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

Abstract

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...

Estrogen-regulated STAT1 activation promotes TLR8 expression to facilitate signaling via microRNA-21 in systemic lupus erythematosus

“…To determine the effect of antagonizing miR-21, primary human macrophages derived from healthy donors were treated with liposomal complexes containing locked nucleic acid (LNA) sequences designed to block miR-21 (LNA antagomiR-21). LNAs are RNA nucleotides containing a modified ribose moiety and were used to antagonize miR functionality here because they are significantly more resistant to enzymatic degradation than RNA [30]. Cells were incubated with autologous serum as an endogenous source of EV-derived miR-21 for 24 h. Relative to nonsense control treatment (LNA-control), inhibition of miR-21 with LNA antagomiR-21 reduced the expression of TLR8 mRNA by 46% ( p < 0.05; Fig.…”

“…Collectively, these data suggest that chloroquine may be binding to miRs to prevent TLR8 induction within endosomal compartments and that multiple miRs are most likely capable of TLR8 activation in this system. To suppress the effect of miR-21, a miR antagonist was designed with a modified LNA backbone, which enhances resistance to degradation when compared to RNA and has been shown to persist in vivo for over a week [30]. Our future investigations will be directed toward the identification of additional EV-derived miR agonists for TLR8 that are associated with SLE and the therapeutic development of LNA-based antagonists to block miR-induced inflammatory responses in vivo using SLE animal models.…”

Quantum Mechanical Studies of DNA and LNA

“…As an example, a single nucleotide modification (e.g., perfect match vs. one base pair mismatch) may lead to major, or ''all or none,'' phenotypic changes. The QM modeling data explains and illustrates reasons underlying some of the unexpected observations relating to major phenotypic changes observed for otherwise ''closely related'' molecules (vide infra) (Seth et al, 2009;Straarup et al, 2010;Laxton et al, 2011;Stanton et al, 2012;Hagedorn et al, 2013).…”

“…For instance, it has been reported that small structural modifications of antisense oligonucleotides (e.g., LNAs) have profound effects on these relations, which are observed as property changes. (Seth et al, 2009;Straarup et al, 2010;Laxton et al, 2011;Stanton et al, 2012). The mechanisms behind this ''property diversity'' are not understood, and it is clear that a reductionist viewpoint, where the complex properties of a given oligonucleotide (e.g., tissue and cellular uptake, pharmacokinetics, toxicity) are assigned by a simple correlation with the nucleobase sequence alone, neither describes nor predicts these structure/activity changes.…”

Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

“…Indeed, different synthetic molecules targeting specific RNA motifs essential for viral RNA multiplication have been exploited as therapeutic agents. [46][47][48][49][50][51] Furthermore, the IRES-inhibitory property of IRAB was observed both in in vitro translation assays using cell free lysates and in cells transfected with infectious RNA revealing that the primary targeted event is viral RNA translation, which is IRES-dependent. 7 In agreement with this, a specific decrease of viral protein synthesis occurred in transfected cells concomitantly to a reduction in virus yield in the presence of IRAB (2 mM).…”