HBsAg Rules at International Liver Congress

Now that the HBV world has gathered extensive clinical experience with interferons and polymerase inhibitors (NUCs) and with the resolve to finally find a cure for a serious disease afflicting hundreds of millions worldwide, the hepatitis B surface antigen (HBsAg) has become recognized as the key determinant for treatment outcome.  This is being confirmed by trial after trial investigating combining the actions of both NUCs and interferons, either one after the other or together at once.

Some of these studies were presented at the International Liver Congress last week in Vienna (ILC2015).  Emerging from them are actionable HBsAg rules which can predict fairly well whether a patient will eventually seroconvert to (or at least lose) HBsAg.  No matter the excitement around CRISPR technologies, HBsAg seroconversion remains the gold standard outcome in HBV treatment in the foreseeable future.

These rules can be divided into pre- and post-IFN treatment onset.

In the pre-IFN setting, it is those patients that have below ~500 IU/ml serum HBsAg as a result of NUC treatment that will most likely respond to interferon treatment/immune stimulation with s-antigen seroconversion (see earlier blog entry).  Since NUCs alone hardly do anything to promote s-antigen seroconversion despite its dramatic lowering of viral HBV titers, it appears to be their slow impact on HBsAg levels ( 0.1 log per year HBsAg reduction) that has the synergistic effect with interferon: with HBsAg lowering you take off the foot on the immune brake, with interferon you step on the immmune gas pedal.

As such, HBsAg knockdown by RNA(i) Therapeutics would seem to do the same for interferons as NUCs do, only in a more rapid and potent manner.  Of course, both could be used concurrently as a run-in to IFN treatment.

However, once on IFNs (post-IFN onset), it is the relative HBsAg decline that has high positive predictive value in prognosticating who will seroconvert.  Of note, the HBsAg decline comes before any adaptive immunity can be detected.  This supports that HBsAg decline in itself contributes to seroconversion rather than it being a mere correlation.  In that setting, it is a 1 log decline in HBsAg the first few weeks after IFN treatment onset that separates the winners from the losers. 

It is uncertain to me, however, whether 1 log is a precondition to s-antigen seroconversion as the non-responders do not even come close to that (maybe 0.3log).  It is therefore possible that anything that pushes HBsAg below say -0.3-0.5log could have a dramatic effect on s-antigen seroconversion rates.

An RNAi Therapeutic for HBV used simultaneously with IFNs may therefore aim at helping IFNs to get to the  0.5-1log reduction threshold, and rapidly at that.

Ergo, there are now a number of obvious strategies that one can apply regarding the use of RNAi Therapeutics in HBV with various knockdown goals, both absolute and relative.  The exact strategy would depend on how the RNA agent is combined with polymerase inhibition/NUCs or immune stimulation. 

While a number of other HBV targets were reported at the conference such as core assembly and entry inhibitors, HBsAg (and HBV mRNA knockdown in general) lowering remains the most distinguished and the mechanism predicted to be most synergistic to existing treatment approaches.  As combination treatment is strongly predicted to be the future of HBV, HBsAg lowering should become a pillar of those treatment regimes.

Disclosure: long ARWR, looking for lower entry in TKMR.

Journal Club: A commonly used treatment for HCV, Interferon Beta, may largely act through microRNAs

While on vacation, an interesting study on the effect of interferon beta on microRNA levels was published in the journal Nature (Pedersen et al.: Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature doi:10.1038/nature06205).

In this study, Pedersen and colleagues were initially interested in whether interferons had the potential to modulate cellular microRNA levels. Not very surprisingly, this potent class of cytokines up- and downregulated a number of microRNAs. Strikingly, however, eight of the interferon beta-induced microRNAs had microRNA seed complementarities with an HCV genome. Moreover, miR-122, a microRNA that has now been shown by a number of laboratories now to facilitate HCV replication, was downregulated by interferon beta.

The link between HCV and interferon-regulated microRNAs is intriguing, since interferon beta is at the center of current HCV treatment regimens. In order to test whether the antiviral activity of interferon beta on HCV replication was indeed mediated by microRNA regulation, the authors asked whether interferon beta could still inhibit HCV replication in the presence of mimics of the upregulated and HCV matching microRNAs and an inhibitor of miR-122. In agreement with the notion that interferon-regulated microRNAs mediate a large part of interferon beta inhibition of HCV, such a mixture of small RNAs alleviated interferon beta inhibition of HCV replication from 90% to around 50% of untreated control in a tissue culture system.

HCV has a long-standing tradition in the RNAi Therapeutics field. As such, a number of drug candidates are expected to enter the clinic in the near future that directly target the HCV genome by RNAi. In addition, since HCV replication is supported by miR-122, it has become the focus of the first wave of microRNA-targeting therapeutic programs. Due to the ability of viruses to escape drug inhibition through mutation, a combination of these approaches appears promising. As much as no other current HCV antiviral alone can reliably get rid of HCV altogether, I do not expect any RNAi-related stand-alone therapy for HCV to be successful. However, when combined with potent agents such as Vertex Pharmaceutical’s late-stage protease inhibitor VX-950, RNAi may be able to further knock down HCV sufficiently so that it can be entirely cleared by the body. Moreover, many patients do not complete interferon therapy due to its severe side-effect profile, and alternatives are desirable. The strategy proposed in the paper may therefore lead to a treatment that works through the same antiviral pathway as interferon beta, but without the side-effects.

Lastly, I would like to briefly comment on the evolutionary aspects of the studies. It is very unlikely, given the rapid evolution of viruses alone, that the sequence of the implicated microRNAs was shaped due to selection based on HCV inhibition. Accordingly, the authors find that the sites complementary to the microRNA seeds are not all conserved in the different HCV genotypes (note: whether this is related to the varying efficacy of interferon beta on different genotypes in the clinic was not discussed). It is only through comparing the modulated microRNAs with a lot of viruses that they found the link with HCV. It is therefore fortuitous that interferon-modulated microRNAs should have anti-HCV activities. Of note, this is similar to a paper published 2 years ago in the journal Science (Lecellier et al.: A cellular microRNA mediates antiviral defense in human cells. Science 308: 557) which showed for the first time that a cellular microRNA may restrict the replication of a mammalian virus through good fortune.

Back to Basics: Does RNAi Exist in Humans?

Whenever I try to explain the promise of RNAi for human biology and medicine to friends and family, I feel torn by a conflict of staying scientifically accurate or running the risk of losing my audience by burdening them with too many details. You may ask “why” now- isn’t RNAi this elegant straightforward mechanism in human cells whereby double-stranded siRNAs silence complementary mRNAs? How would you react when I said that a number, if not the majority of notable molecular biologists hold that RNAi does not exist in humans?

RNAi is most easily explained by starting with long double-stranded RNA (dsRNA) that is then chopped up by Dicer into short interfering RNAs which then get incorporated into the RiSC complex. The siRNAs loaded in RiSC consequently seek out and destroy their targets by a slicing mechanism. However, long double-stranded RNAs are not suitable for specific gene suppression in humans due to an innate immune system called the interferon response. The interferon response recognises dsRNA greater than 30bp in length and consequently shuts down almost all protein translation in a cell. This is a mechanism by which the cell protects itself from viruses which often produce dsRNA intermediates as part of their life-cycle.

I therefore vividly remember myself in an elective class (“RNA World”) as an undergrad student at Edinburgh University, when my lecturer, Dr. David Tollervey, predicted that it would only be a matter of time that RNAi would be discovered in humans- 1 year before Tuschl and colleagues published their seminal paper on siRNAs. In retrospect, it is clear that his confidence must have come from the fact that the human genome was littered with RNAi-related genes, such as some coding for Dicer and Argonaute proteins. He also may have been familiar with recent publications on the discovery of RNAi-related small RNAs in plants (Hamilton and Baulcombe) and the finding that double-stranded RNAs were processed into similarly sized small RNAs in an Drosophila in vitro system (Zamore, Tuschl, Sharp, Bartel).

Of course, we all know now that it was Tuschl who had the genius to conclude that siRNAs would be the natural effectors of RNAi in humans without inducing the interferon response. Except that they were not. Of course, synthetic siRNAs would work, but years of searching for bona fide naturally occurring siRNAs in humans derived from long dsRNA failed to convincingly prove their existence. It turns out that the RNAi enzymes are all present in vertebrates for another, albeit related reason, namely microRNA-mediated gene regulation. MicroRNAs are a major class of small RNAs that are not derived from long dsRNA, but hairpin precursors and that have turned out to be ubiquitous mediators of post-transcriptional gene regulation and that require RNAi-related proteins for their biogenesis and function. It is in this pathway that experimentally introduced siRNAs perform their gene silencing function.

It therefore appears that in humans the interferon system may have rendered antiviral roles for RNAi unnecessary. This is unlike in many invertebrates which lack an interferon system and where the ancient antiviral function is still alive and kicking. A fascinating story of evolving overlapping biological pathways with a happy ending for keeping the therapeutic promise of RNAi alive, but when you ask a scientist about RNAi in humans- beware!

Market Watch: Nastech reported “positive non-clinical study results for [their] siRNA program for treatment of influenza [in tissue culture in vitro studies].” Following an announcement this Monday of a webinar on flu RNAi to be hosted by Nastech this Friday, frantic trading led to big gains in Nastech stock in anticipation of major RNAi news. Nastech and Alnylam vie for a potentially lucrative government contract for flu preparedness and progress in this area will be closely watched as flu RNAi may turn out to be the first commercial RNAi Therapeutics product. It is likely that NSTK will give up some of its gain in the wake of today’s news, while ISIS may gather momentum as their RNA modification IP estate is starting to yield a rich harvest. Alnylam is to follow with their report tomorrow.