Hepatitis B Virus Transgenic Mouse Model Research Articles

Single-cell RNA sequencing reveals reduced intercellular adhesion molecule crosstalk between activated hepatic stellate cells and neutrophils alleviating liver fibrosis in hepatitis B virus transgenic mice post menstrual blood-derived mesenchymal stem cell transplantation.

Liver fibrosis can cause hepatitis B virus (HBV)-associated hepatocellular carcinoma. Menstrual blood-derived mesenchymal stem cells (MenSCs) can ameliorate liver fibrosis through paracrine. Single-cell RNA sequencing (scRNA-seq) may be used to explore the roadmap of activated hepatic stellate cell (aHSC) inactivation to target liver fibrosis. This study established HBV transgenic (HBV-Tg) mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis and demonstrated that MenSCs migrated to the injured liver to improve serological indices and reduce fibrotic accumulation. RNA-bulk analysis revealed that MenSCs mediated extracellular matrix accumulation and cell adhesion. Liver parenchymal cells and nonparenchymal cells were identified by scRNA-seq in the control, CCl4, and MenSC groups, revealing the heterogeneity of fibroblasts/HSCs. A CellChat analysis revealed that diminished intercellular adhesion molecule (ICAM) signaling is vital for MenSC therapy. Specifically, Icam1 in aHSCs acted on Itgal/Itgb2 and Itgam/Itgb2 in neutrophils, causing decreased adhesion. The expression of Itgal, Itgam, and Itgb2 was higher in CCl4 group than in the control group and decreased after MenSC therapy in neutrophil clusters. The Lcn2, Pglyrp1, Wfdc21, and Mmp8 had high expression and may be potential targets in neutrophils. This study highlights interacting cells, corresponding molecules, and underlying targets for MenSCs in treating HBV-associated liver fibrosis.

Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration

ABSTRACT The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.

HMMR alleviates endoplasmic reticulum stress by promoting autophagolysosomal activity during endoplasmic reticulum stress-driven hepatocellular carcinoma progression.

The mechanism of hepatitis B virus (HBV)-induced carcinogenesis remains an area of interest. The accumulation of hepatitis B surface antigen in the endoplasmic reticulum (ER) of hepatocytes stimulates persistent ER stress. Activity of the unfolded protein response (UPR) pathway of ER stress may play an important role in inflammatory cancer transformation. How the protective UPR pathway is hijacked by cells as a tool for malignant transformation in HBV-related hepatocellular carcinoma (HCC) is still unclear. Here, we aimed to define the key molecule hyaluronan-mediated motility receptor (HMMR) in this process and explore its role under ER stress in HCC development. An HBV-transgenic mouse model was used to characterize the pathological changes during the tumor progression. Proteomics and transcriptomics analyses were performed to identify the potential key molecule, screen the E3 ligase, and define the activation pathway. Quantitative real-time PCR and Western blotting were conducted to detect the expression of genes in tissues and cell lines. Luciferase reporter assay, chromatin immunoprecipitation, coimmunoprecipitation, immunoprecipitation, and immunofluorescence were employed to investigate the molecular mechanisms of HMMR under ER stress. Immunohistochemistry was used to clarify the expression patterns of HMMR and related molecules in human tissues. We found sustained activation of ER stress in the HBV-transgenic mouse model of hepatitis-fibrosis-HCC. HMMR was transcribed by c/EBP homologous protein (CHOP) and degraded by tripartite motif containing 29 (TRIM29) after ubiquitination under ER stress, which caused the inconsistent expression of mRNA and protein. Dynamic expression of TRIM29 in the HCC progression regulated the dynamic expression of HMMR. HMMR could alleviate ER stress by increasing autophagic lysosome activity. The negative correlation between HMMR and ER stress, positive correlation between HMMR and autophagy, and negative correlation between ER stress and autophagy were verified in human tissues. This study identified the complicated role of HMMR in autophagy and ER stress, that HMMR controls the intensity of ER stress by regulating autophagy in HCC progression, which could be a novel explanation for HBV-related carcinogenesis.

A hepatitis B virus-derived peptide combined with HBsAg exerts an anti-HBV effect in an HBV transgenic mouse model as a therapeutic vaccine.

For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant. In this study, we explored the potential of Poly6 in combination with HBsAg as a therapeutic vaccine against hepatitis B virus infection. We investigated the immunotherapeutic potential of Poly6 combined with HBsAg vaccination against hepatitis B virus infection in C57BL/6 mice or an HBV transgenic mouse model. In C57BL/6 mice, Poly6 enhanced DC maturation and DC migration capacity in an IFN-I-dependent manner. Moreover, the addition of Poly6 to alum in combination with HBsAg also led to enhanced HBsAg-specific cell-mediated immune (CMI) responses, suggesting its potential as an adjuvant of HBsAg-based vaccines. In HBV transgenic mice, vaccination with Poly6 combined with HBsAg exerted a strong anti-HBV effect via induction of HBV-specific humoral and cell-mediated immune responses. In addition, it also induced HBV-specific effector memory T cells (TEM). Our data indicated that vaccination with Poly6 in combination with HBsAg exerts an anti-HBV effect in HBV transgenic mice, which is mainly mediated by HBV-specific CMI and humoral immune responses via IFN-I-dependent DC activation, suggesting the feasibility of Poly6 as an adjuvant for an HBV therapeutic vaccine.

Release of hepatitis B virions is positively regulated by glucose-regulated protein 78 through direct interaction with preS1.

In this study, we investigated the mechanism of hepatitis B virus (HBV)-enveloped particle release. Specifically, we used preS1 as a bait protein to screen host proteins using mass spectroscopy, with the results of immunofluorescence, western blot, co-immunoprecipitation, isothermal titration calorimetry, and pull-down assays identifying glucose-regulated protein (GRP)78 as a specific target for preS1 binding. We employed transcriptome sequencing, enzyme-linked immunosorbent assays, and particle gel assays to investigate the mechanism of GRP78-mediated positive regulation of HBV-enveloped particle release. Additionally, we performed phage-display, surface plasmon resonance, and molecular-docking assays to assess peptides inhibiting enveloped-particle release. We found that HBV upregulated GRP78 expression in liver cell lines and the serum of patients with chronic hepatitis B. Furthermore, GRP78 promoted the release of HBV-enveloped particles in vitro and in vivo within an HBV transgenic mouse model. Moreover, we identified interactions of preS1 peptides with GRP78 via hydrogen bonding and hydrophobic interactions, which effectively inhibited its interaction with HBV-enveloped particles and their subsequent release. These findings provide novel insights regarding HBV virion release, and demonstrated that GRP78 interacted with preS1 to positively regulate the release of HBV-enveloped particles, suggesting GRP78 as a potential therapeutic target for inhibiting HBV infection.

Improved pharmacokinetics of tenofovir ester prodrugs strengthened the inhibition of HBV replication and the rebalance of hepatocellular metabolism in preclinical models.

Tenofovir (TFV) ester prodrugs, a class of nucleotide analogs (NAs), are the first-line clinical anti-hepatitis B virus (HBV) drugs with potent antiviral efficacy, low resistance rate and high safety. In this work, three marketed TFV ester drugs, tenofovir disoproxil fumarate (TDF), tenofovir alafenamide fumarate (TAF) and tenofovir amibufenamide fumarate (TMF), were used as probes to investigate the relationships among prodrug structures, pharmacokinetic characteristics, metabolic activations, pharmacological responses and to reveal the key factors of TFV ester prodrug design. The results indicated that TMF and TAF exhibited significantly stronger inhibition of HBV DNA replication than did TDF in HBV-positive HepG2.2.15 cells. The anti-HBV activity of TMF was slightly stronger than TAF after 9 days of treatment (EC50 7.29 ± 0.71 nM vs. 12.17 ± 0.56 nM). Similar results were observed in the HBV decline period post drug administration to the HBV transgenic mouse model, although these three TFV prodrugs finally achieved the same anti-HBV effect after 42 days treatments. Furthermore, TFV ester prodrugs showed a correcting effect on disordered host hepatic biochemical metabolism, including TCA cycle, glycolysis, pentose phosphate pathway, purine/pyrimidine metabolism, amino acid metabolism, ketone body metabolism and phospholipid metabolism. The callback effects of the three TFV ester prodrugs were ranked as TMF > TAF > TDF. These advantages of TMF were believed to be attributed to its greater bioavailability in preclinical animals (SD rats, C57BL/6 mice and beagle dogs) and better target loading, especially in terms of the higher hepatic level of the pharmacologically active metabolite TFV-DP, which was tightly related to anti-HBV efficacy. Further analysis indicated that stability in intestinal fluid determined the actual amount of TFV prodrug at the absorption site, and hepatic/intestinal stability determined the maintenance amount of prodrug in circulation, both of which influenced the oral bioavailability of TFV prodrugs. In conclusion, our research revealed that improved pharmacokinetics of TFV ester prodrugs (especially intestinal stability) strengthened the inhibition of HBV replication and the rebalance of hepatocellular metabolism, which provides new insights and a basis for the design, modification and evaluation of new TFV prodrugs in the future.

Heterogeneous phenotypes of Pten-null hepatocellular carcinoma in hepatitis B virus transgenic mice parallels liver lobule zonal gene expression patterns

Chronic HBV infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The phenotypes of HCC are diverse, in part, due to mutations in distinct oncogenes and/or tumor suppressor genes. These genetic drivers of HCC development have generally been considered as major mediators of tumor heterogeneity. Using the liver-specific Pten-null HBV transgenic mouse model of chronic viral infection, a critical role for liver lobule zone-specific gene expression patterns in determining HCC phenotype and β-catenin-dependent HBV biosynthesis is demonstrated. These observations suggest that the position of the hepatocyte within the liver lobule, and hence its intrinsic gene expression pattern at the time of cellular transformation, make critical contributions to the properties of the resulting liver tumor. These results may explain why therapies targeting pathways modulated by specific identified tumor driver genes display variable treatment efficacy.

Anti-HBV therapeutic potential of small molecule 3,5,6,7,3′,4′-Hexamethoxyflavone in vitro and in vivo

Current treatment methods for hepatitis B are mainly antiviral drug therapy and immunotherapy, in which antiviral drugs are mainly nucleoside analogue and interferon. They can significantly reduce the viral load but rarely achieve hepatitis B surface antigen (HBsAg) loss. 3,5,6,7,3′,4′-Hexamethoxyflavone was screened out from a small molecule compound library for its lower cytotoxic effect and greater HBsAg inhibition activity. Meanwhile, we further performed experiments in HepG2.2.15, HepG2-NTCP cells, PHHs and HBV transgenic mouse model to evaluate the anti-HBV effects of 3,5,6,7,3′,4′-Hexamethoxyflavone. Our study found that 3,5,6,7,3′,4′-Hexamethoxyflavone can significantly reduce the level of HBV RNAs, HBV DNA and HBsAg, in addition, the activity of four HBV promoters and the ratio of total RNAs/cccDNA and 3.5 kb RNA/cccDNA were decreased by 3,5,6,7,3′,4′-Hexamethoxyflavone. Mechanistically, we found HNF3α plays important roles in Hex mediated HBV transcription inhibition. Our study indicated Hex was a potential anti-HBV therapeutic drug.

Histone Deacetylase Inhibitors Romidepsin and Vorinostat Promote Hepatitis B Virus Replication by Inducing Cell Cycle Arrest.

Background and AimsChronic hepatitis B virus (HBV) infection is a global public health challenge. HBV reactivation usually occurs in cancer patients after receiving cytotoxic chemotherapy or immunosuppressive therapies. Romidepsin (FK228) and vorinostat (SAHA) are histone deacetylase inhibitors (HDACi) approved by the Food and Drug Administration as novel antitumor agents. The aim of this study was to explore the effects and mechanisms of HDACi treatment on HBV replication.MethodsTo assess these effects, human hepatoma cell lines were cultured and cell viability after FK228 or SAHA treatment was measured by the CCK-8 cell counting kit-8 assay. Then, HBV DNA and RNA were quantified by real-time PCR and Southern blotting. Furthermore, analysis by western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and flow cytometry was performed.ResultsFK228/SAHA treatment significantly promoted HBV replication and biosynthesis in both HBV-replicating cells and HBV-transgenic mouse model. Flow cytometry assay indicated that FK228/SAHA enhanced HBV replication by inducing cell cycle arrest through modulating the expression of cell cycle regulatory proteins. In addition, simultaneous inhibition of HDAC1/2 by FK228 promoted HBV replication more effectively than the broad spectrum HDAC inhibitor SAHA.ConclusionsOverall, our results demonstrate that cell cycle blockage plays an important role in FK228/SAHA-enhanced HBV replication, thus providing a potential avenue for rational use of HDACi in patients with chronic hepatitis B.

Relative DNA Methylation and Demethylation Efficiencies during Postnatal Liver Development Regulate Hepatitis B Virus Biosynthesis.

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.

A hepatitis B virus transgenic mouse model with a conditional, recombinant, episomal genome

Background & AimsDevelopment of new and more effective therapies against hepatitis B virus (HBV) is limited by the lack of suitable small animal models. The HBV transgenic mouse model containing an integrated overlength 1.3-mer construct has yielded crucial insights, but this model unfortunately lacks covalently closed circular DNA (cccDNA), the episomal HBV transcriptional template, and cannot be cured given that HBV is integrated in every cell.MethodsTo solve these 2 problems, we generated a novel transgenic mouse (HBV1.1X), which generates an excisable circular HBV genome using Cre/LoxP technology. This model possesses a HBV1.1-mer cassette knocked into the ROSA26 locus and is designed for stable expression of viral proteins from birth, like the current HBV transgenic mouse model, before genomic excision with the introduction of Cre recombinase.ResultsWe demonstrated induction of recombinant cccDNA (rcccDNA) formation via viral or transgenic Cre expression in HBV1.1X mice, and the ability to regulate HBsAg and HBc expression with Cre in mice. Tamoxifen-inducible Cre could markedly downregulate baseline HBsAg levels from the integrated HBV genome. To demonstrate clearance of HBV from HBV1.1X mice, we administered adenovirus expressing Cre, which permanently and significantly reduced HBsAg and core antigen levels in the murine liver via rcccDNA excision and a subsequent immune response.ConclusionsThe HBV1.1X model is the first Cre-regulatable HBV transgenic mouse model and should be of value to mimic chronic HBV infection, with neonatal expression and tolerance of HBV antigens, and on-demand modulation of HBV expression.Lay summaryHepatitis B virus (HBV) can only naturally infect humans and chimpanzees. Mouse models have been developed with the HBV genome integrated into mouse chromosomes, but this prevents mice from being cured. We developed a new transgenic mouse model that allows for HBV to be excised from mouse chromosomes to form a recombinant circular DNA molecule resembling the natural circular HBV genome. HBV expression could be reduced in these mice, enabling curative therapies to be tested in this new mouse model.

The cyclophilin inhibitor CRV431 inhibits liver HBV DNA and HBsAg in transgenic mice.

Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.

Cisplatin induces autophagy to enhance hepatitis B virus replication via activation of ROS/JNK and inhibition of the Akt/mTOR pathway

Chronic hepatitis B virus (HBV) infection remains a serious global health concern. Cisplatin is a chemotherapeutic agent commonly used to treat various cancers. However, HBV-infected patients receiving chemotherapy are at risk of HBV reactivation via unknown mechanisms, which we aimed to elucidate in this study. We found that autophagy plays a central role in cisplatin-induced HBV replication. Cisplatin treatment induced autophagy in both HBV-replicating cells and an HBV-transgenic mouse model as evident from marked upregulation of microtubule-associated protein 1 light chain 3 (LC3)-II and the accumulation of red fluorescent protein (RFP)-LC3 puncta. Cisplatin induced complete autophagic flux, which was detected via monitoring of p62 degradation and RFP-GFP-LC3 expression. Inhibition of autophagy by chloroquine, 3-methyladenine, or Atg5 knockdown significantly attenuated cisplatin-induced HBV replication. Additionally, cisplatin-induced autophagy could be significantly attenuated by using the ROS scavenger N-acetyl-l-cysteine. Mechanically, cisplatin promoted HBV replication and autophagy through ROS/JNK and AKT/mTOR signaling. Inhibition of JNK or activation of Akt/mTOR signaling reversed cisplatin-mediated autophagy and HBV replication promotion. In contrast, suppression of Akt/mTOR signaling further promoted cisplatin-induced HBV replication. Finally, pharmacotherapeutic inhibition of autophagy or ROS production impaired HBV production induced by cisplatin in vivo. Together, our results indicate that ROS/JNK and mTOR/AKT-mediated autophagy plays an important role in cisplatin-induced HBV reactivation.

Activation of hepatic stem cells compartment during hepatocarcinogenesis in a HBsAg HBV-transgenic mouse model

Chronic infection of Hepatitis B Virus (HBV) is one of the highest risk factors of hepatocellular carcinoma (HCC). The accumulation of HBV surface antigen (HBsAg) into hepatocytes induces inflammation and oxidative stress, impairing their replicative ability and allowing the activation of the hepatic stem cells (SC) compartment. This study aimed to understand the involvement of SC during hepatocarcinogenesis in HBsAg-related liver damage, from early injury until HCC. HBsAg-transgenic (TG) and wild type (WT) mouse were followed at several stages of the liver damage: inflammation, early hepatocytes damage, dysplasia, and HCC. Serum transaminases, liver histology, and diagnostic data were collected. The expressions of SC and cancer stem cells (CSC) markers was analyzed by RT-qPCR, immunohistochemistry and Western blot. Starting from 3 months, TG animals showed a progressive liver damage characterized by transaminases increase. The up-regulations of SCs markers Cd34 and Sca-1 started from the beginning of the inflammatory stage while progressive increase of Krt19 and Sox9 and CSCs markers Epcam and Cd133 from early hepatic injury. The expressions of Cd133, Cd34, and Afp were significantly higher in HCC compared to paired non-HCC tissue, in contrast to Epcam and Krt19. Western blot and IHC confirmed the positivity of Cd34 and Cd133 in small cells subpopulation.

Cisplatin Enhances Hepatitis B Virus Replication and PGC-1\u03b1 Expression through Endoplasmic Reticulum Stress

Chronic hepatitis B infection remains a serious public health issue worldwide. Hepatitis B virus (HBV) reactivation is commonly reported in patients receiving anticancer therapy, immunosuppressive therapy, or organ and tissue transplantation. However, the precise mechanisms underlying chemotherapeutic agent-related HBV reactivation remain unclear. Here, we report that peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) plays a central role in cisplatin-induced HBV transcription and replication. First, cisplatin treatment upregulated the expression levels of PGC-1α and hepatocyte nuclear factor 4 alpha (HNF-4α) in both HBV-replicating cells and an HBV-transgenic mouse model. PGC-1α coactivates with HNF-4α, which interacts with a core promoter and enhancer II region of HBV genome, thereby promoting HBV production. In contrast, knockdown of PGC-1α and HNF-4α by RNA interference in hepatoma cells reversed HBV activation in response to cisplatin. Additionally, PGC-1α upregulation depended on cisplatin-mediated endoplasmic reticulum (ER) stress. We further observed that the recruitment of cyclic AMP-responsive element-binding protein plays a crucial role for PGC-1α transcriptional activation in cisplatin-treated cells. Finally, pharmacologic inhibition of ER stress impaired PGC-1α upregulation and HBV production induced by cisplatin treatment. These findings demonstrate novel molecular mechanisms indicating that ER stress-PGC1α signaling pathway plays a critical role in cisplatin-evoked HBV reactivation.

Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV) transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma.

P022: The hepatitis B virus (HBV) surface antigen (HBsAg) prevents hepadnaviral replication‐dependent interferon responses, which are mediated by non‐parenchymal liver cells in a HBV transgenic mouse model

P022: The hepatitis B virus (HBV) surface antigen (HBsAg) prevents hepadnaviral replication‐dependent interferon responses, which are mediated by non‐parenchymal liver cells in a HBV transgenic mouse model

699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy

A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where sense strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.

Abstract 5346: Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma

Abstract Background and Aim: Hepatocellular carcinoma (HCC) is a highly lethal and prevalent cancer, posing a grave threat to human health globally. Hepatitis B virus (HBV) infection is considered as a major risk factor for this cancer, especially in the Asia-Pacific region. Unfortunately, the molecular mechanisms of hepatocarcinogenesis remain obscure, which hinders the development of effective therapies for the disease. In the present study, we attempted to elucidate the molecular details of HBV-induced hepatocarcinogenesis by investigating differentially regulated genes at multiple developmental stages of HCC in a HBV transgenic mouse model. Materials and Methods: The transgenic mice which overproduced HBV large envelope polypeptide in hepatocytes and developed liver tumors spontaneously were used in this study. To unravel transcriptomics dynamics underlying hepatocarcinogenesis, RNA prepared from livers of both transgenic and wild type mice of different ages (at months 2, 12, 18 and 19) were subjected to RNA sequencing. Selected target genes were first validated by quantitative PCR (qPCR) using a larger set of mouse liver tissues (n=96) collected from 8 time points. Clinical implications of the selected genes were then explored in a set of human liver samples comprising 18 normal, 29 cirrhosis and 96 pairs of HCC. RNA and protein expression levels were determined by qPCR, immunohistochemical staining and Western blotting, respectively. Results: Upon analysis of 20,209 gene transcripts, 2574 and 1035 transcripts were found to be up-regulated (≥2 folds) and down-regulated (≤2 folds) in tumors, respectively, when compared with the wild type controls. Among these, 133 most prominent genes that exhibited concordant differential expression throughout the stages of tumor progression were chosen for validation in mouse liver tissues. Correlation analysis showed a high correlation between RNA sequencing and qPCR data (r=0.7495; P<0.0001), indicating a high validity of the data. Forty-six biologically informative genes were further validated in human liver samples. By Gene Ontology analysis, the target genes were revealed to play roles in a variety of biological processes including stress and inflammation responses, metabolic and apoptotic processes. Immunohistochemical staining and Western blotting demonstrated significant differential expression of these genes between HCC and non-tumorous livers. Statistical analyses revealed their significant correlation with clinicopathological parameters including venous infiltration, tumor size and overall survival, implicating their roles in hepatocarcinogenesis. Conclusion: This study has demonstrated a systematic strategy for identifying crucial genes for HBV-associated HCC, which may have profound implications in combating this deadly cancer. Citation Format: CT Lam, ZF Yang, JC Lau, MN Ng, WC Yu, DW Ho, ST Fan. Identification of essential genes for the development of hepatitis B virus-associated hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2014-5346

Platelets Promote Liver Immunopathology Contributing to Hepatitis B Virus–Mediated Hepatocarcinogenesis

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC). Among the pathogenetic factors triggered by HBV, virus-specific CD8(+) T cells play and important role in disease pathogenesis by promoting necroinflammatory liver damage. Accordingly, amelioration of immune-mediated chronic liver injury may prevent HCC. Platelets facilitate this process by sustaining the hepatic accumulation of virus-specific CD8(+) T cells and subsequently other virus nonspecific inflammatory cells that contribute to liver disease. Importantly, a recent study shows that the long-term use of clinically relevant doses of the anti-platelet drugs aspirin and clopidogrel, administered after the onset of liver disease, in an HBV transgenic mouse model of immune-mediated chronic hepatitis and HCC, can prevent hepatocarcinogenesis improving overall survival. Platelets therefore, act as key players in the pathogenesis of HBV-associated liver cancer supporting the notion that immune-mediated necroinflammatory liver disease is sufficient to trigger HCC and that interference with platelet activation may have clinical implications for HCC prevention.