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A novel orally available small molecule that inhibits hepatitis B virus expression

Open AccessPublished:October 25, 2017DOI:https://doi.org/10.1016/j.jhep.2017.10.014

      Highlights

      • RG7834 is a novel orally bioavailable HBV viral gene expression inhibitor.
      • The effect of RG7834 on HBV expression levels is highly virus-specific.
      • The unique antiviral profile is differentiated from that of nucleos(t)ide analogues.
      • Combination with entecavir and/or PegIFNα improves the antiviral profile and potency.

      Background & Aims

      The hallmarks of chronic HBV infection are a high viral load (HBV DNA) and even higher levels (>100-fold in excess of virions) of non-infectious membranous particles containing the tolerogenic viral S antigen (HBsAg). Currently, standard treatment effectively reduces viremia but only rarely results in a functional cure (defined as sustained HBsAg loss). There is an urgent need to identify novel therapies that reduce HBsAg levels and restore virus-specific immune responsiveness in patients. We report the discovery of a novel, potent and orally bioavailable small molecule inhibitor of HBV gene expression (RG7834).

      Methods

      RG7834 antiviral characteristics and selectivity against HBV were evaluated in HBV natural infection assays and in a urokinase-type plasminogen activator/severe combined immunodeficiency humanized mouse model of HBV infection, either alone or in combination with entecavir.

      Results

      Unlike nucleos(t)ide therapies, which reduce viremia but do not lead to an effective reduction in HBV antigen expression, RG7834 significantly reduced the levels of viral proteins (including HBsAg), as well as lowering viremia. Consistent with its proposed mechanism of action, time course RNA-seq analysis revealed a fast and selective reduction in HBV mRNAs in response to RG7834 treatment. Furthermore, oral treatment of HBV-infected humanized mice with RG7834 led to a mean HBsAg reduction of 1.09 log10 compared to entecavir, which had no significant effect on HBsAg levels. Combination of RG7834, entecavir and pegylated interferon α-2a led to significant reductions of both HBV DNA and HBsAg levels in humanized mice.

      Conclusion

      We have identified a novel oral HBV viral gene expression inhibitor that blocks viral antigen and virion production, that is highly selective for HBV, and has a unique antiviral profile that is clearly differentiated from nucleos(t)ide analogues.

      Lay summary

      We discovered a novel small molecule viral expression inhibitor that is highly selective for HBV and unlike current therapy inhibits the expression of viral proteins by specifically reducing HBV mRNAs. RG7834 can therefore potentially provide anti-HBV benefits and increase HBV cure rates, by direct reduction of viral agents needed to complete the viral life cycle, as well as a reduction of viral agents involved in evasion of the host immune responses.

      Graphical abstract

      Keywords

      Introduction

      Hepatitis B virus (HBV) infection is one of the most common infectious diseases in the world. Chronic hepatitis B (CHB) represents a critical unmet medical need with over 240 million people chronically infected worldwide. Nearly 25% of all chronic HBV carriers develop serious liver diseases such as chronic hepatitis, cirrhosis, and primary hepatocellular carcinoma. More than 686,000 people die every year because of the consequences of CHB.
      • Chisari F.V.
      • Isogawa M.
      • Wieland S.F.
      Pathogenesis of hepatitis B virus infection.

      WHO 2016;Fact sheet no. 204.

      The goal of CHB therapy is to improve quality of life and survival by preventing the progression of the disease to cirrhosis, hepatocellular carcinoma and liver cancer-related death. HBsAg loss, also defined as “functional cure”, and subsequently, seroconversion with detectable anti-HBs antibody levels are considered the goal for new therapies.
      • Zoulim F.
      • Durantel D.
      Antiviral therapies and prospects for a cure of chronic hepatitis B.
      • Ferrari C.
      HBV and the immune response.
      Currently, available treatments for CHB include interferon (i.e. pegylated interferon [PegIFN]) and nucleos(t)ide analogues (i.e. lamivudine, adefovir, entecavir, tenofovir and telbivudine), which are associated with low rates of functional cure.
      • Kwon H.
      • Lok A.S.
      Hepatitis B therapy.
      The hallmarks of chronic HBV infection are high levels of viral load (HBV DNA) and even higher levels of non-infectious HBsAg-containing subviral particles (>100-fold in excess of virions) in the circulation. Evidence suggests that in chronic HBV infection, high levels of dominant viral antigens such as HBsAg in the liver and periphery may contribute to exhaustion of antiviral CD8+ T-cells.
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      • Richter K.
      • Oxenius A.
      How chronic viral infections impact on antigen-specific T-cell responses.
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      Clearing persistent extracellular antigen of hepatitis b virus: an immunomodulatory strategy to reverse tolerance for an effective therapeutic vaccination.
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      CD40 activation rescues antiviral CD8(+) T cells from PD-1-mediated exhaustion.
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      • et al.
      Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.
      Furthermore, several reports describe HBsAg negatively regulating HBV-specific immune responses by direct modulation of dendritic cell, monocyte and natural killer cell functions.
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      Similarly, the most recent studies imply that HBV may interfere with antiviral innate immune responses in patients with CHB.
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      • et al.
      Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
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      • et al.
      The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection.
      Taken together, these studies suggest a potential therapeutic role for antiviral agents that reduce HBsAg levels and restore virus-specific immune responsiveness in patients.
      HBV contains a 3.2 kb circular, partially double-stranded DNA genome that establishes persistent infection as a covalently closed circular DNA (cccDNA) in the nucleus of the infected hepatocytes.
      • Quasdorff M.
      • Protzer U.
      Control of hepatitis B virus at the level of transcription.
      Five major 3′ overlapping transcripts are produced from HBV cccDNA, which in turn result in the expression of seven HBV proteins. These transcripts include the pregenomic RNA (pgRNA), the template for reverse transcription and the mRNA coding for core and polymerase proteins; the precore (pre-C) mRNA which is initiated upstream of the pregenomic RNA and serves as mRNA for the production of hepatitis B e Ag (HBeAg); the 2.4/2.1 kb subgenomic mRNAs that code for HBV large, medium and small envelope (HBsAg) proteins, the most abundant proteins of subviral particles; and the 0.75 kb subgenomic mRNA that encodes the regulatory protein X (HBx).
      Here we describe the discovery of a first-in-class, orally bioavailable small molecule inhibitor of HBV gene expression that selectively reduces viral antigen expression in infected hepatocytes and in the human liver chimeric urokinase-type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mouse model. Unlike the current standard-of-care, nucleos(t)ide therapies which reduce viremia but do not lead to effective reduction in HBV antigen expression, RG7834 significantly reduces the levels of HBsAg, as well as lowering viremia.

      Materials and methods

       Natural infection assays

      Please refer to supplementary materials and methods for details of primary human hepatocytes (PHH) and dHepaRG natural infection assays.

       RNA-seq experiments

      For details, please refer to supplementary materials and methods.

       Humanized mouse studies

      Two separate studies were performed by PhoenixBio Co., Ltd. The use of the animals for these studies had been approved by the Animal Ethics Committee of PhoenixBio (Resolution No.: 1229 + 1426) and adhered to the Guide for the Care and Use of Laboratory Animals. All surgeries were performed under isoflurane anesthesia, and all efforts were made to minimize suffering. These studies were approved and funded by Immunology, Inflammation and Infectious Diseases (I3) department of F. Hoffmann-La Roche Ltd. The ARRIVE (Animal Research: Reporting of In Vivo Experiments) guideline was used to prepare the report for the in vivo studies in this manuscript.
      For further details regarding the materials used, please refer to the CTAT table and supplementary information.

      Results

       RG7834 reduced viral markers in HBV-infected cells

      RG7834 is a small molecule compound belonging to the chemical class of the dihydroquinolizinones (Fig. 1) that was discovered by phenotypic screening for inhibitors of HBsAg secretion from a library containing approximately one million small molecule compounds. We have evaluated the antiviral activity of RG7834 in differentiated HepaRG cells (dHepaRG), a human hepatoma cell line at four days post HBV infection.
      • Gripon P.
      • Rumin S.
      • Urban S.
      • Le Seyec J.
      • Glaise D.
      • Cannie I.
      • et al.
      Infection of a human hepatoma cell line by hepatitis B virus.
      HBV markers were assessed after seven days of drug treatment as explained in materials and methods. RG7834 potently reduced the levels of secreted HBsAg, HBeAg and HBV DNA in a dose-dependent manner with EC50 values ranging from 2.57 to 3.16 nM (Fig. 1). By contrast, the nucleoside analogue entecavir (ETV) only reduced the level of HBV DNA detected in the extracellular supernatant of these cells (EC50 0.06 nM), without affecting the levels of intracellular HBV mRNA levels or secreted viral antigens (Fig. 1). Similar data were obtained when infected dHepaRG cells were treated at day 11 post infection, suggesting RG7834 antiviral activity is independent of early events of HBV infection (Table S1).
      Figure thumbnail gr1
      Fig. 1Antiviral activities of RG7834, RO0321 and ETV in HBV-infected dHepaRG cells. Shown are the chemical structures of the test compounds, and mean dose-response curves of HBV DNA (blue line and squares), HBsAg (red line and circles) and HBeAg (black line and diamonds) inhibition (error bars represent standard deviations from at least four independent experiments). Mean EC50 and CC50 values ± standard deviation from at least four independent experiments are summarized in the table. ETV, entecavir; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; n.d., not determined; SD, standard deviation.
      To explore the mechanism underlying the antigen reduction seen with RG7834 treatment, the total level of intracellular HBV mRNAs was measured by qPCR. RG7834 reduced HBV total mRNA levels in a dose-dependent manner with an EC50 of 5.87 nM, i.e. a value that closely matched that seen with secreted viral parameters. This data suggests that RG7834 may be a potent HBV gene expression inhibitor. As a negative control, RO0321, an enantiomer (“R” configuration) of RG7834, did not show an inhibitory effect on any of the HBV viral parameters analyzed (Fig. 1 and Table S1).
      To test if the antiviral potency of RG7834 is specific to a particular cell line or HBV genotype, we evaluated the compound potency in primary human hepatocytes (PHH) infected with clinical isolates representing the most prevalent HBV genotypes A, B, C, and D. Like entecavir, RG7834 inhibited all four HBV genotypes with similar activity, reducing HBV DNA levels with EC50 values in the range of 2.2 to 6.0 nM (Table 1).
      Table 1Inhibition of extracellular HBV DNA by RG7834 in PHHs infected with different HBV genotypes.
      HBV genotypeEC50 ± SD (nM)
      Mean EC50 values ± standard deviation for the inhibition of HBV DNA by RG7834 and entecavir were determined in PHH in three independent experiments.
      RG7834Entecavir
      A6.00 ± 5.420.005 ± 0.0001
      B2.37 ± 0.290.003 ± 0.0006
      C2.21 ± 0.760.002 ± 0.001
      D5.25 ± 3.990.005 ± 0.002
      HBV, hepatitis B virus; PHH, primary human hepatocytes.
      a Mean EC50 values ± standard deviation for the inhibition of HBV DNA by RG7834 and entecavir were determined in PHH in three independent experiments.
      The selectivity of RG7834 was further investigated by evaluating its activity in in vitro antiviral assays against a panel of 15 DNA and RNA viruses (Table S2). RG7834 was highly selective for HBV since it did not impact infection by any of these other viruses. Furthermore, RG7834 did not influence cell viability up to the highest tested concentration of 25 µM in a broad panel of different human and rodent primary cells and cell lines representing various organs and tissues (Table S3). Taken together, these studies demonstrate that RG7834 is a novel HBV-specific antiviral molecule that blocks viral antigen and virion production.

       Gene expression analysis with RNA-seq revealed fast and selective reduction of HBV RNA in response to RG7834 treatment

      To determine the effect of RG7834 on HBV vs. cellular mRNAs a time course gene expression study was performed using next generation RNA sequencing. HBV-infected dHepaRG cells were treated either with RG7834 or DMSO as a negative control, and RNA samples were collected at 6, 9, 12 and 24 h post-treatment. HBV gene expression was quantified by counting sequencing reads mapped to the HBV genome normalized by the reads mapped to the human genome (Fig. 2A). RG7834 induced a specific reduction of HBV gene expression as early as six hours post-treatment (multiple-testing adjusted p = 0.0024), an effect, that persisted until the end of the experiment at 24 h post-treatment (adjusted p <0.01) (Fig. 2A). By mapping sequencing reads to individual nucleotides of the viral genome, it was revealed that RG7834 lowered the expression of both pgRNA and subgenomic RNAs (Fig. 2B). By contrast, expression of host genes was only moderately affected by RG7834 treatment (Fig. 2C). The log2FC distribution of most genes was restricted between -1 and 1. On average less than 0.1% of human genes were modulated more than twofold, with a false discovery rate smaller than 0.05. Most notably, RG7834 had no significant impact on the gene expression levels of ubiquitous or liver-enriched transcription factors that bind to HBV promoter/enhancer elements and are critical for regulation of both HBV and hepatocytes-specific genes expression (Fig. S1).
      • Quasdorff M.
      • Protzer U.
      Control of hepatitis B virus at the level of transcription.
      Importantly, the expression levels of hepatocyte-specific genes were unchanged confirming RG7834 does not interfere with the functions or expression levels of major transcription factors in hepatocytes (Fig. 2D). To further rule out the possibility of a role for transcription factors in RG7834-mediated inhibition of HBV expression, we showed that RG7834 did not alter the expression levels of a reporter gene driven by either pgRNA/core or HBsAg (PreS2) promoters (Fig. S2). Thus, the effect of RG7834 on HBV RNA levels was highly virus-specific.
      Figure thumbnail gr2
      Fig. 2RNA deep sequencing analysis of the effect of RG7834 on HBV RNA transcripts and on host gene expression in HBV-infected dHepaRG cells. (A) Viral RNA transcript levels in the presence and absence of RG7834 (100 nM) at different time points of post-treatment initiation (error bars represent standard deviations). (B) HBV transcript-level quantification by nucleotide mapping in the presence and absence of RG7834 (100 nM) after 6 h (upper panel) and 24 h (lower panel) of treatment. Localization of HBV RNA transcripts mapped to the genome is shown above. (C) Host gene expression quantification in the presence of RG7834 after 6 h, 9 h, 12 h and 24 h compared to non-treated samples. Fold changes are shown as log2 scale. (D) Heatmap of expression of genes that are preferentially expressed in liver compared with other human organs and tissues. These ‘liver-enriched genes’ were identified from an integrative analysis of multiple large-scale transcriptomic datasets in our previous study.
      • Zhang J.D.
      • Hatje K.
      • Sturm G.
      • Broger C.
      • Ebeling M.
      • Burtin M.
      • et al.
      Detect tissue heterogeneity in gene expression data with BioQC.
      Mean expression of liver-enriched genes in three biological replicates are visualized with heatmap using units of log2-transformed Fragments Per Kilobase Million (FPKM). Each row represents one liver-enriched gene and each column one condition, i.e. 6 h, 9 h, 12 h, or 24 h after vehicle or RG7834 treatment, respectively. HBV, hepatitis B virus.
      RG7834 demonstrated a higher degree of selectivity for subgenomic RNAs than pgRNA, as those reads that uniquely mapped to pgRNA were less reduced than those of other regions in the HBV genome (Fig. 2B). To further explore the selective reduction of subgenomic RNAs by RG7834, the differential gene expression score (DGE scores), defined as log10-transformed p value was calculated, using the negative-binomial model multiplied by the sign of differential expression. RG7834-treated samples were compared with DMSO treated samples for each nucleotide of the HBV genome at each time point (Fig. 3A). DGE scores further revealed a less pronounced reduction of viral pgRNA when compared to other regions of the HBV genome at 24 h post-treatment. To further validate our finding, we treated HBV-infected PHH with RG7834 or transfected with a siRNA that targets a region of HBV mRNAs common to all viral transcripts (siHBx) or an unspecific scrambled siRNA as a negative control. RNA samples were collected at 6, 9, 12 and 24 h post-treatment and were subjected to northern blot analysis of HBV RNA (Fig. 3B).
      Figure thumbnail gr3
      Fig. 3RG7834 preferentially degrades subgenomic HBV RNA transcripts. (A) Differential expression scores between RG7834 (100 nM) and non-treated, after 6 h (upper panel) and 24 h (lower panel) of treatment, are visualized in grey. A cubic smoothing line with 10 degrees of freedom is fitted for each time point and is shown in red. P, HBV polymerase; S, HBsAg; X, HBx; C, HBV core. (B) Northern blot analysis of 2 μg of RNA extracted from PHH treated five days after HBV infection with RG7834, HBx siRNA (siHBx) or control siRNA (siControl) for the number of hours indicated above the blot. HBV RNA was specifically detected with a genome length HBV (-) strand RNA probe. The migrations of the pgRNA, preS1 and preS2 RNA species are indicated on the right. rRNA was stained on the blotting membrane with methylene blue, the 18S rRNA band served as a loading control. HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; pgRNA, pregenomic RNA; PHH, primary human hepatocytes; rRNA, ribosomal RNA.
      Interestingly, RG7834 treatment altered the gel electrophoretic mobility of HBV RNA as early as 6 h post-treatment and reduced viral RNAs levels. Similar to our RNA-seq analysis, the pattern of HBV RNA reduction was disproportional and preferential for subgenomic RNAs at 24 h post-treatment. As expected, siHBx potently reduced all HBV mRNAs compared to the negative control. However, in contrast to RG7834, siHBx reduced both pgRNA and subgenomic RNAs proportionally. Furthermore, neither siHBx nor the negative control induced any changes to electrophoretic mobility of HBV RNAs in our assays. These data, in combination with above RNA-seq analysis suggest RG7834 preferentially reduces subgenomic RNAs and its mechanism of action is distinct from that of siRNA.

       RG7834 reduced HBsAg levels and other viral markers in HBV-infected human liver chimeric uPA-SCID mice

      Human liver chimeric uPA/SCID mice have previously been used to evaluate the potency of antiviral compounds interfering with various stages of the HBV life cycle.
      • Belloni L.
      • Allweiss L.
      • Guerrieri F.
      • Pediconi N.
      • Volz T.
      • Pollicino T.
      • et al.
      IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome.
      • Volz T.
      • Allweiss L.
      • Ben M.M.
      • Warlich M.
      • Lohse A.W.
      • Pollok J.M.
      • et al.
      The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus spreading in humanized mice previously infected with hepatitis B virus.
      A pharmacokinetic/pharmacodynamic study in HBV-infected human liver chimeric mice was designed with the aim of evaluating RG7834 efficacy in a chronic HBV replication model. RG7834 doses were chosen based on a compartmental model fitted to the plasma concentrations measured after administering oral doses at 4 mg/kg/day and 40 mg/kg/day to uninfected mice (Tables S4 and S5). Model simulations were then used to select dose regimens necessary to achieve free plasma concentrations continuously above the upper range measured in vitro HBsAg EC50 of 5 nM (2.8 ng/ml). The chosen doses, which were both expected to be efficacious, were 4 mg/kg and 10 mg/kg given twice daily. During the efficacy study, blood samples that were taken for plasma concentration measurements confirmed that both regimens had reached exposure levels above the EC50 (Table S6). The duration of dosing was 21 days and ETV was used as a control at 0.03 mg/kg once daily. At day 21, two animals from each group were sacrificed and the livers were used for hepatic HBV DNA and cccDNA determination. The remaining animals were monitored for an additional two-week period. Serum viral markers and human albumin were measured as controls for human hepatocyte function throughout the in-life phase, up to day 35. Treatment with 4 mg/kg RG7834 resulted in a 0.91 log10 (p <0.001) reduction of HBsAg at the end of treatment (day 21). A similar result was obtained with the 10 mg/kg dose (1.09 log10 reduction; p <0.001), suggesting that the maximal reduction of HBsAg compared to baseline in this model is near 1.0 log10 (Fig. 4). HBsAg levels restored to baseline levels after cessation of treatment. HBeAg was reduced by 0.43 log10 and 0.53 log10 with 4 mg/kg and 10 mg/kg of RG7834, respectively. RG7834 also reduced serum HBV DNA by a mean value of 0.60 log10 and 0.76 log10 in the 4 mg/kg and 10 mg/kg treated animals, respectively. By contrast, ETV only reduced HBV DNA levels (1.97 log10 reduction), as expected (Fig. 4). Neither intrahepatic HBV DNA nor cccDNA was modulated significantly by RG7834 which is consistent with the rapid viral antigen rebound that has been seen during the off-treatment follow-up period. Blood human albumin levels were only weakly reduced, which was similarly in all treated groups, including the ETV group. We did not observe any significant changes to individual body weights of animals during and after treatment with RG7834 or ETV (Fig. 4 and Table S7).
      Figure thumbnail gr4
      Fig. 4Effect of RG7834 and ETV on HBV viral markers in serum and liver from HBV genotype C infected uPA/SCID mice. Kinetics of HBV DNA, HBsAg, HBeAg and blood human albumin were determined in serum from vehicle or drug treated HBV-infected uPA/SCID mice during the treatment and follow-up period up to 35 days. Shown are mean values ± standard errors from four to six mice. At day 21, two animals from each group were sacrificed and the livers were harvested for hepatic HBV DNA and cccDNA determination. The remaining animals were monitored for the follow-up period. Log10 reduction p values were calculated using a linear mixed effect model using R software and p values were globally adjusted for all performed comparisons (Holm’s method). cccDNA, covalently closed circular DNA; ETV, entecavir; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; uPA/SCID, urokinase-type plasminogen activator/severe combined immunodeficiency; n.a., not applicable; QD, once daily; BID, twice daily. aMean log10 reduction ± standard error from baseline; ***p <0.001; *p <0.05; n.s., not significant. bMean log10 reduction ± standard error from vehicle; **p <0.01; n.s., not significant.

       Combination treatment with RG7834 and ETV or interferon induced a balanced and profound reduction of both viremia and HBsAg levels

      To test the effect of combination treatments, a series of in vitro and in vivo studies were conducted. Initially, pairwise drug combinations of RG7834 and ETV, or RG7834 and interferon (IFN), were tested for anti-HBV activity in HBV-infected dHepaRG cells (Fig. S2). The analysis was performed using two independent drug-drug combination analysis models, namely the Loewe additivity (CalcuSyn and isobolograms) and the Bliss independence (MacSynergy II). The combinations of RG7834 with ETV or IFNα-2a were found to be at least additive in the analysis models for inhibition of both HBV DNA and HBsAg. Importantly, antagonism was not observed in any of the in vitro studies.
      Next, the antiviral efficacy of RG7834 in combination with entecavir and/or PegIFNα-2a (PegIFNα) was evaluated in a repeat-dose study in the human liver chimeric mouse model. The infected mice were treated with vehicle, with RG7834 at 10 mg/kg once daily, with ETV at 0.03 mg/kg once daily, and with PegIFNα at 0.025 mg/kg injected subcutaneously once weekly, as previously described.
      • Allweiss L.
      • Volz T.
      • Lutgehetmann M.
      • Giersch K.
      • Bornscheuer T.
      • Lohse A.W.
      • et al.
      Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration.
      The duration of treatment was five weeks, followed by a two week off-treatment follow-up period and HBV DNA, HBsAg, HBeAg and human albumin levels in mouse serum were measured throughout the study (Fig. 5). At day 35, two animals from each group were sacrificed and the livers were harvested for intrahepatic HBV DNA and cccDNA determination and immunohistochemistry studies for the qualitative detection of intrahepatic HBsAg and HBV core protein levels (Fig. 6 and Table 2). When given once daily, RG7834 alone significantly reduced both circulating HBsAg (0.76 log10; p <0.001) and HBV DNA levels (0.68 log10; p <0.001). As before, ETV alone reduced HBV DNA (2.25 log10; p <0.001) with no significant effect on HBsAg levels compared to baseline. Similar to a previous report, PegIFNα alone reduced HBV DNA (1.79 log10; p <0.001) and circulating HBsAg (0.41 log10; p <0.01) and the combination of ETV and PegIFNα resulted in even more pronounced reduction of both HBV DNA and HBsAg levels (3.30 log10; p <0.001, 0.41 log10; p <0.01, respectively) compared to baseline.
      • Allweiss L.
      • Volz T.
      • Lutgehetmann M.
      • Giersch K.
      • Bornscheuer T.
      • Lohse A.W.
      • et al.
      Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration.
      The combinations of both RG7834 and ETV, and RG7834 and PegIFNα led to profound and significant reductions in both HBV DNA (2.72 log10, and 2.23 log10, respectively; p <0.001) and HBsAg levels (1.12 log10, and 1.09 log10, respectively; p <0.001) compared to baseline. Most notably, combination of RG7834, ETV and PegIFNα resulted in the most pronounced reduction of HBV DNA and HBsAg levels (3.38 log10 and 1.18 log10, respectively; p <0.001) compared to any other group in this study (Fig. 5 and Table 2). A similar effect was observed for reduction of intracellular total HBV DNA, while in two animals sacrificed at the end of treatment, on day 35, cccDNA loads did not differ significantly in comparison to the vehicle treated controls (Table 2).
      Figure thumbnail gr5
      Fig. 5Effect of RG7834, ETV and PegIFNα alone and in combination on HBV viral markers in serum and liver from HBV-infected uPA/SCID mice. Kinetics of HBV DNA, HBsAg, HBeAg and blood human albumin were determined in serum from vehicle or drug treated HBV-infected uPA/SCID mice during the treatment (five mice per group) and follow-up period (three mice per group) up to 49 days. Mean values ± standard errors are shown. ETV, entecavir; HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; PegIFNα, pegylated interferon α-2a; uPA/SCID, urokinase-type plasminogen activator/severe combined immunodeficiency.
      Figure thumbnail gr6
      Fig. 6Effect of RG7834, ETV and PegIFNα alone or in combination on HBV viral markers in livers from HBV-infected uPA/SCID mice. HBsAg (red) and cell nuclei (blue) (upper panel), and HBcAg (green), cell nuclei (blue) and human hepatocytes (red) (lower panel) were detected by immunohistochemistry in the mouse liver samples at the end of treatment (day 35). ETV, entecavir; HBV, hepatitis B virus; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; PegIFNα, pegylated interferon α-2a; uPA/SCID, urokinase-type plasminogen activator/severe combined immunodeficiency.
      Table 2Effect of RG7834, entecavir and pegylated interferon α-2 a alone and in combination on HBV viral markers in serum and liver from HBV genotype C infected uPA/SCID mice.
      DrugSerum day 35
      Mean log10 reduction ± standard error from baseline (five animals per group); ***p <0.001; **p <0.01; *p <0.05.
      Liver day 35
      Mean log10 reduction ± standard error from vehicle (two animals per group); **p <0.01.
      HBV DNAHBsAgHBeAgAlbuminHBV DNAcccDNA
      Vehicle0.04 ± 0.13n.s.−0.001 ± 0.11n.s.−0.09 ± 0.07n.s.0.05 ± 0.03n.s.n.an.a
      RG78340.68 ± 0.12***0.76 ± 0.10***0.47 ± 0.07***0.11 ± 0.02***0.49 ± 0.36n.s.0.11 ± 0.29n.s.
      ETV2.25 ± 0.13***0.20 ± 0.11n.s.0.25 ± 0.07*0.05 ± 0.03n.s.1.66 ± 0.36**0.50 ± 0.29n.s.
      PegIFNα1.79 ± 0.12***0.41 ± 0.10**0.75 ± 0.07***0.19 ± 0.02***1.75 ± 0.36**0.85 ± 0.29n.s.
      RG7834 + ETV2.72 ± 0.12***1.12 ± 0.10***0.75 ± 0.07***0.14 ± 0.02***1.83 ± 0.36**0.34 ± 0.29n.s.
      RG7834 + PegIFNα2.23 ± 0.12***1.09 ± 0.10***1.04 ± 0.07***0.20 ± 0.02***1.48 ± 0.36**0.49 ± 0.29n.s.
      ETV + PegIFNα3.30 ± 0.12***0.41 ± 0.10**0.72 ± 0.07***0.12 ± 0.02***2.20 ± 0.36**0.62 ± 0.29n.s.
      RG7834 + ETV + PegIFNα3.38 ± 0.12***1.18 ± 0.10***1.04 ± 0.07***0.20 ± 0.02***2.16 ± 0.36**0.54 ± 0.29n.s.
      Vehicle, RG7834 (10 mg/kg), and ETV (0.03 mg/kg) were dosed once daily, and PegIFN (0.025 mg/kg) was dosed twice weekly.
      ETV, entecavir; HBV, hepatitis B virus; PegIFNα, pegylated interferon α-2a; n.a., not applicable; n.s., not significant; uPA/SCID, urokinase-type plasminogen activator/severe combined immunodeficiency.
      a Mean log10 reduction ± standard error from baseline (five animals per group); ***p <0.001; **p <0.01; *p <0.05.
      b Mean log10 reduction ± standard error from vehicle (two animals per group); **p <0.01.
      Furthermore, intracellular HBsAg and HBV core levels of individual mouse livers from different groups with similar viral and antigen load at the baseline were evaluated (Tables S8 and S9). The immunofluorescence analysis confirmed a remarkable reduction of intrahepatic HBsAg and HBV core protein levels for the RG7834-treated animals compared to the vehicle, ETV or PegIFNα treated animals (Fig. 6). However, HBsAg levels were reduced more significantly when compared to core protein levels. This observation is in line with our in vitro RNA analysis that suggests RG7834 preferentially reduce subgenomic RNAs. Furthermore, the combination of RG7834 and ETV resulted in a similar reduction of intracellular antigens compared to RG7834 alone. As expected, PegIFNα reduced intracellular antigen levels, with bigger reductions for HBV core protein than HBsAg. This observation agrees with previous reports suggesting IFN accelerates pgRNA and core particle degradation in transgenic mice.
      • Lutgehetmann M.
      • Volzt T.
      • Quaas A.
      • Zankel M.
      • Fischer C.
      • Dandri M.
      • et al.
      Sequential combination therapy leads to biochemical and histological improvement despite low ongoing intrahepatic hepatitis B virus replication.
      • Wieland S.F.
      • Eustaquio A.
      • Whitten-Bauer C.
      • Boyd B.
      • Chisari F.V.
      Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids.
      More importantly, combination of RG7834 and PegIFNα or triple therapy complemented the RG7834 antiviral profile and induced a near-complete depletion of intracellular antigens pools.
      Taken together, our results confirm that in the infected mice, RG7834 and PegIFNα are the key drivers for reducing antigen levels. Moreover, the combination therapy resulted in a robust and balanced reduction of all viral markers and intracellular antigen levels tested in our study.

      Discussion

      Immune control of chronic HBV requires profound HBV-specific immune responses involving both the innate and adaptive arms of the immune system, similar to that observed in acute HBV infection.
      • Bauer T.
      • Sprinzl M.
      • Protzer U.
      Immune control of hepatitis B virus.
      • Tan A.
      • Koh S.
      • Bertoletti A.
      Immune response in hepatitis B virus infection.
      • Shin E.C.
      • Sung P.S.
      • Park S.H.
      Immune responses and immunopathology in acute and chronic viral hepatitis.
      Given the well-described contribution of high HBsAg levels to HBV-specific T-cell exhaustion, the direct negative regulation of immune cells and the potential role in regulating antiviral innate immune response in CHB, it is highly plausible that the initial goal of any future therapy should include reduction of the intracellular and circulating HBsAg for reversal of antiviral immunity.
      • Ferrari C.
      HBV and the immune response.
      • Ochel A.
      • Cebula M.
      • Riehn M.
      • Hillebrand U.
      • Lipps C.
      • Schirmbeck R.
      • et al.
      Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.
      • Lebosse F.
      • Testoni B.
      • Fresquet J.
      • Facchetti F.
      • Galmozzi E.
      • Fournier M.
      • et al.
      Intrahepatic innate immune response pathways are downregulated in untreated chronic hepatitis B.
      • Gane E.J.
      • Lim Y.S.
      • Gordon S.C.
      • Visvanathan K.
      • Sicard E.
      • Fedorak R.N.
      • et al.
      The oral toll-like receptor-7 agonist GS-9620 in patients with chronic hepatitis B virus infection.
      Nucleos(t)ide therapy effectively reduces viral load but does not affect HBV gene expression and viral antigen production. On the other hand, IFNα is unique in a manner that it possesses both direct antiviral and immunomodulatory properties.
      • Schoggins J.W.
      • Rice C.M.
      Interferon-stimulated genes and their antiviral effector functions.
      • Swiecki M.
      • Colonna M.
      Type I interferons: diversity of sources, production pathways and effects on immune responses.
      Although the direct antiviral activity of IFNα is inefficient, as only a modest reduction in viral antigen levels by PegIFNα were observed in HBV-infected humanized mice that lack immune cells, both in our study and in an earlier report.
      • Allweiss L.
      • Volz T.
      • Lutgehetmann M.
      • Giersch K.
      • Bornscheuer T.
      • Lohse A.W.
      • et al.
      Immune cell responses are not required to induce substantial hepatitis B virus antigen decline during pegylated interferon-alpha administration.
      Previous attempts to reduce both intracellular and circulating HBsAg have been limited to injectable RNA therapeutics targeting various regions of the intracellular HBV transcripts that in turn leads to the inhibition of viral replication and production of HBsAg.
      • Billioud G.
      • Kruse R.L.
      • Carrillo M.
      • Whitten-Bauer C.
      • Gao D.
      • Kim A.
      • et al.
      In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.
      • Sebestyen M.G.
      • Wong S.C.
      • Trubetskoy V.
      • Lewis D.L.
      • Wooddell C.I.
      Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.
      In this communication, we report the discovery and characterization of a novel small molecule viral gene expression inhibitor. To the best of our knowledge, RG7834 is the first oral viral expression inhibitor with a high degree of selectivity towards HBV. The anti-HBV potency of RG7834 is well-demonstrated in both in vitro dHepaRG and PHH infection assays. Several lines of evidence suggest RG7834 is a highly selective inhibitor of HBV gene expression. Firstly, RG7834 is more than 100-fold more selective for HBV than its R configuration enantiomer, RO0321. Secondly, RG7834 had no impact on infection by several other DNA and RNA viruses, and did not exhibit any cytotoxicity in a large panel of primary cells and cell lines, demonstrating a therapeutic index of more than 2,000-fold. Thirdly, transcriptome-wide RNA-seq analysis confirmed no major changes to global gene expression associated with RG7834 treatment. Lastly, RG7834 reduced viral subgenomic RNA levels more profoundly than pregenomic RNA, demonstrating another layer of selectivity among the different viral mRNA species. Despite the observed preferential reduction of subgenomic RNAs, our methods did not differentiate between the 2.4/2.1 kb and 0.7 kb subgenomic mRNA species because of the overlapping nature of HBV mRNAs (Fig. 2A). Similarly, we were unable to detect intracellular HBx protein as the limit of detection for commercially available anti-HBx antibodies is restricted to HBx over-expression by transfection or transduction methods in cell culture.
      The selectivity of RG7834 for HBV was further confirmed in vivo using the human chimeric mouse model of HBV infection. Efficacy of RG7834 against HBV was shown in this model by demonstrating a significant reduction of the viral markers with no significant dose-dependent effect on human albumin, which is continuously secreted from the transplanted human hepatocytes. Interestingly, both extracellular and intracellular HBsAg levels were reduced more profoundly than HBV DNA, HBeAg and intracellular HBV core levels, which is highly consistent with the early finding that subgenomic RNAs are reduced more significantly than precore/pregenomic RNAs (template for DNA synthesis and mRNAs for HBeAg and HBV core protein). Furthermore, we observed a rebound of viral loads and HBsAg levels to baseline after cessation of therapy, indicating that reduction of viral expression is dependent on the continued presence of RG7834 and is rapidly reversible. Consistent with this observation and the RG7834 mechanism of action, no significant changes were observed in cccDNA pools in RG7834-treated infected livers of human chimeric mice.
      Combination therapy has been a successful strategy for treatment of viral diseases including HCV and HIV.
      • Chahine E.B.
      • Sucher A.J.
      • Hemstreet B.A.
      Sofosbuvir/velpatasvir: the first pangenotypic direct-acting antiviral combination for hepatitis C.
      • Greig S.L.
      • Deeks E.D.
      Abacavir/dolutegravir/lamivudine single-tablet regimen: a review of its use in HIV-1 infection.
      Combination of interventions targeting various key steps in the virus replication cycle is potentially more efficient and effective than monotherapy. Thus, combination therapy may be able to address the deficiencies of current chronic HBV therapy. Clearly the antiviral properties and the mechanism of action of RG7834 is differentiated from that of nucleos(t)ide analogues. In our studies, RG7834 and PegIFNα are the main drivers of HBsAg reduction, while both ETV and PegIFNα were superior in reducing HBV DNA levels. Combination of these agents with their complementary mechanism of action resulted in a balanced antiviral profile that effectively reduced both HBV DNA and levels of intracellular and circulating antigens (HBsAg and HBeAg) in both in vitro and in vivo models of chronic HBV infection.
      HBV infection and replication does not induce significant changes to global host transcriptional response in PHH.
      • Niu C.
      • Livingston C.M.
      • Li L.
      • Beran R.K.
      • Daffis S.
      • Ramakrishnan D.
      • et al.
      The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.
      Therefore, we do not expect considerable modulations in host global gene expression levels in response to RG7834 and/or entecavir treatment. Consistently in our study, the reduction of HBV RNA by RG7834 treatment did not induce prominent changes to host global gene expression levels. However, it is unclear if inhibition of viral polymerase by treatment with entecavir or any other nucleos(t)ide analogue polymerase inhibitors directly or indirectly modulates host global gene expression levels. Therefore, future studies will be directed at studying changes to host global gene expression levels that are associated with treatment with nucleoside or nucleotide analogues polymerase inhibitors alone and in combination with RG7834 in HBV-infected PHH.
      Previous studies suggest that high levels of antigen expression in liver impairs antiviral innate and adaptive immune responses.
      • Isogawa M.
      • Chung J.
      • Murata Y.
      • Kakimi K.
      • Chisari F.V.
      CD40 activation rescues antiviral CD8(+) T cells from PD-1-mediated exhaustion.
      • Ochel A.
      • Cebula M.
      • Riehn M.
      • Hillebrand U.
      • Lipps C.
      • Schirmbeck R.
      • et al.
      Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes.
      Therefore, we expect that combining novel HBV expression inhibitors such as RG7834 with immunomodulators may enhance the host immune responses against HBV and that such combinations are more likely to achieve functional cure. Surprisingly, the combination of RG7834 and PegIFNα resulted in an additive anti-HBV response and did not potentiate PegIFNα antiviral response significantly in our infection assay, or in the human chimeric mouse model. Nonetheless, our data are consistent with recent reports indicating that HBV is neither sensed nor interferes with intrinsic innate immunity of the infected hepatocytes.
      • Niu C.
      • Livingston C.M.
      • Li L.
      • Beran R.K.
      • Daffis S.
      • Ramakrishnan D.
      • et al.
      The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.
      • Cheng X.
      • Xia Y.
      • Serti E.
      • Block P.D.
      • Chung M.
      • Chayama K.
      • et al.
      Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages.
      Notably, the human chimeric mouse model used in our study lacks immune cells and a functional adaptive immune system. Thus, it was not possible to assess the ability of RG7834 to restore HBV-specific immunity in this model or to fully evaluate the effect of a combination of RG7834 with immunomodulators such as PegIFNα. Future preclinical studies will be directed at exploring RG7834 in other combination regimens with existing and novel direct acting antivirals and immunomodulators
      • Durantel D.
      • Zoulim F.
      New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus.
      • Lempp F.A.
      • Urban S.
      Inhibitors of hepatitis B virus attachment and entry.
      in immune competent animal models of HBV infection.
      • Allweiss L.
      • Dandri M.
      Experimental in vitro and in vivo models for the study of human hepatitis B virus infection.
      Our data suggest that RG7834 does not interfere with the gene expression levels and functions of transcription factors in infected hepatocytes. Based on our RNA-seq and northern blot analysis studies, RG7834 may directly or indirectly modify viral RNAs and promote their degradation. While the target and the exact mechanism of action of RG7834 remains to be elucidated, we are conducting a number of experiments involving proteomics and genomics to identify pharmacological and physical targets of RG7834. The observed selectivity, the availability of the inactive enantiomer and the potential role of putative targets that play a role in viral RNA fate may further help us to accelerate and focus our target identification and characterization efforts.
      In summary, we have identified RG7834 as a novel, orally available inhibitor of HBV viral gene expression. Further characterization of this molecule, combined with a better understanding of its mechanism of action may lead to new therapeutic approaches for chronic HBV infection.

      Conflict of interest

      HM, SW, SL, SY, JW, AL, PT, GO, WL, NJP, JDZ, RS, TR, JCH, EK, FP, XZ, GS, JY and HJ are employees of F. Hoffmann-La Roche Ltd. MD received research support from Hoffmann-La Roche, Basel (Grant N. 14-490) and from the German Research Foundation (DFG) by a Heisenberg Professorship D (DA1063/3-2).
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      HJ, JY and SY initiated and supervised the studies. HJ, HM, SW, SL JW, AL, PT, GO, WL, NJP, JDZ, RS, TR, EK, JCH, FP, XZ, GS, GR, TV, MD designed and performed experiments or contributed to data analysis. HJ, SW, HM, JDZ and JY wrote the manuscript.

      Acknowledgements

      We are grateful to Miriam Triyatni and Brian Leonard for providing HBV clinical isolates genotype A to C.

      Supplementary data

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