Antiviral Research Volume 121, September 2015, Pages 69–81

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Review
Chronic hepatitis B: A wave of new therapies on the horizon

Timothy M. Blocka, , , Siddhartha Rawata, Carol L. Brosgartb
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doi:10.1016/j.antiviral.2015.06.014
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Highlights
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This articles introduces a symposium marking the 50th anniversary of the discovery of the Australia antigen.
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Current antiviral therapies for chronic hepatitis B suppress viral replication, but rarely eradicate the virus.
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The persistence of viral cccDNA in the nucleus of hepatocytes is the principal obstacle to curative therapy.
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Many new treatment approaches are now under development, and some may be more effective in eliminating cccDNA.
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As for hepatitis C, optimal therapy for hepatitis B may consist of a combination of drugs with different targets.

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Abstract
This year marks the 50th anniversary of the discovery of the Australia antigen (Blumberg et al., 1965), which in 1967 was identified to be the hepatitis B virus (HBV) surface antigen. Even though several antiviral medications have been in use for the management of chronic HBV infection for more than 20 years, sustained clearance of HBsAg, similar to the sustained viral response (SVR) or cure in chronic hepatitis C, occurs in only a minority of treated patients. Moreover, even after 10 years of effective suppression of HBV viremia with current therapy, there is only a 40–70% reduction in deaths from liver cancer. Recent success in developing antivirals for hepatitis C that are effective across all genotypes has renewed interest in a similar cure for chronic HBV infection. In this article, we review a wave of newly identified drug targets, investigational compounds and experimental strategies that are now under clinical evaluation or in preclinical development. The paper forms part of a symposium in Antiviral Research on “An unfinished story: From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B.”

Keywords

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Keywords
Hepatitis B virus; Chronic hepatitis B; Antiviral therapy; Clinical trials
This year marks the 50th anniversary of the discovery of the Australia antigen, which in 1967 was identified to be the hepatitis B virus (HBV) surface antigen (Blumberg et al., 1965 and Blumberg et al., 1967). Even though several antiviral medications have been in use for the management of chronic HBV infection for more than 20 years, sustained clearance of HBsAg, similar to the sustained viral response (SVR) or cure in chronic hepatitis C (HCV), occurs in only a minority of treated patients. Moreover, even after 10 years of effective suppression of HBV viremia with current therapy, there is only a 40–70% reduction in deaths from liver cancer. Recent success in developing combination antiviral therapies for hepatitis C that are effective across all genotypes has renewed interest in a similar cure for chronic HBV infection.

This article introduces a symposium in Antiviral Research on “An unfinished story: From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B.” This collection of some 15 invited papers describes a wave of newly identified drug targets, investigational compounds and experimental strategies that are now under clinical evaluation or in preclinical development. In this article, we provide a general overview of these new approaches, and refer readers to papers in the symposium in which additional information on each type of novel therapy can be found.

1. Introduction

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1. Introduction
More than 350 million people are chronically infected with hepatitis B virus (HBV), with some 600,000 deaths per year attributed to the virus (El-Serag and Rudolph, 2007 and Kanwal et al., 2015). Chronic HBV infection is associated with significant morbidity and mortality, secondary to acute and chronic hepatitis, fibrosis, cirrhosis, end-stage liver diseases and primary hepatocellular carcinoma (HCC) (see forthcoming review by Gish et al. in this symposium). It is estimated that, if left untreated, approximately 15–25% of chronically infected individuals would develop liver cirrhosis and HCC, after decades of infection (Block et al., 2007 and Block et al., 2003). Although the precise mechanisms involved in the virus-mediated pathology are not completely known, it is generally assumed that suppression of HBV replication and antigen production is beneficial. Indeed, there is now considerable evidence that suppression of HBV DNA replication can arrest, and even reverse liver fibrotic disease and decrease the incidence of HCC (Bedossa, 2015, Lok, 2004 and Lok and McMahon, 2009).

The first drug to be approved for managing chronic HBV infection was interferon-α2b (Intron A®), in 1991. Since then, seven drugs have been approved, the most recent being tenofovir disoproxil fumarate (Viread®) in 2008 (Hoofnagle et al., 2007) (Fig. 1). Broadly, these drugs can be classified as either host-targeting antivirals (HTA) or direct-acting antivirals (DAA) (Fig. 2). HTAs target host gene products, while DAAs target viral gene products.

Time-line of approval of hepatitis B therapeutics by the US Food and Drug ...
Fig. 1.
Time-line of approval of hepatitis B therapeutics by the US Food and Drug Administration. Green: immunomodulators (interferons); blue: direct-acting antivirals (polymerase inhibitors).
Figure options
Categorization of therapeutics for management of chronic HBV infection. ...
Fig. 2.
Categorization of therapeutics for management of chronic HBV infection. Direct-acting antivirals (DAAs) interfere with a specific step in viral replication. Host-targeted antivirals (HTA) inhibit viral replication by modifying host cell function.
Figure options
To date, the only approved HTAs are the interferons, and th

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To date, the only approved HTAs are the interferons, and there are two approved for use in the United States: interferon-α2b (Intron A®) and peginterferon-α2a (Pegasys®). The pegylated form of interferon-α is considered to be an improvement over the non-pegylated form, with a decreased renal clearance rate, longer half-life, and increased bioavailability, thereby reducing the number of times per week that it needs to be injected, to once weekly. However, pegylated interferon-α showed no improvement in the side-effects profile caused by the first-generation interferon-α (Chae and Hann, 2007).

There are currently five approved DAAs for chronic hepatitis B in the US, all of which are nucleot(s)ide analogues: lamivudine (Epivir-HBV®), adefovir dipivoxil (Hepsera®), entecavir (Baraclude®), telbivudine (Tyzeka™) and tenofovir disoproxil fumarate (Viread®). All of them inhibit the reverse transcriptase/polymerase activity, resulting in a decrease in viral replication as measured by reductions in serum HBV DNA (see review by Gish et al. in this symposium). Use of the current therapeutics has been widely reviewed (Hoofnagle et al., 2007, Liaw et al., 2008 and Lok and McMahon, 2009). Many other DAAs and HTAs can be contemplated (Fig. 3).

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Is a cure for hepatitis B possible? Hepatitis C patients are now routinely cured of their chronic viral infection with the use of combination all-oral DAA regimens or DAA regimens in combination with pegylated interferon, with or without ribavirin. Current HCV combination regimens can achieve an off-therapy sustained viral response at 12 weeks post completing therapy (SVR 12) in ∼90–100% of patients, across all genotypes and all stages of chronic hepatitis C (Hoofnagle and Sherker, 2014). It is often reasoned that management of chronic HBV infection is likely to be more refractory than chronic HCV infection, because HBV persists with a nuclear phase, and can reactivate, even after decades of indolence (Hoofnagle, 2009, Lok et al., 2012 and Seto et al., 2014) or with immunosuppression. This is mainly due to the presence of covalently closed circular DNA (cccDNA) of HBV present in the nucleus of infected hepatocytes (see forthcoming review by Guo and Guo in this symposium). cccDNA is a highly stable structure that acts as a minichromosome for all HBV transcripts. HBV also appears to be less responsive than HCV to interferons (Aspinall et al., 2011, Brouwer et al., 2015, Chan et al., 2011 and Locarnini, 2004). However, the most effective management of chronic HCV infection today is with interferon-free, all-oral DAA regimens (Afdhal et al., 2014, Curry et al., 2015 and Kowdley et al., 2014).

Complete suppression of the HBV polymerase should, in theory, reduce viremia and intrahepatic levels of replicative forms of HBV DNA to zero, and even cccDNA should be eliminated, as the infected cells are eventually replaced (Block et al., 2013). Therefore, according to this model, people with chronic HBV infection should be cured with DAAs alone. However, this has not been the case with most patients. Perhaps complete inhibition of the HBV polymerase has not been routinely achieved, since the degree of suppression of viral replication has been inadequate. We note that the most impressive and curative hepatitis C suppression has not been achieved with single DAAs, but with powerful combinations that target different steps in the viral replication cycle ( Kowdley et al., 2014 and Pawlotsky, 2014). However, combinations for HBV that are curative or even reliably superior to monotherapy have not been demonstrated with current medications, and even the most effective currently available DAAs for HBV do not drive intrahepatic levels of HBV DNA down by more than 2 log10 (despite 5–10 log10 reductions in serum viremia) ( Block et al., 2013, Coffin et al., 2011, Lau et al., 2005, Lee et al., 2013, Pellicelli et al., 2008, Werle-Lapostolle et al., 2004 and Yang et al., 2012).

The sustained, and substantial, intra-hepatic HBV DNA levels, even after more than a year of >5 log10 reductions in viremia, suggest that the current polymerase inhibitors are not inhibiting the enzyme by more than 99%. Thus, although these reductions in viremia are very impressive, they are still incomplete. Enzyme inhibition principles dictate that every additional ten-fold suppression in polymerase activity will require significantly greater amounts of inhibitor. However increasing the amounts of currently used polymerase inhibitors may not be safely tolerated in humans. The current polymerase inhibitors were optimized, and dosed to reduce serum HBV DNA levels to below the limits of detection, but not intra-hepatic viral DNA. Perhaps, if there were more potent polymerase inhibitors, or alternative means to decrease intra-hepatic levels of HBV DNA, including cccDNA, it would be possible to cure people with chronic HBV infection, with DAAs, in the same way that HCV is currently cured. In this review, we consider this possibility and identify several DAAs that might serve as alternatives or be complimentary, as part of new combination regimens, to the current portfolio of approved single-agent medications.

But how effective are current therapies for chronic hepatitis B? Clinically, chronically infected individuals have been divided into two groups, based on the presence or absence of the HBV gene product, HBeAg, which is derived from the capsid protein gene (Ganem and Prince, 2004 and Hoofnagle et al., 2007). Although the significance of these subcategories continues to be debated, HBeAg-positive individuals generally have greater serum viral loads. Clinical trials have customarily aimed to sero-convert HBeAg-positive individuals into becoming HBeAg-negative, and HBe-antibody (HBeAb) positive (Gish et al., 2010 and Hoofnagle et al., 2007). Both interferons and polymerase inhibitors are able to achieve HBeAg/Ab sero-conversion in approximately one-third of cases (Gordon et al., 2014 and Lok and McMahon, 2009). These also represent the subset of those experiencing viremia reduction, which is routinely achieved in almost everyone treated with polymerase inhibitors. Indeed, at least 90% will have serum HBV DNA levels reduced by 4–6 orders of magnitude, often reaching undetectable or nearly undetectable levels by current methods (Liaw and Crawford, 1999, Liaw et al., 2004 and Zeisel et al., 2015). However, reductions of intrahepatic viral DNA are far more modest, usually only 2 log10, even after two years of treatment, and this may be responsible for sequelae of virus reactivation and promote persistent liver disease (Lok, 2011, Zoulim and Durantel, 2015 and Zoulim and Locarnini, 2009).

Currently, the treatment of chronic hepatitis B must be life-long for the majority of patients, because virus rebound occurs, often within weeks to months after cessation of treatment (Cho et al., 2014 and Yuen and Lai, 2011). More concerning, even after 5–10 years of viremia suppression, the reduction in deaths due to liver disease is only 40–70% (Arends et al., 2014, Chang et al., 2006, Gordon et al., 2014, Lok, 2011 and Yapali et al., 2014). Finally, current recommendations advise therapy only for those with elevated viremia and serum transaminases, leaving at least half of patients at significant risk of liver disease without any medical options (EASL, 2012, Liaw et al., 2012, Lok and McMahon, 2009, Uribe et al., 2014 and Yapali et al., 2014). New approaches are clearly needed.

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2. A new wave of hepatitis B therapies
In addition to the currently approved interferons and nucleot(s)ide inhibitors, there is the possibility of developing new agents to inhibit viral replication, with mechanisms of action that have not yet been explored. As shown in Fig. 3, a number of critical steps in the HBV life cycle can potentially be targeted to decrease HBV replication. These steps use both host pathways/proteins and HBV-specific proteins, so that new HTAs and DAAs could be developed. In the following sections, we briefly profile several promising investigational HTAs and DAAs under clinical development, many of which target steps in the HBV life cycle not previously exploited. We also refer readers to symposium articles in which each novel approach to therapy is reviewed. Readers may also wish to see a forthcoming review by Liang and colleagues in Hepatology.

The most immediate new wave of therapies is likely to come from investigational agents currently in later stages of clinical development. As shown in Fig. 4A and B, using publically available sources such as scholarly publications, the US government website www.clinical trials.gov, and individual pharmaceutical company websites, we found at least 38 new investigational agents under development for the management of chronic hepatitis B, of which 19 have reached human trials. Of the 38 investigational agents, we have designated 21 as DAAs and 17 as HTAs. The latter can be further sub-categorized as either immunomodulators (HTA-i) or targeting other host functions (HTA-hf) needed by the virus. Of the 19 drugs reported to have reached human trials, 3 have failed or been discontinued for commercial reasons. When we were unable to determine a drug’s status, it is designated “status uncertain”.

Therapeutics in development for the management of chronic HBV infection. (A) ...
Fig. 4.
Therapeutics in development for the management of chronic HBV infection. (A) Investigational agents in Phase 1 clinical trials at the time of writing this review. The NCT number (wherever applicable) after the company’s name is the clinicaltrials.gov identifier. (B) Investigational agents in preclinical stages. The stage of development is indicated, from in vitro identification through animal efficacy and ultimately human clinical trials. DAAs are highlighted in green and HTAs in blue. Investigational agents that have failed or have been stopped are shown in red. See text for citations. When we were unable to find a published reference, we cite the sponsor’s website.

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3. Direct-acting antivirals
3.1. New investigational agents now in clinical trials

3.1.1. Prodrugs of HBV polymerase inhibitors

Prodrugs are chemical or molecular precursors of active drugs (Hostetler, 2009 and Smith, 2007). Typically, a prodrug is designed to improve the performance of the active drug substance, usually by decreasing toxicity, improving solubility, enhancing tissue absorption and/or increasing the half-life, so that the agent can be dosed no more frequently than once daily. Thus, as long as the prodrug can be efficiently and safely converted to the active agent, it may have better efficacy and safety profile. Prodrugs that reduce toxicity and improve pharmacokinetics are certainly welcome, although their contributions to care may be more incremental than transformational due to the persistence of cccDNA in the nucleus of infected hepatocytes and incomplete inhibition of HBV polymerase.

Prodrugs of tenofovir are in the most advanced stages of development (Fig. 4A). As mentioned, earlier, the five approved polymerase inhibitors are currently used at doses that impressively reduce viremia, but have a much more modest impact on intracellular HBV DNA levels (Lau et al., 2005, Peters et al., 2004 and Werle-Lapostolle et al., 2004). These drugs achieve effective suppression of viremia by 5–9 log10 or greater, but intrahepatic HBV viral DNA, although reduced, remains at significant levels. These reduced levels of replication are apparently sufficient to restore nuclear pools of cccDNA through intracellular recycling of nucleocapsids, even in people for who are serologically “PCR negative” (aviremic) for HBV.

That said, if a prodrug of a polymerase inhibitor could be safely used at higher doses or could achieve increased bioavailability of the active compound within hepatocytes, so as to achieve greater inhibition of the HBV polymerase, it could have a greater than incremental value. Indeed, since current polymerase inhibitors are associated with the loss of HBsAg and the appearance of HBsAb in as many as 10% of those treated over 5 years (Zoulim and Durantel, 2015), it is possible that a prodrug that inhibits intra-hepatic DNA replication could significantly increase seroconversion. Several prodrugs at various clinical stages of development target the HBV polymerase.

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3.1.1.1. AGX1009 and TAF

AGX1009 (Agenix) and TAF (Gilead), are prodrugs of tenofovir in Phase 3 clinical trials, although TAF is in Phase 3 for HIV indications and Phase 1/2 for hepatitis B (Menendez-Arias et al., 2014). They are predicted to have reduced long-term toxicity compared to tenofovir.

3.1.1.2. Besifovir, elvucitabine, pradefovir mesylate & MIV210

Besifovir (LBO80380/ANA380) 9-[1-(Phosphonomethoxycyclopropyl) methyl] guanine (PMCG) by Idong Pharma (Korea) is in Phase 3 clinical trial (Yuen et al., 2010). Achillion’s elvucitabine is a l-cytosine nucleoside analog reverse transcriptase inhibitor that demonstrated potent antiviral activity against HBV and HIV. Phase 2 clinical studies showed that elvucitabine is well tolerated in patients with chronic HBV infection, with an antiviral potency is similar to that of lamivudine (Achillion Pharmaceuticals, 2010). No information is available regarding the efficacy of elvucitabine against lamivudine resistant HBV.

On the other hand, pradefovir mesylate, a propylated adefovir that depends upon CYP3A mediated activation, looked promising in Phase 2 studies, but was put on hold because of tumor formation in animals (Reddy et al., 2008). The development of MIV210 (Michalak et al., 2009) has also been abandoned (Grogan, 2013). Thus, even for prodrugs that have an established mechanism of action that is clearly beneficial, it is impossible to predict the “winners and losers” prior to preclinical and/or clinical evaluation.

3.1.1.3. CMX157

CMX157 (Chimerix/Contravir) is a lipid conjugate (hexadecycloxypropyl adenine) of tenofovir diphosphate that was designed to exploit lipid uptake pathways (Painter et al., 2007). CMX 157 delivers tenofovir diphosphate at high concentration in the hepatocytes, thus increasing the bioavailability of tenofovir diphosphate and at the same time decreasing circulating tenofovir levels to minimize potential renal side effects (Contravir). It has utility for both HIV and HBV, and is now entering Phase I/2 clinical trials for HBV.

3.1.2. siRNA

In principle, siRNA-acting drugs, which target HBV transcripts, should be able to shut down all HBV gene product production. This approach has had great promise, but has been frustrated by the inefficiency in delivery of the nucleic acid oligomers to human hepatocytes, despite extremely compelling results in experimental animals (Wooddell et al., 2013). Thus, if the delivery problem could be solved, the potential for siRNA and similar nucleic acid-directed suppressive molecules, is tremendous. It is with this hope and expectation, that a new wave of siRNA molecules is greeted (see forthcoming review by Gish and colleagues in this symposium).

3.1.2.1. ARC-520

ARC-520 is the siRNA from Arrowhead, which is lipid conjugated and uses nanoparticle-assisted delivery system. ARC-520 demonstrated good efficacy in reducing the levels of serum HBsAg, HBeAg and HBV DNA levels, in non-transgenic mouse model for HBV infection (Wooddell et al., 2013). It also showed promising results in HBV-infected chimpanzee model system. ARC-520 reached Phase 2 trials, but was placed on a clinical hold, and only recently it has been allowed to proceed with the Phase 2 studies (Arrowhead, 2015a).

3.1.2.2. ALN-HBV, TKM HBV

The ALN-HBV by Alnylam and TKM-HBV by Tekmira use lipid nanoparticle technology for delivering their siRNA. The Alnylam siRNA candidate demonstrated significant suppression of circulating HBV DNA and HBsAg levels in chimpanzee model system (Alnylam, 2014). The siRNA therapeutic candidates from these companies will have reached clinical phase by the time of this review’s publication. The Tekmira siRNA agent is likely to be in at least Phase 1 clinical trial. Thus, despite recent reports of disappointing Phase 2 results for the Arrowhead compound (Arrowhead, 2015b and Wooddell et al., 2013) it still appears that, after the prodrugs, the siRNA technologies are the furthest along in development.

3.1.3. HBsAg-reducing agents

Rep2139, from Replicor, is a nucleic acid-based polymer (NAP), comprised of phosphorothioated nucleic acids (Noordeen et al., 2013) (see forthcoming review by Vaillant and colleagues in this symposium). The sponsor reports that the agent is safe, and in small overseas human trials in Bangladesh, to have beneficial activity in combination with interferons or Zadaxin (Mahtab and Vaillant, 2015). The mechanism of action is unclear, but the sponsor reports it acts on HBsAg. As stated, compounds that act on HBsAg are particularly interesting because they also have the potential for direct activity against hepatitis delta virus (HDV), since HDV infection is dependent upon the HBsAg (Seeger and Mason, 2000).

3.1.4. Inhibitors of capsid formation

Of the DAAs that have reached clinical phase, at least three inhibit the formation of the HBV capsid. Since the first report of a capsid inhibitor more than 10 years ago (Stray et al., 2005, Stray and Zlotnick, 2006 and Weber et al., 2002), there have been a number of new examples of this approach (see forthcoming review by Zlotnick et al. in this symposium). Capsid formation is an essential viral process that does not occur in the uninfected cell, and thus would be expected to provide a virus-selective target. Moreover, capsid proteins are readily detected in the nucleus of infected cells, far from the site of nucleocapsid formation in the cytoplasm. This is consistent with evidence that capsid proteins play a role in regulating HBV cccDNA expression and stability, as well as in regulating host innate immune response genes. Therefore, even though investigational agents may have a phenotypically similar effect on capsid assembly, they may modulate these other processes differently, thus affecting the overall ability of the agent to manage chronic HBV infection.

Three capsid inhibitors have reached clinical phase development: BAY4109 (AiCuris), NV1221 (Novira) and GLS 4 (Sunshine).

3.1.4.1. BAY4109

The capsid inhibitor BAY4109 came on the scene with a great deal of fanfare, as an innovative, first in class capsid inhibitor (Deres et al., 2003 and Weber et al., 2002). It is reported to be highly species specific, for the virus, with activity against only the human HBV. This rendered pre-clinical efficacy testing more limited, since testing in woodchucks would not be possible, as it does not have activity against WHV. It was evaluated in Phase 1 clinical trials but its current development status is unclear.

3.1.4.2. NV1221

In the US, the Novira agent, NV1221, is probably the most advanced of the new capsid inhibitors, now in Phase 1 studies in New Zealand. There is no structural or specific functional information available about the compound, but it appears to prevent HBV capsid formation in a way analogous, but (importantly) not identical to the BAY4109.

3.1.4.3. GLS-4

GLS4 is a heteroaryl pyrimidine analogue that was derived from BAY4109 after structural optimization. GLS4 has a unique mechanism of action by which it causes aberrant capsid protein formation. GLS 4 was shown to inhibit nucleoside-analogue resistant HBV mutants in preclinical studies (Wang et al., 2012 and Wu et al., 2013). HEC pharma group reports that Phase 1 studies of GLS4 are completed (HEC).

3.2. Direct-acting antivirals in preclinical development

3.2.1. Inhibitors of capsid morphogenesis

Preclinical phase capsid inhibitor candidates include CpAMs (Assembly Biosciences), DVR (Oncore-Tekmira), and DSS (Oncore-Tekmira). All these capsid inhibitors are small molecules that interfere with HBV capsid morphogenesis, but not necessarily at the same step. CpAMs are HBV core protein allosteric modulators that accelerate a dysfunctional capsid protein dimerization (Katen et al., 2013). DVRs prevent the association of HBV pregenomic RNA with the capsid (Campagna et al., 2013). The mechanism of action of the DSS compounds is not yet reported.

3.2.2. Inhibitors of HBsAg secretion

The development and current status of inhibitors of HBsAg secretion are reviewed by Cuconati and colleagues in a forthcoming article in this symposium. TTP is a small molecule that has been shown to prevent the secretion of HBsAg and viral DNA in vitro, possibly by interfering with the ability of HBsAg to associate with the LDL secretion machinery ( Dougherty et al., 2007 and Yu et al., 2011). HBsAg may also have immunosuppressive functions ( Jaroszewicz et al., 2010 and Xu et al., 2009). The TTPs are at an early preclinical stage of development, but are the only small molecule inhibitors of HBsAg secretion.

3.2.3. RNase H inhibitors

Unlike other DNA viruses HBV replication depends upon the RNAseH activity of HBV polymerase to degrade pregenomic RNA (Seeger and Mason, 2000). RNAseH enzymatic activity should, in principle, be a viable antiviral target as is the reverse transcriptase/DNA polymerase activity of HBV polymerase. A group in St Louis University (Cai et al., 2014 and Tavis and Lomonosova, 2015) has reported identifying “hit” compounds, some based on those that are validated HIV drugs, that are selective inhibitors of the HBV polymerase RNAseH activity (see review by Tavis and Lomonosova in this symposium). These compounds need further development and could be a welcome addition to the HBV antiviral arsenal. They can prove be very effective when used in combination with the existing nucleot(s)ide analogues and may help to achieve long-term inhibition of HBV replication at a level that is not achieved by current nucleot(s)ide analogues alone.

3.2.4. CRISPR/Cas9 system

The bacterial clustered regularly interspaced short palindromic repeats associated systems (CRSPR/Cas9) loci encode RNA guided endonucleases, derived from bacterial immune response against foreign genetic elements such as bacteriophages (Kennedy et al., 2015 and Seeger and Sohn, 2014) and have been adapted for mammalian systems (see forthcoming review by Cullen and colleagues in this symposium). In principle, they can be used to target destruction of specific DNA sequences, and thus hold a great potential for specific degradation of HBV cccDNA. The challenges of getting these complex systems into hepatocytes, let alone into the nucleus, are clear. However, lentiviruses expressing CRISPR/Cas9 guide RNAs that are specific for HBV DNA have been transduced into HBV cccDNA-producing cells and shown to be suppressive (Dong et al., 2015, Kennedy et al., 2015 and Lin et al., 2014). HepG2 cells expressing the receptor were infected with HBV and the CRSPR/Cas9 system was used to induce degradation of cccDNA. This also suggested that the targeted DNA is degraded rather than repaired following Cas9 nuclease digestion. Thus, there is some progress with these systems, although clinical investigation is probably a long way off due to the difficulties in the delivery process.

3.2.5. siRNA

ddRNAi from Benitech is another therapeutic approach to directly target HBV transcripts using RNA interference technology. This program was initiated by Benitech in 2009 and is currently in preclinical stages. The studies are being conducted in collaboration with Chinese-based “Biomics biotechnologies”, and are aimed at targeting HBV polymerase transcript using three different short-hairpin RNA (shRNA) to target different regions of the polymerase transcript (Biopharma).

3.2.6. cccDNA formation inhibitor

cccDNA can be repressed by preventing its’ formation, expression, or stability. Cai et al. (2012) screened a small compound library and described identification of a di-substituted sulfonamide (DSS) which significantly and selectively inhibited cccDNA. The mechanism appears to be at the formation step. This molecule, and its derivatives, are currently in pre-clinical development by Oncore-Tekmira.