the U.S. population, and an estimated 170 million people worldwide, are HCV carriers (2). The only approved therapy for chronic hepatitis C is alpha interferon (IFN-␣), either alone or in combination with ribavirin. Anemia is the most common adverse effect associated with ribavirin treatment, and neuropsychiatric adverse effects of IFN-␣ lead to premature cessation of therapy in 10 to 20% of patients (9, 13).As additional treatment options are urgently needed, there is an ongoing search for more potent antiviral compounds with fewer adverse effects. However, the search for improved antiviral agents is hampered by the limited and cumbersome propagation of HCV in vitro (4). Therefore, surrogate models such as the HCV RNA replicon that replicates in the human hepatoma cell line Huh7 have been developed (6,29). Improved versions of these HCV replicons contain adaptive mutations (25), and their use has facilitated the evaluation of candidate anti-HCV drugs.Bovine viral diarrhea virus (BVDV) is one of the best characterized members of the Flaviviridae family and has one of the largest RNA genomes (12.5 kb) in this family (8). This virus has the remarkable property of existing as noncytopathic and cytopathic (cpBVDV) biotypes, with cpBVDV strains showing insertions or viral genome rearrangements at the junction site between nonstructural protein 2 (NS2) and NS3 (32). BVDV may provide a surrogate model for HCV, both for the molecular study of viral proteins (33) and for the evaluation of antiviral compounds (3,7,47).In the search for therapeutic agents, any element that is essential for viral (or replicon) RNA replication may be considered a drug discovery target. Such elements can be either viral proteins (NS2-NS3 protease, NS3-NS4A serine proteinase, NS3 RNA helicase, or RNA-dependent RNA polymerase [3,24,34,36] [18,31,41,43]). Current knowledge of the human genome, combined with array technology and pathogen infection models, will likely lead to more defined host-pathogen-related targets for future drug design (17, 23). Today, however, the most successful classes of antiviral compounds with clinical utility in combat against other human viral pathogens (human immunodeficiency virus type 1 [HIV-1], hepatitis B virus [HBV], herpes simplex virus [HSV], and cytomegalovirus) are the protease, the nonnucleoside analogue, and the nucleoside analogue inhibitors. As the latter class of compounds is crucial in controlling herpesvirus, HIV-1, and HBV infections, it is likely and anticipated that

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“…The conserved HCV IRES is a likely target for effective therapeutic intervention. Extensive effort has been devoted to the development of antisense oligonucleotide, [1][2][3][4][5][6][7] ribozyme, [8][9][10] and DNA ribonuclease 11 inhibitors targeting HCV IRES sequences that are accessible for nucleic acid hybridization.…”

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confidence: 99%

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice

et al. 2003

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C ap-independent translation of the hepatitis C virus (HCV) polyprotein initiates at a highly structured internal ribosome entry site (IRES) at the 5Ј end of the genomic RNA. The conserved HCV IRES is a likely target for effective therapeutic intervention. Extensive effort has been devoted to the development of antisense oligonucleotide, 1-7 ribozyme, [8][9][10] and DNA ribonuclease 11 inhibitors targeting HCV IRES sequences that are accessible for nucleic acid hybridization.Although in vitro assays have been invaluable in the early evaluation of inhibitors, work by our group 12 and that of others 13,14 suggests that cell-free systems may not faithfully recapitulate translation from the HCV IRES. A recent report that HCV IRES translation varies with cell cycling 15 also raises concern that rapidly dividing cells in tissue culture may not accurately model the normally quiescent human liver. Therefore, model systems that allow evaluation of pharmacokinetic parameters such as inhibitor bioavailability, toxicity, and systemic clearance in the complex milieu of a mammalian organism would be useful. Preclinical small animal models for testing nucleic acid inhibitors of HCV will help move candidate inhibitors into the clinic. Recently, a mouse xenotransplantation model was reported that supports HCV viral replication in transplanted human liver cells. 16 Although very exciting, this system requires access to fresh human liver tissue. A vaccinia virus expressing a HCV IRES luciferase fusion also has been used as a model system for evaluating antisense inhibitors in vivo. 4 In this model, a phosphorothioate and a 5-methylcytidine antisense oligonucleotide moderately reduced expression from the HCV IRES (ϳ50% and 80%). However, nonspecific effects

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Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translation

Abstract: These data demonstrate that terminally modified B-ODN 4 is a potent inhibitor of HCV gene expression in vitro and in HepG2 cell culture and may be valuable for future antiviral treatment.

Cited by 33 publications

References 53 publications

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Selection, Optimization, and Pharmacokinetic Properties of a Novel, Potent Antiviral Locked Nucleic Acid-Based Antisense Oligomer Targeting Hepatitis C Virus Internal Ribosome Entry Site

Ribonucleoside Analogue That Blocks Replication of Bovine Viral Diarrhea and Hepatitis C Viruses in Culture

A potent and specific morpholino antisense inhibitor of hepatitis C translation in mice

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