Treating hepatitis C by blocking a cellular microRNA

HCV UTRMiravirsen is a drug that binds to and blocks the function of a cellular microRNA called miR-122 that is required for the replication of hepatitis C virus (HCV). Treatment of chimpanzees chronically infected with HCV with this drug leads to suppression of viral replication. The results of a phase 2b human clinical trial in HCV infected humans indicate that Miravirsen reduces levels of viral RNA without evidence for viral resistance. I asked virologist Stan Lemon (who appeared recently on TWiV 235) his opinion of these findings.

Are you surprised that the antiviral effect of Miravirsen is long lasting?

The Janssen study published in NEJM basically recapitulated what Lanford had observed in HCV-infected chimps treated with the compound, with a very slow onset of antiviral effect, and then a very slow rebound as well. This probably reflects the pharmacokinetics and very high stability of the locked nucleic acid compound, and the time required to sequester endogenous miR-122 – changes in serum cholesterol also move very slowly. I think this is why the antiviral effect (and cholesterol effect) are long-lasting.

Is it surprising that no resistance to Miravirsen was observed?

As for the lack of resistance, it doesn’t surprise me much. This was observed in the chimps as well. The virus is really dependent upon miR-122 for its replication, and can’t readily mutate around it – the requirement for miR-122 reflects more than just the stabilizing effect of miR-122 on the viral genome, as we showed in a recent PNAS paper (Li et al., Proc. Nat’l. Acad. Sci U.S.A., 110:1881-6, 2013) written in follow-up to our earlier demonstration of the stabilizing effect of the miRNA on the HCV genome (Shimakami et al., Proc. Nat’l. Acad. Sci U.S.A. 109: 941-6, 2012, that you reviewed in TWIV 180) – what we know and don’t know about the mechanism of action is summarized in an “opinion” piece now in press in RNA Biology.

Do you think this drug will ultimately get FDA approval?

Given issues of resistance, relapse, and poor pan-genotype coverage with direct-acting antivirals for HCV, all of this should bode well for Miravirsen. However, it has issues like almost all the new therapies under evaluation.

First, the spaghetti plots in the Janssen paper show large variation in the response of individual patients, with some having little effect when receiving Miravirsen. This is unlike studies with enzyme inhibitor antivirals, and I am not aware of any good reason for it other than potential variation in endogenous miR-122 abundance.

A second and greater issue is the cancer concern. Most hepatocellular carcinomas (except those associated with HCV, interestingly enough) demonstrate significant reductions in miR-122 abundance, and miR-122 can reverse some malignancy-associated features of transformed hepatocytes in vitro – thus, miR-122 seems to act much like a tumor suppressor in the liver.  miR-122 knockout mice develop normally but have a high incidence of hepatocellular carcinoma. I think this poses real problems for the development of Miravirsen. While one could reasonably argue that short-term exposure to the antagomir is very different than gene knockout, the patients being treated are those at the highest risk for HCC – particularly if there is advanced fibrosis or cirrhosis, which characterizes those most in need of treatment. It is also clear that HCC can manifest itself in patients AFTER therapeutic elimination of the virus. The risk is most certainly greatly reduced, but it is not zero (HCC develops very slowly, and in a multi-centric fashion), and with the evidence that the drug has relatively long-lasting effects on cholesterol (as well as the virus), I think the developers of Miravirsen may find it difficult to defend against future claims that the drug contributed to the development of HCC in some cases. There isn’t a good way to de-risk this, to show that this theoretical concern is not real, and this must be worrying the regulatory authorities – especially since there are now many alternative therapies under evaluation that don’t carry this risk, some of which are looking very good in combination with each other (e.g., advanced NS3 inhibitors, NS5A inhibitors, and nucs).

New hepatitis C antiviral drugs

chlorocebus-pygerythrusNearly 3% of the world’s population – about 175,000,000 individuals – are infected with hepatitis C virus (HCV), and 3-4 million new infections are added each year. A high percentage of these become life long, chronic infections, which may lead to cirrhosis, hepatocellular carcinoma, and death. No vaccine is available, and the only antiviral treatment, pegylated interferon plus ribavirin, is often ineffective and has adverse side effects. The extent and severity of hepatitis C has stimulated the development of many new antiviral compounds, including protease and polymerase inhibitors. Perhaps one of the most unusual treatments being considered is one that directly targets a small RNA, known as a microRNA (miRNA), that is abundant in the liver.

Micro RNAs are single-stranded RNA molecules 21-23 nucleotides long which regulate the expression of about 30% of all mammalian protein-coding genes. They act by binding to the 3′-non coding region of mRNAs which leads to degradation of the mRNA or inhibition of translation. When miRNAs were first discovered, many virologists searched for target sequences in viral genomes to discover new ways to inhibit viral replication. A binding site for a liver-specific miRNA, called miR-122, was found in the RNA genome of HCV. However the target site for miR-122 was found in the 5′-noncoding region of the mRNA, not in the 3′-end. To the investigators’ surprise, binding of miR-122 to the HCV RNA stimulated viral replication. HCV replication in cells lacking miR-122 is significantly reduced compared with cells that produce miR-122.

The requirement of miR-122 for HCV replication suggested that this miRNA could be a target for therapeutic intervention. One approach has been the use of antagomirs, which are small synthetic RNAs that are complementary to the miRNA target. By binding to the miRNA, antagomirs block their ability to bind the target sequence and inhibit their function. An antagomir to miR-122 was found to impair HCV replication in cultured liver cells. Last year, a phase I clinical trial of an miR-122 antagomir was initiated in humans.

An important question is whether inhibition of miR-122 influences the expression of cellular genes which might lead to adverse side effects. If the antagomir is ever licensed, it will have another benefit besides inhibiting HCV replication: reduced cholesterol levels. miR-122 is believed to be involved in the regulation of cholesterol, fatty acid, and lipid metabolism, and African green monkeys who were administered the miR-122 antagomir had reduced plasma cholesterol.

C. L. Jopling, MinKyung Yi, Alissa M. Lancaster, Stanley M. Lemon, Peter Sarnow (2005). Modulation of Hepatitis C Virus RNA Abundance by a Liver-Specific MicroRNA Science, 309 (5740), 1577-1581 DOI: 10.1126/science.1113329

Jan Krützfeldt, Nikolaus Rajewsky, Ravi Braich, Kallanthottathil G. Rajeev, Thomas Tuschl, Muthiah Manoharan, Markus Stoffel (2005). Silencing of microRNAs in vivo with ‘antagomirs’ Nature, 438 (7068), 685-689 DOI: 10.1038/nature04303

Y SHAN, J ZHENG, R LAMBRECHT, H BONKOVSKY (2007). Reciprocal Effects of Micro-RNA-122 on Expression of Heme Oxygenase-1 and Hepatitis C Virus Genes in Human Hepatocytes Gastroenterology, 133 (4), 1166-1174 DOI: 10.1053/j.gastro.2007.08.002

Joacim Elmén, Morten Lindow, Sylvia Schütz, Matthew Lawrence, Andreas Petri, Susanna Obad, Marie Lindholm, Maj Hedtjärn, Henrik Frydenlund Hansen, Urs Berger, Steven Gullans, Phil Kearney, Peter Sarnow, Ellen Marie Straarup, Sakari Kauppinen (2008). LNA-mediated microRNA silencing in non-human primates Nature, 452 (7189), 896-899 DOI: 10.1038/nature06783