Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg-positive patients with functional cure following PEG-IFN-based therapy.

BACKGROUND: Hepatitis B surface antigen (HBsAg) seroclearance marks regression of hepatitis B virus (HBV) infection. However, more than one-fifth of patients with functional cure following pegylated interferon-based therapy may experience HBsAg seroreversion. The mechanisms causing the HBV relapse remain unclear. AIM: To investigate the level and origin of HBV transcripts in patients with functional cure and their role in predicting relapse. METHODS: Liver tissue obtained from patients with functional cure, as well as uncured and treatment-naïve HBeAg-negative patients with chronic hepatitis B (CHB) were analysed for intrahepatic HBV markers. HBV capture and RNA sequencing were used to detect HBV integration and chimeric transcripts. RESULTS: Covalently closed circular DNA (cccDNA) levels and the proportion of HBsAg-positive hepatocytes in functionally cured patients were significantly lower than those in uncured and treatment-naïve HBeAg-negative patients. Integrated HBV DNA and chimeric transcripts declined in functionally cured patients compared to uncured patients. HBsAg-positive hepatocytes present in 25.5% of functionally cured patients, while intrahepatic HBV RNA remained in 72.2%. The levels of intrahepatic HBV RNA, integrated HBV DNA, and chimeric transcripts were higher in functionally cured patients with intrahepatic HBsAg than in those without. The residual intrahepatic HBsAg in functionally cured patients was mainly derived from transcriptionally active integrated HBV DNA; meanwhile, trace transcriptional activity of cccDNA could also remain. Two out of four functionally cured patients with intrahepatic HBsAg and trace active cccDNA experienced HBV relapse. CONCLUSION: Integrated HBV DNA and cccDNA maintain transcriptional activity and maybe involved in HBsAg seroreversion in intrahepatic HBsAg-positive patients with functional cure and linked to virological relapse.

The premise of capsid assembly modulators towards eliminating HBV persistence.

INTRODUCTION: The burden of chronic hepatitis B virus (HBV) results in almost a million deaths per year. The most common treatment for chronic hepatitis B infection is long-term nucleoside analogs (NUC) or one-year interferon-alpha (pegylated or non-pegylated) therapy before or after NUC therapy. Unfortunately, these therapies rarely result in HBV functional cure because they do not eradicate HBV from the nucleus of the hepatocytes, where the covalently closed circular DNA (cccDNA) is formed and/or where the integrated HBV DNA persists in the host genome. Hence, the search continues for novel antiviral therapies that target different steps of the HBV replication cycle to cure chronically infected HBV individuals and eliminate HBV from the liver reservoirs. AREAS COVERED: The authors focus on capsid assembly modulators (CAMs). These molecules are unique because they impact not only one but several steps of HBV viral replication, including capsid assembly, capsid trafficking into the nucleus, reverse transcription, pre-genomic RNA (pgRNA), and polymerase protein co-packaging. EXPERT OPINION: Mono- or combination therapy, including CAMs with other HBV drugs, may potentially eliminate hepatitis B infections. Nevertheless, more data on their potential effect on HBV elimination is needed, especially when used daily for 6-12 months.

[A conceptual framework for dynamics of cccDNA in hepatitis B virus].

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.

Targeting hepatitis B virus cccDNA levels: Recent progress in seeking small molecule drug candidates.

Hepatitis B virus (HBV) infection is a major global health problem that puts people at high risk of death from cirrhosis and liver cancer. The presence of covalently closed circular DNA (cccDNA) in infected cells is considered to be the main obstacle to curing chronic hepatitis B. At present, the cccDNA cannot be completely eliminated by standard treatments. There is an urgent need to develop drugs or therapies that can reduce HBV cccDNA levels in infected cells. We summarize the discovery and optimization of small molecules that target cccDNA synthesis and degradation. These compounds are cccDNA synthesis inhibitors, cccDNA reducers, core protein allosteric modulators, ribonuclease H inhibitors, cccDNA transcriptional modulators, HBx inhibitors and other small molecules that reduce cccDNA levels.

Liver-Humanized NSG-PiZ Mice Support the Study of Chronic Hepatitis B Virus Infection and Antiviral Therapies.

Hepatitis B virus (HBV) is a pathogen of major public health importance that is largely incurable once a chronic infection is established. Only humans and great apes are fully permissive to HBV infection, and this species restriction has impacted HBV research by limiting the utility of small animal models. To combat HBV species restrictions and enable more in vivo studies, liver-humanized mouse models have been developed that are permissive to HBV infection and replication. Unfortunately, these models can be difficult to establish and are expensive commercially, which has limited their academic use. As an alternative mouse model to study HBV, we evaluated liver-humanized NSG-PiZ mice and showed that they are fully permissive to HBV. HBV selectively replicates in human hepatocytes within chimeric livers, and HBV-positive (HBV+) mice secrete infectious virions and hepatitis B surface antigen (HBsAg) into blood while also harboring covalently closed circular DNA (cccDNA). HBV+ mice develop chronic infections lasting at least 169 days, which should enable the study of new curative therapies targeting chronic HBV, and respond to entecavir therapy. Furthermore, HBV+ human hepatocytes in NSG-PiZ mice can be transduced by AAV3b and AAV.LK03 vectors, which should enable the study of gene therapies that target HBV. In summary, our data demonstrate that liver-humanized NSG-PiZ mice can be used as a robust and cost-effective alternative to existing chronic hepatitis B (CHB) models and may enable more academic research labs to study HBV disease pathogenesis and antiviral therapy. IMPORTANCE Liver-humanized mouse models have become the gold standard for the in vivo study of hepatitis B virus (HBV), yet their complexity and cost have prohibited widespread use of existing models in research. Here, we show that the NSG-PiZ liver-humanized mouse model, which is relatively inexpensive and simple to establish, can support chronic HBV infection. Infected mice are fully permissive to hepatitis B, supporting both active replication and spread, and can be used to study novel antiviral therapies. This model is a viable and cost-effective alternative to other liver-humanized mouse models that are used to study HBV.

Serum HBsAg and HBcrAg is associated with inflammation in HBeAg-positive chronic hepatitis B patients.

Backgrounds & aims: Liver inflammation is the main risk factor for developing liver fibrosis, cirrhosis, and even hepatocellular carcinoma in chronic hepatitis B (CHB) patients. To replace biopsy, additional non-invasive biomarkers to diagnose and grade liver necroinflammation are urgently required in clinical practice. Method: Ninety-four CHB patients, including 74 HBeAg-positive and 20 HBeAg-negative patients, were enrolled and started entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg, hepatitis B core-related antigen (HBcrAg), ALT and AST levels, as well as intrahepatic HBV DNA and cccDNA were measured at baseline and during treatment. Liver inflammation was assessed at baseline and month 60 by liver biopsy. Inflammation regression was defined as a ≥1-grade decrease according to the Scheuer scoring system. Results: In HBeAg-positive CHB patients, at baseline, serum HBsAg and HBcrAg levels negatively correlated with inflammation grade, while ALT and AST levels positively correlated with inflammation grade. AST plus HBsAg exhibited excellent diagnostic ability for significant inflammation with an AUROC of 0.896. After 60 months of antiviral treatment, almost all the patients' liver inflammation ameliorated to G1, and no patients had inflammation progression. Conclusion: Besides ALT and AST, serum HBsAg and HBcrAg correlated with inflammation grade in HBeAg-positive CHB patients before NAs treatment. Moreover, the combination of HBsAg and AST exhibited excellent diagnostic ability for significant inflammation.

The progress of molecules and strategies for the treatment of HBV infection.

Hepatitis B virus infections have always been associated with high levels of mortality. In 2019, hepatitis B virus (HBV)-related diseases resulted in approximately 555,000 deaths globally. In view of its high lethality, the treatment of HBV infections has always presented a huge challenge. The World Health Organization (WHO) came up with ambitious targets for the elimination of hepatitis B as a major public health threat by 2030. To accomplish this goal, one of the WHO's strategies is to develop curative treatments for HBV infections. Current treatments in a clinical setting included 1 year of pegylated interferon alpha (PEG-IFNα) and long-term nucleoside analogues (NAs). Although both treatments have demonstrated outstanding antiviral effects, it has been difficult to develop a cure for HBV. The reason for this is that covalently closed circular DNA (cccDNA), integrated HBV DNA, the high viral burden, and the impaired host immune responses all hinder the development of a cure for HBV. To overcome these problems, there are clinical trials on a number of antiviral molecules being carried out, all -showing promising results so far. In this review, we summarize the functions and mechanisms of action of various synthetic molecules, natural products, traditional Chinese herbal medicines, as clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas)-based systems, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), all of which could destroy the stability of the HBV life cycle. In addition, we discuss the functions of immune modulators, which can enhance or activate the host immune system, as well some representative natural products with anti-HBV effects.

Chronic Hepatitis B Infection: Current and Emerging Therapeutic Strategies.

The chronic infection of the hepatitis B virus (CHB) represents a major public health problem worldwide. Despite the availability of an effective prophylactic vaccine, millions of hepatitis B patients are at increased risk of developing chronic liver disease. The currently available treatments for HBV infection include interferon and nucleos(t)ide analogues that are effective at suppressing viral load and preventing or delaying the progression of liver disease. However, these treatments offer somewhat unsatisfactory clinical cures due to the persistence of the intrahepatic pool of covalently closed circular DNA (cccDNA) that serves as a reservoir for viral progenies and a potential source of recurring infections. Elimination of viral cccDNA remains a challenge for scientists and pharmaceutical industries in order to achieve the eradication and control of HBV infection. This would involve a detailed understanding of the molecular mechanisms of cccDNA formation, its intracellular stability, and regulation during replication and transcription. Recent advances in drug therapy have heralded a new horizon of novel therapeutic approaches for CHB infection, with several promising antiviral and immunomodulatory agents currently in preclinical or clinical testing. However, approval of any new curative therapy would involve rigorous evaluation of the efficacy and safety of each treatment and defining correct endpoints associated with improved clinical outcomes. This article summarizes the current landscape of HBV treatments, and drugs in clinical trials and highlights the most recent anti-HBV small molecules designed to directly target HBV or to improve immune response during chronic infection.

Emerging Therapies for Chronic Hepatitis B and the Potential for a Functional Cure.

Worldwide, an estimated 296 million people are living with chronic hepatitis B virus (HBV) infection, with a significant risk of morbidity and mortality. Current therapy with pegylated interferon (Peg-IFN) and indefinite or finite therapy with nucleoside/nucleotide analogues (Nucs) are effective in HBV suppression, hepatitis resolution, and prevention of disease progression. However, few achieve hepatitis B surface antigen (HBsAg) loss (functional cure), and relapse often occurs after the end of therapy (EOT) because these agents have no direct effect on durable template: covalently closed circular DNA (cccDNA) and integrated HBV DNA. Hepatitis B surface antigen loss rate increases slightly by adding or switching to Peg-IFN in Nuc-treated patients and this loss rate greatly increases up to 39% in 5 years with finite Nuc therapy with currently available Nuc(s). For this, great effort has been made to develop novel direct-acting antivirals (DAAs) and immunomodulators. Among the DAAs, entry inhibitors and capsid assembly modulators have little effect on reducing HBsAg levels; small interfering RNA, antisense oligonucleotides, and nucleic acid polymers in combination with Peg-IFN and Nuc may reduce HBsAg levels significantly, even a rate of HBsAg loss sustained for > 24 weeks after EOT up to 40%. Novel immunomodulators, including T-cell receptor agonists, check-point inhibitors, therapeutic vaccines, and monoclonal antibodies may restore HBV-specific T-cell response but not sustained HBsAg loss. The safety issues and the durability of HBsAg loss warrant further investigation. Combining agents of different classes has the potential to enhance HBsAg loss. Compounds directly targeting cccDNA would be more effective but are still in the early stage of development. More effort is required to achieve this goal.

Discovery of a first-in-class orally available HBV cccDNA inhibitor.

BACKGROUND & AIMS: The persistence of covalently closed circular DNA (cccDNA) in infected hepatocytes is the major barrier preventing viral eradication with existing therapies in patients with chronic hepatitis B. Therapeutic agents that can eliminate cccDNA are urgently needed to achieve viral eradication and thus HBV cure. METHODS: A phenotypic assay with HBV-infected primary human hepatocytes (PHHs) was employed to screen for novel cccDNA inhibitors. A HBVcircle mouse model and a uPA-SCID (urokinase-type plasminogen activator-severe combined immunodeficiency) humanized liver mouse model were used to evaluate the anti-HBV efficacy of the discovered cccDNA inhibitors. RESULTS: Potent and dose-dependent reductions in extracellular HBV DNA, HBsAg, and HBeAg levels were achieved upon the initiation of ccc_R08 treatment two days after the HBV infection of PHHs. More importantly, the level of cccDNA was specifically reduced by ccc_R08, while it did not obviously affect mitochondrial DNA. Additionally, ccc_R08 showed no significant cytotoxicity in PHHs or in multiple proliferating cell lines. The twice daily oral administration of ccc_R08 to HBVcircle model mice, which contained surrogate cccDNA molecules, significantly decreased the serum levels of HBV DNA and antigens, and these effects were sustained during the off-treatment follow-up period. Moreover, at the end of follow-up, the levels of surrogate cccDNA molecules in the livers of ccc_R08-treated HBVcircle mice were reduced to below the lower limit of quantification. CONCLUSIONS: We have discovered a small-molecule cccDNA inhibitor that reduces HBV cccDNA levels. cccDNA inhibitors potentially represent a new approach to completely cure patients chronically infected with HBV. IMPACT AND IMPLICATIONS: Covalently closed circular DNA (cccDNA) persistence in HBV-infected hepatocytes is the root cause of chronic hepatitis B. We discovered a novel small-molecule cccDNA inhibitor that can specifically reduce cccDNA levels in HBV-infected hepatocytes. This type of molecule could offer a new approach to completely cure patients chronically infected with HBV.

Hepatitis B RNA and Core-Related Antigen Provide Value Beyond DNA in Evaluating e But Not Surface Antigen Clearance.

In chronic hepatitis B virus (HBV) infection, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) clearance are important milestones toward immune control.1 A drop in HBV DNA is an established correlate of both HBeAg and HBsAg clearance.2 We evaluated changes in HBV RNA and hepatitis B core-related antigen (HBcrAg) levels, markers of transcriptional activity of covalently closed circular DNA (cccDNA),3,4 with HBeAg and HBsAg clearance, and compared them with changes in HBV DNA level among adult participants in the Hepatitis B Research Network (HBRN).

A conceptual framework for dynamics of cccDNA in hepatitis B virus / 中华肝脏病杂志

The resolution of the hepatitis C issue has raised expectations for a chronic hepatitis B cure, driving the industry to expand investment in research and development efforts to strengthen functional cure strategies. These strategies have a wide variety of types, and the published research findings are heterogeneous. The theoretical analysis of these strategies is of great significance for determining prioritized research orientations as well as sensibly allocating research and development resources. However, due to a paucity of necessary conceptual models, current theoretical analysis has not been able to unify various therapeutic strategies into a proper theoretical framework. In view of the fact that the decrease in the quantity of cccDNA is an inevitable core event accompanied by the process of functional cure, this paper intends to analyze several chronic hepatitis B cure strategies using cccDNA dynamics as a framework. Furthermore, there are currently few studies on the dynamics of the cccDNA field, hoping that this article can promote recognition and research in this field.

Active site polymerase inhibitor nucleotides (ASPINs): Potential agents for chronic HBV cure regimens.

Chronic hepatitis B virus (HBV) infection affects 240 to 300 million people worldwide. In the nucleus of infected hepatocytes, the HBV genome is converted to covalently closed circular DNA (cccDNA), which persists and serves as a transcriptional template for viral progeny. Therefore, a long-term cure for chronic HBV infection will require elimination of cccDNA. Although currently available nucleos(t)ide analogues (eg, tenofovir disoproxil fumarate, tenofovir alafenamide, entecavir) effectively control HBV replication, they are seldom curative (functional cure rate ∼10%) and require lifelong treatment for most patients. As such, antiviral agents with novel mechanisms of action are needed. Active site polymerase inhibitor nucleotides (ASPINs) noncompetitively distort the HBV polymerase active site to completely inhibit all polymerase functions, unlike traditional chain-terminating nucleos(t)ide analogues, which only target select polymerase functions and are consumed in the process. Clevudine, a first-generation ASPIN, demonstrated potent and prolonged HBV suppression in phase 2 and 3 clinical studies, but long-term treatment was associated with reversible myopathy in a small number of patients. ATI-2173, a novel next-generation ASPIN, is structurally similar to clevudine but targets the liver and demonstrates potent anti-HBV activity on and off treatment, and may ultimately demonstrate an improved pharmacokinetic and safety profile by significantly reducing systemic clevudine exposure. Thus, ATI-2173 is currently in clinical development as an agent for HBV cure. Here, we review the mechanism of action and preclinical and clinical profiles of clevudine and ATI-2173 to support the role of ASPINs as part of curative regimens for chronic HBV infection.

Integrated hepatitis B virus DNA maintains surface antigen production during antiviral treatment.

The focus of hepatitis B functional cure, defined as sustained loss of hepatitis B virus (HBV) surface antigen (HBsAg) and HBV DNA from blood, is on eliminating or silencing the intranuclear template for HBV replication, covalently closed circular DNA (cccDNA). However, HBsAg also derives from HBV DNA integrated into the host genome (iDNA). Little is known about the contribution of iDNA to circulating HBsAg with current therapeutics. We applied a multiplex droplet digital PCR assay to demonstrate that iDNA is responsible for maintaining HBsAg quantities in some individuals. Using paired bulk liver tissue from 16 HIV/HBV-coinfected persons on nucleos(t)ide analog (NUC) therapy, we demonstrate that people with larger HBsAg declines between biopsies derive HBsAg from cccDNA, whereas people with stable HBsAg levels derive predominantly from iDNA. We applied our assay to individual hepatocytes in paired tissues from 3 people and demonstrated that the individual with significant HBsAg decline had a commensurate loss of infected cells with transcriptionally active cccDNA, while individuals without HBsAg decline had stable or increasing numbers of cells producing HBsAg from iDNA. We demonstrate that while NUC therapy may be effective at controlling cccDNA replication and transcription, innovative treatments are required to address iDNA transcription that sustains HBsAg production.

Serum HBV DNA plus RNA reflecting cccDNA level before and during NAs treatment in HBeAg positive CHB patients.

Background & Aims: Correlations between serum viral markers and intrahepatic cccDNA in patients undergoing long-term nucleos(t)ide analogues (NAs) treatment haven't been fully explored. In this study, we evaluate the correlation between intrahepatic cccDNA and other serum viral markers and intrahepatic HBV DNA in HBeAg positive chronic hepatitis B (CHB) patients during 60-month treatment with NAs. Methods: Fifty-four HBeAg positive CHB patients received long-term NAs treatment were included in this study. Serial serum samples were regularly collected and quantitatively analyzed for HBsAg, HBV DNA, HBV RNA and HBcrAg. Histological samples from liver biopsy at baseline and month 60 were analyzed for intrahepatic HBV DNA and cccDNA. Results: At baseline, serum HBV DNA plus RNA was positively associated with intrahepatic cccDNA in multivariate regression analysis (ß=0.205, P<0.001). In the correlation analysis between cccDNA and serum viral markers, HBV DNA plus RNA had the highest correlation coefficient (r=0.698, P<0.001), followed by serum HBV DNA (r=0.641, P<0.001), HBV RNA (r=0.590, P<0.001), and HBcrAg (r=0.564, P<0.001). At month 60, correlations between these serum viral markers and cccDNA were not observed (P>0.05). Multivariate regression analysis showed that only the decreased HBV DNA plus RNA was positively associated with cccDNA decline (ß=0.172, P =0.006). Changes of HBV DNA plus RNA (r=0.525, P=0.001) was better correlated with cccDNA decline as compared to HBV RNA (r=0.384, P=0.008), HBV DNA (r=0.431, P=0.003), and HBsAg (r=0.342, P=0.029). Conclusions: Serum HBV DNA plus RNA better correlated with intrahepatic cccDNA than other viral makers before and during NAs treatment in HBeAg positive CHB patients.

Novel therapeutic strategies for chronic hepatitis B.

The last few years have seen a resurgence of activity in the hepatitis B drug pipeline, with many compounds in various stages of development. This review aims to provide a comprehensive overview of the latest advances in therapeutics for chronic hepatitis B (CHB). We will discuss the broad spectrum of direct-acting antivirals in clinical development, including capsids inhibitors, siRNA, HBsAg and polymerase inhibitors. In addition, host-targeted therapies (HTT) will be extensively reviewed, focusing on the latest progress in immunotherapeutics such as toll-like receptors and RIG-1 agonists, therapeutic vaccines and immune checkpoints modulators. A growing number of HTT in pre-clinical development directly target the key to HBV persistence, namely the covalently closed circular DNA (cccDNA) and hold great promise for HBV cure. This exciting area of HBV research will be highlighted, and molecules such as cyclophilins inhibitors, APOBEC3 deaminases and epigenetic modifiers will be discussed.

Reactivation of γ-globin expression using a minicircle DNA system to treat ß-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for ß-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of ß-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of ß-thalassemia.

Single-stranded circular DNA theranostics.

The past decade has witnessed the blossom of nucleic acid therapeutics and diagnostics (theranostics). Unlike conventional small molecule medicines or protein biologics, nucleic acid theranostics have characteristic features such as the intrinsic ability as "information drugs" to code and execute genetic and theranostic information, ready programmability for nucleic acid engineering, intrinsic stimulatory or regulatory immunomodulation, versatile functionalities, and easy conformational recovery upon thermal or chemical denaturation. Single-stranded circular DNA (circDNA) are a class of single-stranded DNAs (ssDNA) featured with their covalently-closed topology. In addition to the basic advantages of nucleic acids-based materials, such as low cost, biocompatibility, and simplicity of chemical modification, the lack of terminals in circDNA prevents exonuclease degradation, resulting in enhanced biostability relative to the corresponding linear ssDNA. circDNA has been explored for versatile theranostic applications. For instance, circDNA has been extensively studied as templates for bioanalytical signal amplification and the synthesis of nano-/micro-/macro- biomaterials via rolling circle amplification (RCA) and rolling circle transcription (RCT) technologies. circDNA has also been commonly used as the scaffolds for the self-assembly of versatile DNA origami. Finally, circDNA has been implemented as theranostic aptamers, miRNA inhibitors, as well as clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) gene editing donors. In this review article, we will discuss the chemistry, characteristic properties, and the theranostic applications of circDNA (excluding double-stranded circular DNA such as plasmids); we will also envision the challenges and opportunities in this research field.

Toward a complete cure for chronic hepatitis B: Novel therapeutic targets for hepatitis B virus.

Hepatitis B virus (HBV) affects approximately 250 million patients worldwide, resulting in the progression to cirrhosis and hepatocellular carcinoma, which are serious public health problems. Although universal vaccination programs exist, they are only prophylactic and not curative. In the HBV life cycle, HBV forms covalently closed circular DNA (cccDNA), which is the viral minichromosome, in the nuclei of human hepatocytes and makes it difficult to achieve a complete cure with the current nucleos(t)ide analogs and interferon therapies. Current antiviral therapies rarely eliminate cccDNA; therefore, lifelong antiviral treatment is necessary. Recent trials for antiviral treatment of chronic hepatitis B have been focused on establishing a functional cure, defined by either the loss of hepatitis B surface antigen, undetectable serum HBV DNA levels, and/or seroconversion to hepatitis B surface antibody. Novel therapeutic targets and molecules are in the pipeline for early clinical trials aiming to cure HBV infection. The ideal strategy for achieving a long-lasting functional or complete cure might be using combination therapies targeting different steps of the HBV life cycle and immunomodulators. This review summarizes the current knowledge about novel treatments and combination treatments for a complete HBV cure.