Search Results (11)

In a previous study, eight chronically HBV-infected nucleos (t)ide analog (NA)-naïve patients began receiving entecavir (ETV) concomitant with a single ARC-520 HBV siRNA injection. This single dose of ARC-520 (SD) was followed by 6–8 months of ETV alone before the patients received 4–9 monthly doses of ARC-520, the multi-dose (MD) period, while continuing ETV. Quantities of HBV DNA, RNA, and antigens were measured from serum and a liver biopsy collected ~30 months after the last MD from five patients. All full-length HBV transcripts from the livers were characterized. Viral parameters and HBV transcripts from patients were compared to these measurements collected at multiple points in ARC-520 + ETV-treated chronically HBV-infected chimpanzees. Multiple forms of HBx mRNA were observed, and these differed between chimpanzees and patients. Products of cccDNA were greatly decreased in patients who were previously highly viremic and HBeAg+, although a biopsied patient had similar amounts of cccDNA to the highly viremic HBeAg+ chimpanzees. The comparison of all HBV transcripts and cccDNA levels between patients and chimpanzees demonstrate the transcriptional silencing of cccDNA following the siRNA treatment of patients but not the chimpanzees that received a different treatment regimen. Results from this small study suggest that continued NA treatment during and between periods of HBV antigen re-expression post-siRNA treatment enhanced viral parameter reductions. Full article

Full-length hepatitis B virus (HBV) transcripts of chimpanzees and patients treated with multidose (MD) HBV siRNA ARC-520 and entecavir (ETV) were characterized by single-molecule real-time (SMRT) sequencing, identifying multiple types of transcripts with the potential to encode HBx, HBsAg, HBeAg, core, and polymerase, as well as transcripts likely to be derived from dimers of dslDNA, and these differed between HBeAg-positive (HBeAg+) and HBeAg-negative (HBeAg−) individuals. HBV transcripts from the last follow-up ~30 months post-ARC-520 treatment were categorized from one HBeAg+ (one of two previously highly viremic patients that became HBeAg− upon treatment and had greatly reduced cccDNA products) and four HBeAg− patients. The previously HBeAg+ patient received a biopsy that revealed that he had 3.4 copies/cell cccDNA (two to three orders of magnitude more cccDNA than HBeAg− chimpanzees) but expressed primarily truncated X and HBsAg from iDNA, like two patients that were HBeAg− at the start of the study and had one copy/cell cccDNA. No HBV transcripts were detected in two other HBeAg− patients that had ~0.3 copies/cell cccDNA, one of which had seroconverted for HBsAg. The paucity of cccDNA-derived transcripts in the presence of high cccDNA demonstrates the transcriptional silencing of HBV following MD siRNA treatment with ETV. Full article

Background: Despite an existing safe and effective vaccine for hepatitis B virus (HBV), it is still a major public health concern. Nowadays, several drugs are used to treat chronic hepatitis B; however, full healing remains controversial. The viral covalently closed circular DNA (cccDNA) formed by HBV forms a major challenge in its treatment, as does the ability of HBV to integrate itself into the host genome, which enables infection reactivation. Interfering RNA (RNAi) is a gene-silencing post-transcriptional mechanism which forms as a promising alternative to treat chronic hepatitis B. The aim of the present review is to assess the evolution of hepatitis B treatment approaches based on using RNA interference. Methods: Data published between 2016 and 2023 in scientific databases (PubMed, PMC, LILACS, and Bireme) were assessed. Results: In total, 76,949 articles were initially identified and quality-checked, and 226 eligible reports were analyzed in depth. The main genomic targets, delivery systems, and major HBV therapy innovations are discussed in this review. This review reinforces the therapeutic potential of RNAi and identifies the need for conducting further studies to fill the remaining gaps between bench and clinical practice. Full article

Cells have developed various mechanisms to counteract viral infections. In an evolutionary arms race, cells mobilize cellular restriction factors to fight off viruses, targeted by viral factors to facilitate their own replication. The hepatitis B virus (HBV) is a small dsDNA virus that causes acute and chronic infections of the liver. Its genome persists in the nuclei of infected hepatocytes as a covalently closed circular DNA (cccDNA) minichromosome, thus building up an episomal persistence reservoir. The chromosomal maintenance complex SMC5/6 acts as a restriction factor hindering cccDNA transcription, whereas the viral regulatory protein HBx targets SMC5/6 for proteasomal degradation, thus relieving transcriptional suppression of the HBV minichromosome. To date, no curative therapies are available for chronic HBV carriers. Knowledge of the factors regulating the cccDNA and the development of therapies involving silencing the minichromosome or specifically interfering with the HBx-SMC5/6 axis holds promise in achieving sustained viral control. Here, we summarize the current knowledge of the mechanism of SMC5/6-mediated HBV restriction. We also give an overview of SMC5/6 cellular functions and how this compares to the restriction of other DNA viruses. We further discuss the therapeutic potential of available and investigational drugs interfering with the HBx-SMC5/6 axis. Full article

The hepatitis B virus (HBV) infects hepatocytes and hijacks host cellular mechanisms for its replication. Host proteins can be frontline effectors of the cell’s defense and restrict viral replication by impeding multiple steps during its intracellular lifecycle. This review summarizes many of the well-described restriction factors, their mechanisms of restriction, and counteractive measures of HBV, with a special focus on viral transcription. We discuss some of the limitations and knowledge gaps about the restriction factors, highlighting how these factors may be harnessed to facilitate therapeutic strategies against HBV. Full article

Chronic hepatitis B (CHB) virus infection is a major public health burden and the leading cause of hepatocellular carcinoma. Despite the efficacy of current treatments, hepatitis B virus (HBV) cannot be fully eradicated due to the persistence of its minichromosome, or covalently closed circular DNA (cccDNA). The HBV community is investing large human and financial resources to develop new therapeutic strategies that either silence or ideally degrade cccDNA, to cure HBV completely or functionally. cccDNA transcription is considered to be the key step for HBV replication. Transcription not only influences the levels of viral RNA produced, but also directly impacts their quality, generating multiple variants. Growing evidence advocates for the role of the co-transcriptional regulation of HBV RNAs during CHB and viral replication, paving the way for the development of novel therapies targeting these processes. This review focuses on the mechanisms controlling the different co-transcriptional processes that HBV RNAs undergo, and their contribution to both viral replication and HBV-induced liver pathogenesis. Full article

Persistent hepatitis B virus (HBV) infection remains a serious medical problem worldwide, with an estimated global burden of 257 million carriers. Prophylactic and therapeutic interventions, in the form of a vaccine, immunomodulators, and nucleotide and nucleoside analogs, are available. Vaccination, however, offers no therapeutic benefit to chronic sufferers and has had a limited impact on infection rates. Although immunomodulators and nucleotide and nucleoside analogs have been licensed for treatment of chronic HBV, cure rates remain low. Transcription activator-like effector nucleases (TALENs) designed to bind and cleave viral DNA offer a novel therapeutic approach. Importantly, TALENs can target covalently closed circular DNA (cccDNA) directly with the potential of permanently disabling this important viral replicative intermediate. Potential off-target cleavage by engineered nucleases leading to toxicity presents a limitation of this technology. To address this, in the context of HBV gene therapy, existing TALENs targeting the viral core and surface open reading frames were modified with second- and third-generation FokI nuclease domains. As obligate heterodimers these TALENs prevent target cleavage as a result of FokI homodimerization. Second-generation obligate heterodimeric TALENs were as effective at silencing viral gene expression as first-generation counterparts and demonstrated an improved specificity in a mouse model of HBV replication. Full article

Hepatitis B virus (HBV) infection is a major global health problem causing acute and chronic liver disease that can lead to liver cirrhosis and hepatocellular carcinoma (HCC). HBV covalently closed circular DNA (cccDNA) is essential for viral replication and the establishment of a persistent infection. Integrated HBV DNA represents another stable form of viral DNA regularly observed in the livers of infected patients. HBV DNA integration into the host genome occurs early after HBV infection. It is a common occurrence during the HBV life cycle, and it has been detected in all the phases of chronic infection. HBV DNA integration has long been considered to be the main contributor to liver tumorigenesis. The recent development of highly sensitive detection methods and research models has led to the clarification of some molecular and pathogenic aspects of HBV integration. Though HBV integration does not lead to replication-competent transcripts, it can act as a stable source of viral RNA and proteins, which may contribute in determining HBV-specific T-cell exhaustion and favoring virus persistence. The relationship between HBV DNA integration and the immune response in the liver microenvironment might be closely related to the development and progression of HBV-related diseases. While many new antiviral agents aimed at cccDNA elimination or silencing have been developed, integrated HBV DNA remains a difficult therapeutic challenge. Full article

Background: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence in patients with chronic HBV infection. Understanding the mechanisms underlying stability and persistence of HBV cccDNA in hepatocytes is critical for developing novel therapeutics and managing chronic hepatitis B. In this study, we observed an unexpected increase in HBV cccDNA levels upon suppression of transcription by de novo DNA methyltransferase DNMT3A and uncovered additional mechanisms potentially involved in HBV cccDNA maintenance. Methods: HBV-expressing cell lines were transfected with a DNMT3A-expressing plasmid. Real-time PCR and HBsAg assays were used to assess the HBV replication rate. Cell cycling was analyzed by fluorescent cell sorting. CRISPR/Cas9 was utilized to abrogate expression of APOBEC3A and APOBEC3B. Alterations in the expression of target genes were measured by real-time PCR. Results: Similar to previous studies, HBV replication induced DNMT3A expression, which in turn, led to reduced HBV transcription but elevated HBV cccDNA levels (4- to 6-fold increase). Increased levels of HBV cccDNA were not related to cell cycling, as DNMT3A accelerated proliferation of infected cells and could not contribute to HBV cccDNA expansion by arresting cells in a quiescent state. At the same time, DNMT3A suppressed transcription of innate immunity factors including cytidine deaminases APOBEC3A and APOBEC3B. CRISPR/Cas9-mediated silencing of APOBEC3A and APOBEC3B transcription had minor effects on HBV transcription, but significantly increased HBV cccDNA levels, similar to DNMT3A. In an attempt to further analyze the detrimental effects of HBV and DNMT3A on infected cells, we visualized γ-H2AX foci and demonstrated that HBV inflicts and DNMT3A aggravates DNA damage, possibly by downregulating DNA damage response factors. Additionally, suppression of HBV replication by DNMT3A may be related to reduced ATM/ATR expression. Conclusion: Formation and maintenance of HBV cccDNA pools may be partially suppressed by the baseline expression of host inhibitory factors including APOBEC3A and APOBEC3B. HBV inflicts DNA damage both directly and by inducing DNMT3A expression. Full article

Hepatitis B virus (HBV) infection is a major health problem affecting about 300 million people globally. Although successful administration of a prophylactic vaccine has reduced new infections, a cure for chronic hepatitis B (CHB) is still unavailable. Current anti-HBV therapies slow down disease progression but are not curative as they cannot eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The cccDNA minichromosome persists in the nuclei of infected hepatocytes where it forms the template for all viral transcription. Interactions between host factors and cccDNA are crucial for its formation, stability, and transcriptional activity. Here, we summarize the reported interactions between HBV cccDNA and various host factors and their implications on HBV replication. While the virus hijacks certain cellular processes to complete its life cycle, there are also host factors that restrict HBV infection. Therefore, we review both positive and negative regulation of HBV cccDNA by host factors and the use of small molecule drugs or sequence-specific nucleases to target these interactions or cccDNA directly. We also discuss several reporter-based surrogate systems that mimic cccDNA biology which can be used for drug library screening of cccDNA-targeting compounds as well as identification of cccDNA-related targets. Full article

Chronic infection with the hepatitis B virus (HBV) is a global health concern and accounts for approximately 1 million deaths annually. Amongst other limitations of current anti-HBV treatment, failure to eliminate the viral covalently closed circular DNA (cccDNA) and emergence of resistance remain the most worrisome. Viral rebound from latent episomal cccDNA reservoirs occurs following cessation of therapy, patient non-compliance, or the development of escape mutants. Simultaneous viral co-infections, such as by HIV-1, further complicate therapeutic interventions. These challenges have prompted development of novel targeted hepatitis B therapies. Given the ease with which highly specific and potent nucleic acid therapeutics can be rationally designed, gene therapy has generated interest for antiviral application. Gene therapy strategies developed for HBV include gene silencing by harnessing RNA interference, transcriptional inhibition through epigenetic modification of target DNA, genome editing by designer nucleases, and immune modulation with cytokines. DNA-binding domains and effectors based on the zinc finger (ZF), transcription activator-like effector (TALE), and clustered regularly interspaced short palindromic repeat (CRISPR) systems are remarkably well suited to targeting episomal cccDNA. This review discusses recent developments and challenges facing the field of anti-HBV gene therapy, its potential curative significance and the progress towards clinical application. Full article