Antagonizing MicroRNA-122 and treatment of hepatitis C virus infection
Authors: Sébastien Pfeffer, Thomas F. Baumert, Kris Kowdley, Geoffrey McCaughan, Christian Trautwein
Journal: Hepatology (2010)

Lanford RE, Hildebrandt-Eriksen ES, Petri A, Persson R, Lindow M, Munk ME, et al. Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 2010;327:198-201. (Reproduced with permission.) The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance. Hepatitis C virus (HCV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma worldwide. Current antiviral treatment consisting of pegylated interferon-alpha (IFN-α) and ribavirin is limited by resistance, adverse effects, and high costs.1 Although the clinical development of novel antiviral compounds that target HCV protein processing has been shown to markedly improve sustained virological response, toxicity of the individual compounds and development of viral resistance remain major challenges.2 Thus, novel antiviral strategies are urgently needed. Micro-RNAs (miRNAs) are key regulators of gene expression at a posttranscriptional level.3 Their biogenesis is now well characterized and involves the processing of a large primary transcript into a stem-loop pre-miRNA, ultimately leading to the mature single-stranded ∼22-nucleotide miRNA. This functional miRNA is assembled into an RNA-induced silencing complex (RISC) that invariably contains a member of the Argonaute protein family (Fig. 1). Once loaded, the active RISC can be directed toward its messenger RNA target to regulate, predominantly in a negative manner, its translation.4 Besides targeting cellular messenger RNAs, miRNAs were recently shown to interact with transcripts of viral origin.5 The first description of such interactions revealed that miRNAs of cellular origin could negatively regulate viral messenger RNAs. Furthermore, mammalian viruses have been shown to usurp the cellular miRNA repertoire. One remarkable example of such usurpation is provided by HCV, which recruits the liver-specific miR-122 to enhance its replication.6 In vivo, the impact of miRNA for pathogenesis of HCV infection is more complex: analyzing liver biopsies from subjects with chronic hepatitis C who are undergoing IFN therapy, Sarasin-Filipowicz et al. showed no correlation of miR-122 expression with viral load but markedly decreased pretreatment miR-122 levels in subjects who had no virological response during later IFN therapy.7 Model of the mode of action of the LNA oligonucleotide SPC3649. The oligonucleotide binds to the 5′ part of the mature miR-122 with strong affinity due to the incorporation of some modified bases containing a ribose locked by an extra covalent liaison (shown above the LNA). The binding of the LNA prevents the incorporation of miR-122 into its effector RISC complex, which it normally uses to regulate both cellular genes and HCV RNA. Therefore, when the LNA is present, cellular targets of miR-122 have increased expression (which results in a decrease of cholesterol levels), whereas HCV replication goes down. The two effects of the inhibition of miR-122 are not interrelated. To truly assess the importance of miRNAs as a therapeutic target requires the use of chemically modified antisense oligonucleotides complementary to the miRNA to prevent its interaction with the target RNA. This approach was first established in vitro,8 before it was shown that it was also very effective in preventing miRNA action in mouse models.9 The later study was carried out using miR-122 as a model, and it enabled the identification of several cellular targets, most of which are involved in the cholesterol biogenesis pathway, e.g., 3-hydroxy-3-methylglutaryl-coenzymeA reductase. Primarily, two types of chemistries have been used to modify the antisense oligonucleotide and increase its stability and/or uptake by cells: 2′-O-methylated oligonucleotides, usually coupled to a cholesterol group9 and, more recently, oligonucleotides containing locked nucleic acid (LNA) residues (Fig. 1). A LNA antisense oligonucleotide binding to the 5′ part of miR-122 (SPC3649; Santaris Pharma, Hoersholm, Denmark) has been shown to be efficient via mouse models in confirming that blocking miR-122 results in a decrease in cellular targets involved in cholesterol biogenesis.10 Next, the inhibition of miR-122 and its effect on cholesterol levels was confirmed in nonhuman primates.11 The stage was set for testing the real effect of blocking miR-122 on HCV infection in a primate model. Following up on these investigations, miRNA-122 has now been shown to be a target for antiviral intervention. In a report this month in Science, Robert Lanford and colleagues showed that the inhibition of miR-122 in chimpanzees leads to long-lasting suppression of HCV viremia (Fig. 1).12 Using high and low doses of the SPC3649 oligonucleotide, they demonstrated that treatment of chronically HCV-infected animals with the LNA oligonucleotide results in a marked and sustained decrease of HCV RNA in both serum and liver. The sequestration of miR-122 by the LNA oligonucleotide was confirmed, as well as the strong reduction of free miR-122 levels for the high-dose animals. No rebound in viremia was observed during the treatment, and no adaptive mutations were found in the miR-122–binding sites. The analysis of the liver transcriptome revealed a marked down-regulation of IFN-regulated genes, which confirmed that the endogenous IFN pathway in the liver was normalized after inhibition of HCV RNA. Finally, as expected, the antagonism of miR-122 resulted in a strong decrease in serum cholesterol (Fig. 1). Besides that effect, no measurable toxic effect in the liver could be attributed to SPC3649. What are the clinical implications of this landmark study? The results of Lanford et al. clearly show that antagonism of miR-122 by the LNA oligonucleotide SPC3649 leads to marked suppression of viremia in chronically HCV-infected chimpanzees and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound suggests that targeting miRNA-122 by antagonists holds promise as a novel antiviral therapy. A potential advantage compared to therapeutic strategies that target viral factors may be the high barrier to resistance, as demonstrated by the lack of rebound in viremia during the treatment and the lack of adaptive mutations in the two miR-122 seed sites of the HCV 5′ noncoding region. Furthermore, conservation of both miR-122 seed sites in all HCV genotypes and subtypes suggests that such therapy will most likely be genotype-independent. A potential limitation of the therapeutic approach for the HCV-infected patient may be the delivery of the oligonucleotide, via frequent intravenous injections. Addressing this point in their discussion, the authors suggest that the safety profile and high stability of the LNA oligonucleotide in vivo combined with the prolonged suppression of viremia beyond treatment could result in less-frequent dosing after viral suppression is attained. Safety is another potential concern for an approach that targets a host factor involved in regulation of several genes. Although an extensive analysis of clinical symptoms, clinical chemistry, and histopathology in this small cohort of chimpanzees did not reveal any evidence of serious adverse effects, more-detailed investigations in humans are required to rule out potential adverse effects. Taken together, this study demonstrates the feasibility and safety of prolonged administration of an LNA oligonucleotide drug that antagonizes the function of a specific miRNA in a clinically relevant infectious disease model. Clinical trials are the next step to demonstrate the efficacy and safety of this approach in the HCV-infected patient.