Elsevier

Journal of Hepatology

Volume 64, Issue 4, April 2016, Pages 781-789
Journal of Hepatology

Research Article
In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides

https://doi.org/10.1016/j.jhep.2015.11.032Get rights and content

Background & Aims

Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting.

Methods

A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively.

Results

ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro.

Conclusion

Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Introduction

Hepatitis B virus (HBV) represents an important human pathogen with about 350 million chronic carriers worldwide [1], [2] in which many cases will develop liver cirrhosis and hepatocellular carcinoma (HCC), accounting for more than 50% of all HCC cases worldwide [3]. Despite the availability of an effective vaccine, between 0.5 and 1 million die every year as a result of HCC or liver failure caused by chronic HBV (CHB).

HBV, a member of the Hepadnaviridae, is a hepatotropic virus with a partially double stranded DNA genome. The viral genome is converted to a covalently closed circular DNA molecule (cccDNA) that serves for the production of several viral transcripts used for viral gene expression and reverse transcription for progeny virus production [4]. Subviral particles composed of viral envelope proteins (hepatitis B virus surface antigens [HBsAg] particles) or hepatitis B virus e antigens (HBeAg) are secreted from infected cells and far outnumber infectious virus in the blood of infected patients and are associated with poor prognosis of chronic HBV infection [5]. The large amount of circulating HBsAg in patients has been implicated in HBV-specific T cell anergy, acting as a high-dose tolerogen during HBV infection [6], [7]. Similarly, circulating HBeAg is believed to contribute to viral persistence by promoting anergy of virus specific T cells [8], [9]. Finally, HBsAg and HBeAg have recently been implicated in the evasion of HBV to the immune system by limiting type-I IFN antiviral activity [10]. Thus, therapeutic reduction of circulating viral antigens might be a key factor in restoring a functional and effective adaptive immune response to HBV that might clear the infection.

Currently approved therapies for CHB such as the widely used nucleos(t)ide analogues (NUCs) however, fail to significantly reduce antigenemia [11], [12] or promote HBeAg or HBsAg seroconversion [11], [13]. Only recently, efficient viral antigen reduction has been achieved using RNA interference (RNAi)-based methodology in in vivo HBV model systems [14], [15]. Its impact on chronic HBV infection in patients, however, remains to be determined. Recently, also a novel HBV entry inhibitor has been shown to be safe and well tolerated and is being currently tested in CHB patients [16].

In this study we evaluated the potential of antisense oligonucleotide (ASO)-mediated antiviral therapy to specifically reduce HBV antigenemia. ASOs are small single-stranded nucleic acids (8–50 nucleotides) complementary to their target RNA, which they bind through Watson-Crick base pairing resulting in degradation of the target RNA via a RNase H-dependent pathway [17]. ASOs are exceptionally target-specific, with single nucleotide mismatches affecting their activity, thus reducing the possibility of off-target effects [18].

Earlier studies have described the in vitro antiviral effect of unmodified first generation ASOs against HBV, either by directly targeting HBV sequences [19], [20], [21], [22], [23], [24], [25], [26], or indirectly by targeting host proteins [27], [28], [29] in HBV-expressing cell lines. In vivo antiviral studies using first generation ASOs included the Pekin duck model of duck hepatitis B virus infection [30], [31], [32], [33] and different mouse models [34], [35], [36]. However, instability and toxicity of first generation ASOs severely limited their potency and use in vivo [17], [37]. These limitations have been overcome with the development of second generation ASOs containing 2′-O-methoxyethyl (MOE) sugar modifications in their 5′ and 3′-regions that greatly improved ASO potency, stability, specificity and safety [17], [37], [38], [39]. It is this second generation ASO technology that serves as the basis for developing the novel HBV antiviral therapy described in this report.

We now demonstrate efficient HBV antiviral activity of a second generation ASO in in vitro and in vivo model systems of HBV gene expression and replication. A lead ASO identified in vitro, efficiently decreased HBV gene expression, replication, viremia and antigenemia in HBV transgenic mice. The same ASO also displayed pan-genotype antiviral activity in hydrodynamic transfection experiments. Finally, the lead ASO also inhibited HBV gene expression derived from cccDNA during HBV infection in vitro. Together, these results demonstrate the utility of second generation ASO technology as a novel and alternative therapeutic approach for the treatment of chronic HBV infection.

Section snippets

Antisense oligonucleotides

ASO-HBV (5′-GTGAAGCGAAGTGCACACGG-3′) is 100% complementary to the reference sequences for HBV genotypes A through H (Supplementary Table 1). Control ASO-Ctrl1 (5′-GTGCGCGCGAGCCCGAAATC-3′), ASO-Ctrl2 (5′-TAGTGCGGACCTACCCACGA-3′), and ASO-Ctrl3 (5′-CCTTCCCTGAAGGTTCCTCC-3′) have no known targets in HBV and the mouse or human transcriptome.

Cell culture dose response

HepG2.2.15 cells were plated at 28,000 cells per well in a 96-well plate and transfected with ASOs using Lipofectamine 2000 (Life technologies, Carlsbad, CA).

Dose-dependent antiviral activity of ASO-HBV in cell culture

The ASO-HBV was designed to target all HBV RNA species in virtually all human HBV genotypes (Supplementary Table 1). Furthermore, the ASO-HBV target site is distant from all known NUC-induced resistance mutations. The ability of ASO-HBV to inhibit virus replication and antigen production was demonstrated in vitro in HepG2.2.15 cells that express and replicate HBV and secrete HBV virus and subviral particles [40]. HepG2.2.15 cells were transfected with ASO-HBV and control ASO (ASO-Ctrl2).

Discussion

In this study, we demonstrated the efficacy of 2′MOE-modified phosphorothioate ASO to efficiently reduce HBV replication and virus production similar to the effects of currently approved antivirals [47]. However, unlike NUCs, ASO-HBV treatment also significantly reduced HBV antigenemia. In vitro screening of ASO identified several highly effective ASO candidates that target highly conserved regions of the HBV genome. In this study we used one of the ASO candidates (ASO-HBV) to demonstrate ASO

Financial support

This work was supported by a grant from the National Institutes of Health to S.W. (AI094409). L.C. was supported by NIH grants HL-51586 and L.C. and K.D.B. by P30-DK079638.

Conflict of interest

E.S., M.L.MC., J.R.C., A.K. and D.G. are employed by and have a financial interest in Ionis Pharmaceuticals. R.H. and Z.H. are employed by and have a financial interest in Glaxo Smith Kline. All other authors have nothing to disclose.

Authors’ contributions

The project was conceived by E.S., R.H., Z.H., M.L.MC., J.R.C., KD.B. and S.W.; experiments were designed and analyzed by S.W., G.B., E.S., M.L.MC., KD.B. and U.G.; experiments were performed by G.B., M.C., C.W-B., R.L.K., D.G., A.K. L.C., KD.B., S.W. and L.C.; and the paper was written by G.B., E.S., KD.B. and S.W.

Acknowledgments

We thank Francis V. Chisari (The Scripps Research Institute, La Jolla, CA, USA) for providing the HBV transgenic mice and JoSan Chung for excellent technical assistance. The authors thank Hiroaki Okamoto (Jichi Medical University, Tochigi, Japan) for the HBV genotype A, B and C plasmids. This work was supported by a grant from the National Institutes of Health to S.W. (AI094409). L.C. was supported by NIH grants HL-51586 and L.C. and K.D.B. by P30-DK079638. This is manuscript no. 28041 of The

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