Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo.

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL in vivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Ongoing viral replication and production of infectious virus in patients with chronic hepatitis B virus suppressed below the limit of quantitation on long-term nucleos(t)ide therapy.

Nucleos(t)ide analogs are standard-of-care for the treatment of chronic hepatitis B and can effectively reduce hepatitis B virus (HBV) replication but rarely leads to cure. Nucleos(t)ide analogs do not directly eliminate the viral episome, therefore treatment cessation typically leads to rapid viral rebound. While treatment is effective, HBV DNA is still detectable (although not quantifiable) in the periphery of the majority of nucleos(t)ide analog treated HBV patients, even after prolonged treatment. Addressing whether the detectable HBV DNA represents infectious virus is a key unknown and has important implications for the development of a curative treatment for HBV. The minimum HBV genome equivalents required to establish infection in human liver chimeric mice was determined by titration of HBV patient sera and the infectivity in chimeric mice of serum from patients (n = 7) suppressed to the limit of detection on nucleos(t)ide analog therapy was evaluated. A minimum of 5 HBV genome equivalents were required to establish infection in the chimeric mice, confirming this model has sufficient sensitivity to determine whether serum from virally suppressed patients contains infectious virus. Strikingly, serum from 75% (n = 3 out of 4) of nucleos(t)ide-treated HBV patients with DNA that was detectable, but below the lower limit of quantitation, also established infection in the chimeric mice. These results demonstrate that infectious virus is still present in some HBV patients on suppressive nucleos(t)ide therapy. This residual virus may support viral persistence via continuous infection and explain the ongoing risk for HBV-related complications despite long-term suppression on therapy. Thus, additional treatment intensification may facilitate HBV cure.

Anti-HBV activity of retinoid drugs in vitro versus in vivo.

We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC50 values ranging from 0.4 to 2.6 µM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.

Development of a robust luciferase reporter 1b/2a hepatitis C virus (HCV) for characterization of early stage HCV life cycle inhibitors.

The development of JFH1 based intergenotypic recombinants that exploit the unique replication characteristics of JFH1 has made it possible to study infectious hepatitis C virus (HCV) encoding the structural genes of additional HCV genotypes. To facilitate the study of 1b structural proteins, we aimed to develop a robust 1b/2a chimera encoding a humanized Renilla luciferase reporter gene (1b/2a hRluc). The unadapted genome replicated efficiently but produced very low titers of infectious virus. Adaptation by continuous passage over a novel Huh-7 Lunet clone improved viral titers approximately 100-fold but caused an unexpected decline in luciferase activity, limiting the utility of the reporter-containing virus. Genotypic analysis revealed 17 adenosine to guanosine (A to G) nucleotide mutations in the luciferase gene and two potential adaptive mutations. To overcome the problems of low viral titers and editing of the luciferase gene during viral adaptation, six adaptive mutations previously identified in a non-reporter 1b/2a HCV genome were introduced into the 1b/2a hRluc genome. This resulted in the immediate production of high-titer viral stocks (approximately 1000-fold greater than the parental virus) that could efficiently infect naïve cells and generate robust luciferase signals. The improved sensitivity of the luciferase reporter also facilitated time of addition studies validating the utility of this system for characterizing the early steps of HCV infection. Thus, the development of the 1b/2a hRluc reporter virus described here provides a versatile tool for discovery of inhibitors targeting the early steps of the viral life cycle and genotype 1b structural genes.