CRISPR/Cas9 delivery by NIR-responsive biomimetic nanoparticles for targeted HBV therapy

Background Currently, there are no curative drugs for hepatitis B virus (HBV). Complete elimination of HBV covalently closed circular DNA (cccDNA) is key to the complete cure of hepatitis B virus infection. The CRISPR/Cas9 system can directly destroy HBV cccDNA. However, a CRISPR/Cas9 delivery system with low immunogenicity and high efficiency has not yet been established. Moreover, effective implementation of precise remote spatiotemporal operations in CRISPR/Cas9 is a major limitation. Results In this work, we designed NIR-responsive biomimetic nanoparticles (UCNPs-Cas9@CM), which could effectively deliver Cas9 RNP to achieve effective genome editing for HBV therapy. HBsAg, HBeAg, HBV pgRNA and HBV DNA along with cccDNA in HBV-infected cells were found to be inhibited. These findings were confirmed in HBV-Tg mice, which did not exhibit significant cytotoxicity and minimal off-target DNA damage. Conclusions The UCNPs-based biomimetic nanoplatforms achieved the inhibition of HBV replication via CRISPR therapy and it is a potential system for efficient treatment of human HBV diseases. Graphical Abstract

Mechanism of Hepatitis B Virus cccDNA Formation

Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.

SIRT7 restricts HBV transcription and replication through catalyzing desuccinylation of histone H3 associated with cccDNA minichromosome

Chronic HBV infection is a significant public health burden worldwide. HBV cccDNA organized as a minichromosome in nucleus is responsible for viral persistence and is the key obstacle for a cure of chronic hepatitis B. Recent studies suggest cccDNA transcription is epigenetically regulated by histone modifications, especially histone acetylation and methylation. In this study, we identified transcriptionally active histone succinylation (H3K122succ) as a new histone modification on cccDNA minichromosome by using cccDNA ChIP-Seq approach. SIRT7, as a NAD+-dependent histone desuccinylase, could bind to cccDNA through interaction with HBV core protein where it catalysed histone H3K122 desuccinylation. Moreover, SIRT7 acts cooperatively with histone methyltransferase SUV39H1 and SETD2 to induce silencing of HBV transcription through modulation of chromatin structure. Our data improved the understanding of histone modifications of the cccDNA minichromosome, thus transcriptional silencing of cccDNA may represent a novel antiviral strategy for the prevention or treatment of HBV infection.

Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis

Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA is the key molecule responsible for viral persistence within infected hepatocytes. The evaluation of HBV cccDNA is crucial for the management of patients with chronic HBV infection and for the personalization of treatment. However, the need for liver biopsy is the principal obstacle for the assessment of intrahepatic HBV cccDNA. In the last decade, several studies have investigated the performance of hepatitis B core-related antigen (HBcrAg) as a surrogate of HBV cccDNA amount in the liver. In this meta-analysis, we collected 14 studies (1271 patients) investigating the correlation between serum HBcrAg and intrahepatic HBV cccDNA. Serum HBcrAg showed a high correlation with intrahepatic HBV cccDNA (r = 0.641, 95% confidence interval (CI) 0.510–0.743, p < 0.001). In a head-to-head comparison, we observed that the performance of HBcrAg was significantly superior to that of hepatitis B surface antigen (r = 0.665 vs. r = 0.475, respectively, p < 0.001). Subgroup analysis showed that the correlation between HBcrAg and intrahepatic HBV cccDNA was high, both in hepatitis B e antigen-positive and -negative patients (r = 0.678, 95% CI 0.403–0.840, p < 0.001, and r = 0.578, 95% CI 0.344–0.744, p < 0.001, respectively). In conclusion, the measurement of serum HBcrAg qualifies as a reliable non-invasive surrogate for the assessment of an intrahepatic HBV cccDNA reservoir.

Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus

ABSTRACTWorldwide, ∼250 million people are chronically infected with the hepatitis B virus (HBV) and are at increased risk of cirrhosis and hepatocellular carcinoma. The HBV persists as covalently closed circular DNA (cccDNA), which acts as the template for all HBV mRNA transcripts. Nucleos(t)ide analogs do not directly target the HBV cccDNA and cannot eradicate the HBV. We have discovered a unique structural motif, a G-quadruplex in HBV’s pre-core promoter region that is conserved amongst nearly all genotypes, and is central to critical steps in the viral life-cycle including the production of pre-genomic RNA, core and polymerase proteins, and encapsidation. Thus, an increased understanding of the HBV pre-core may lead to the development of novel anti-HBV cccDNA targets. We utilized biophysical methods to characterize the presence of the G-quadruplex, employed assays using a known quadruplex-binding protein (DHX36) to pull-down HBV cccDNA, and compared HBV infection in HepG2 cells transfected with wild-type and mutant HBV plasmids. This study provides insights into the presence of a G-quadruplex in the HBV pre-core promoter region essential for HBV replication. The evaluation of this critical host-protein interaction site in the HBV cccDNA may ultimately facilitate the development of novel anti-HBV therapeutics against the resilient cccDNA template.

A polypeptide D-TTK001 targeting HBx controls cccDNA against HBV

Chronic infection of HBV is a serious global health issue. However, the HBV covalently closed circular DNA (cccDNA) cannot be completely cleaned in liver. Here, we engineer a novel polypeptide, termed D-TTK001, targeting HBV X protein (HBx) against HBV cccDNA. D-TTK001 is a D-type polypeptide, which has a high binding affinity to HBx. Interestingly, it is able to against HBV efficiently in primary human hepatocytes (PHHs) from three donors. D-TTK001 displays high activities against HBV, remarkably reducing the levels of cccDNA in the HBV-infected human liver-chimeric mice. Mechanically, D-TTK001 depresses cccDNA transcription by regulating the epigenetic modification of cccDNA minichromosome and disturbing the interaction of HBx with DDB1 in the cells. D-TTK001 decreases the levels of cccDNA by down-regulating MSL2, leading to the increase of APOBEC3B in liver. When combined with entecavir or interferon-α, D-TTK001 shows additive antiviral effects. Clinically, D-TTK001 is a strong candidate to clear cccDNA against HBV.

3D landscape of Hepatitis B virus interactions with human chromatins

Hepatitis B viral (HBV) DNAs, including covalently closed circular DNA (cccDNA) and integrated HBV DNA forms, are considered to be primary contributors to the development and progression of HBV-associated liver diseases. However, it remains largely unclear how HBV DNAs communicate with human chromatin. Here we employed a highly sensitive technology, 3C-high-throughput genome-wide translocation sequencing (3C-HTGTS), to globally identify HBV DNA–host DNA contacts in cellular models of HBV infection. HBV DNA does not randomly position in host genome but instead preferentially establishes contacts with the host DNA at active chromatin regions. HBV DNA–host DNA contacts are significantly enriched at H3K4me1-marked regions modified by KMT2C/D; this histone modification is also observed in the HBV cccDNA mini-chromosome and strongly influences HBV transcription. On the other hand, chromatin loop formed by integrated HBV DNA with host genomic DNA was found in transcriptionally active regions. Furthermore, HBV infection influences host gene expression accompanied with HBV DNA–host DNA contacts. Our study provides a 3D landscape of spatial organization of cccDNA and integrated HBV DNA within the human genome, which lays the foundation for a better understanding of the mechanisms how HBV involves in liver disease development and progression.