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

Presence of sodium taurocholate co-transporting polypeptide and Hepatitis B replication marker on placenta: Another home for the virus

Background: The role of sodium taurocholate co-transporting polypeptide (NTCP), in facilitating the binding of Hepatitis B virus (HBV) on surface of hepatocytes is well documented. Expression of NTCP in extra hepatic cells may make these cells susceptible to HBV infection and support cellular proliferation akin to hepatocytes. Placental replication of HBV is not well explored. In this study we have assessed the expression of NTCP and HBV replication markers (HBeAg, HBcAg, and HBV DNA) in placental cells, to investigate if these cells act as host for HBV. Methods: Fourty one HBsAg+ve pregnant women along with 10 healthy controls were enrolled after obtaining informed consent. The HBV DNA in placenta was detected by qPCR using primers for X and core ORF. Expression of NTCP in placenta was analyzed by qRT-PCR and further investigated by immunohistochemistry (IHC) along with HBV replication biomarkers, HBeAg, and HBcAg. Results: HBsAg positive subjects were divided in two groups on the basis of viral load [High Viral Load (HVL) Group; viral load ≥2000IU/ml, Low Viral Load (LVL) Group; viral load <2000IU/ml] according to INASL guidelines 2018. HBV infected females showed increased expression of NTCP in trophoblasts of placenta compared to control group (HVL 3.69,SE 0.13 Vs Control 1.74,SE 0.15, p=0.0117). Furthermore, significant difference in NTCP expression was also observed between HVL and LVL group (HVL 3.69,SE 0.13 Vs LVL 1.98,SE 0.17, p=0.022) and positively correlated with the maternal HBV DNA load. Membranous and/or cytoplasmic immunostaining of NTCP, and cytoplasmic staining of HBeAg and HBcAg in trophoblasts along with presence of HBV DNA indicated that trophoblasts are not only susceptible to HBV infection but may also be a site for viral replication. Conclusions: This is the pioneer study, which demonstrates expression of NTCP on placenta which may facilitate the entry of HBV. Furthermore, the study establishes the presence of HBeAg in placenta of patients without circulating HBeAg, indicating these cells may act as replication host/reservoir. This pioneering finding hints at the possibility of exploring the potential of NTCP blocking strategies in preventing vertical transmission of HBV.

The Association of Hepatitis B Virus Replication During Pregnancy with Perinatal Outcomes: A Retrospective Cohort Study

Background The effect of hepatitis B virus (HBV) replication during pregnancy on the outcomes of singleton pregnancies is not fully understood. In this study, we investigated the association between HBV replication and poor maternal and infant outcomes. Methods We retrospectively analyzed the clinical data of 836 pregnant inpatients with hepatitis B surface antigen positivity who delivered at two provincial tertiary grade A hospitals in Fujian Province from June 2016 to October 2020. The patients were divided into the HBV replication (n = 283) and non-HBV replication groups (n = 553). Chi-squared test of adverse maternal and infant outcomes was performed using SPSS 26.0 software, and univariate analysis of variance of basic clinical indexes of pregnant women and newborns was performed. P<0.05 was considered statistically significant. Results The incidences of perinatal outcomes of intrahepatic cholestasis of pregnancy, hypertensive syndrome complicating pregnancy, gestational diabetes mellitus, preterm birth, macrosomia, growth restriction, and vaginal infection in the HBV and non-HBV replication groups were not significantly different (P>0.05); however, there were significant differences between the two groups in the rate of cesarean section (53.8% vs. 45.0%; P=0.017) and neonatal jaundice (15.5% vs. 7.2%; P=0.000). After using propensity score analysis and multivariable modeling to adjust for glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels in the two groups, the replication group was found to have an increased risk for cesarean section (54.3% vs. 33.5%; P=0.000) and vaginal infection (3% vs. 0.4%; P=0.038), and their infants had a higher rate of newborn jaundice (16% vs.1.5%; P=0.000). Conclusion The findings provide further understanding of the association between maternal HBV replication status and perinatal outcomes. Pregnant women with viral replication have an increased risk of vaginal infection and cesarean section, and their infants appear to be at a higher risk for neonatal jaundice.

Hepatocyte Growth Factor-Dependent Antiviral Activity of Activated cdc42-Associated Kinase 1 Against Hepatitis B Virus

Activated cdc42-associated kinase 1 (ACK1) is a well-known non-receptor tyrosine kinase that regulates cell proliferation and growth through activation of cellular signaling pathways, including mitogen-activated protein kinase (MAPK). However, the anti-HBV activity of ACK1 has not been elucidated. This study aimed to investigate the role of ACK1 in the HBV life cycle and the mechanism underlying the anti-HBV activity of ACK1. To examine the antiviral activity of ACK1, we established HepG2-ACK1 cells stably overexpressing ACK1. The HBV life cycle, including HBeAg/HBsAg secretion, HBV DNA/transcription, and enhancer activity, was analyzed in HepG2 and HepG2-ACK1 cells with HBV replication-competent HBV 1.2mer (HBV 1.2). Finally, the anti-HBV activity of ACK1 was examined in an HBV infection system. ACK1 suppressed HBV gene expression and transcription in HepG2 and HepG2-ACK1 cells. Furthermore, ACK1 inhibited HBV replication by decreasing viral enhancer activity. ACK1 exhibited its anti-HBV activity via activation of Erk1/2, which consequently downregulated the expression of HNF4α binding to HBV enhancers. Furthermore, hepatocyte growth factor (HGF) induced ACK1 expression at an early stage. Finally, ACK1 mediated the antiviral effect of HGF in the HBV infection system. These results indicated that ACK1 induced by HGF inhibited HBV replication at the transcriptional level by activating the MAPK-HNF signaling pathway. Our findings suggest that ACK1 is a potentially novel upstream molecule of MAPK-mediated anti-HBV activity.

Virtual Docking of VBP1 with HBX and NFκB Protein to Study the Activation of NFκB in the Regulatory Mechanism of Liver Cancer

Molecular docking is an efficient way to study protein-protein and protein-ligand interactions in virtual mode, this provides structural annotations of molecular interactions, required in the drug discovery process. The Cartesian FFT approach in ‘Hex’ spherical polar Fourier (SPF) uses rotational correlations, this method is used here to study protein-protein interactions. Hepatitis B virus (HBV) X protein (HBx) is essential for virus infection and has been used in the development of therapeutics for liver cancer. It can interact with many cellular proteins. It interferes with cell viability and stimulates HBV replication. The von Hippel-Lindau binding protein 1(VBP1) has an important role in HBx-mediated nuclear factor kappa B (NFkB) stimulation. VBP1 and HBx function as coactivators in the activation of NFκB binding. Docking results revealed that HBx and NFkB bind with VBP1 at the common site on amino acids positions Arg 161, Glu 92, and Arg 82, which may have a role in HBx-mediated NFκB activation. Lowest energy complex VBP1- NFkB1 was obtained at -883.70 Kcal/mol. The amino acids involved in interaction among HBx, VBP1, and NFκB proteins, may be involved in transcriptional regulation and has significance in normal and abnormal regulation. These amino acid interactions may be associated with the manifestation of Liver cancer.

The HBV Core Protein and Core Particle Both Bind to the PPiase Par14 and Par17 to Enhance Their Stabilities and HBV Replication

We recently reported that the PPIase Par14 and Par17 encoded by PIN4 upregulate HBV replication in an HBx-dependent manner by binding to conserved arginine–proline (RP) motifs of HBx. HBV core protein (HBc) has a conserved 133RP134 motif; therefore, we investigated whether Par14/Par17 bind to HBc and/or core particles. Native agarose gel electrophoresis (NAGE) and immunoblotting and co-immunoprecipitation were used. Chromatin immunoprecipitation from HBV-infected HepG2-hNTCP-C9 cells was performed. NAGE and immunoblotting revealed that Par14/Par17 bound to core particles and co-immunoprecipitation revealed that Par14/Par17 interacted with core particle assembly-defective, and dimer-positive HBc-Y132A. Thus, core particles and HBc interact with Par14/Par17. Par14/Par17 interacted with the HBc 133RP134 motif possibly via substrate-binding E46/D74 and E71/D99 motifs. Although Par14/Par17 dissociated from core particles upon heat treatment, they were detected in 0.2 N NaOH-treated opened-up core particles, demonstrating that Par14/Par17 bind outside and inside core particles. Furthermore, these interactions enhanced the stabilities of HBc and core particles. Like HBc-Y132A, HBc-R133D and HBc-R133E were core particle assembly-defective and dimer-positive, demonstrating that a negatively charged residue at position 133 cannot be tolerated for particle assembly. Although positively charged R133 is solely important for Par14/17 interactions, the 133RP134 motif is important for efficient HBV replication. Chromatin immunoprecipitation from HBV-infected cells revealed that the S19 and E46/D74 residues of Par14 and S44 and E71/D99 residues of Par17 were involved in recruitment of 133RP134 motif-containing HBc into cccDNA. Our results demonstrate that interactions of HBc, Par14/Par17, and cccDNA in the nucleus and core particle–Par14/Par17 interactions in the cytoplasm are important for HBV replication.

Hepatitis C virus core protein inhibits hepatitis B virus replication by downregulating HBx levels via Siah-1-mediated proteasomal degradation during coinfection

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the mechanism remains unclear. Here, we found that HCV core protein inhibits HBV replication by downregulating HBx levels during coinfection in human hepatoma cells. For this effect, HCV core protein increased reactive oxygen species levels in the mitochondria and activated the ataxia telangiectasia mutated-checkpoint kinase two pathway in the nucleus, resulting in an upregulation of p53 levels. Accordingly, HCV core protein induced p53-dependent activation of seven in absentia homolog one expression, an E3 ligase of HBx, resulting in the ubiquitination and proteasomal degradation of HBx. The effect of the HCV core protein on HBx levels was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, providing evidence for the inhibition of HBV replication by HCV core protein. The present study may provide insights into the mechanism of HCV dominance in HBV- and HCV-coinfected patients.

ATM-Dependent Phosphorylation of Hepatitis B Core Protein in Response to Genotoxic Stress

Chronic hepatitis caused by infection with the Hepatitis B virus is a life-threatening condition. In fact, 1 million people die annually due to liver cirrhosis or hepatocellular carcinoma. Recently, several studies demonstrated a molecular connection between the host DNA damage response (DDR) pathway and HBV replication and reactivation. Here, we investigated the role of Ataxia-telangiectasia-mutated (ATM) and Ataxia telangiectasia and Rad3-related (ATR) PI3-kinases in phosphorylation of the HBV core protein (HBc). We determined that treatment of HBc-expressing hepatocytes with genotoxic agents, e.g., etoposide or hydrogen peroxide, activated the host ATM-Chk2 pathway, as determined by increased phosphorylation of ATM at Ser1981 and Chk2 at Thr68. The activation of ATM led, in turn, to increased phosphorylation of cytoplasmic HBc at serine-glutamine (SQ) motifs located in its C-terminal domain. Conversely, down-regulation of ATM using ATM-specific siRNAs or inhibitor effectively reduced etoposide-induced HBc phosphorylation. Detailed mutation analysis of S-to-A HBc mutants revealed that S170 (S168 in a 183-aa HBc variant) is the primary site targeted by ATM-regulated phosphorylation. Interestingly, mutation of two major phosphorylation sites involving serines at positions 157 and 164 (S155 and S162 in a 183-aa HBc variant) resulted in decreased etoposide-induced phosphorylation, suggesting that the priming phosphorylation at these serine-proline (SP) sites is vital for efficient phosphorylation of SQ motifs. Notably, the mutation of S172 (S170 in a 183-aa HBc variant) had the opposite effect and resulted in massively up-regulated phosphorylation of HBc, particularly at S170. Etoposide treatment of HBV infected HepG2-NTCP cells led to increased levels of secreted HBe antigen and intracellular HBc protein. Together, our studies identified HBc as a substrate for ATM-mediated phosphorylation and mapped the phosphorylation sites. The increased expression of HBc and HBe antigens in response to genotoxic stress supports the idea that the ATM pathway may provide growth advantage to the replicating virus.

Advances in designing Adeno-associated viral vectors for development of anti-HBV gene therapeutics

Despite the five decades having passed since discovery of the hepatitis B virus (HBV), together with development of an effective anti-HBV vaccine, infection with the virus remains a serious public health problem and results in nearly 900,000 annual deaths worldwide. Current therapies do not eliminate the virus and viral replication typically reactivates after treatment withdrawal. Hence, current endeavours are aimed at developing novel therapies to achieve a functional cure. Nucleic acid-based therapeutic approaches are promising, with several candidates showing excellent potencies in preclinical and early stages of clinical development. However, this class of therapeutics is yet to become part of standard anti-HBV treatment regimens. Obstacles delaying development of gene-based therapies include lack of clinically relevant delivery methods and a paucity of good animal models for preclinical characterisation. Recent studies have demonstrated safety and efficiency of Adeno-associated viral vectors (AAVs) in gene therapy. However, AAVs do have flaws and this has prompted research aimed at improving design of novel and artificially synthesised AAVs. Main goals are to improve liver transduction efficiencies and avoiding immune clearance. Application of AAVs to model HBV replication in vivo is also useful for characterising anti-HBV gene therapeutics. This review summarises recent advances in AAV engineering and their contributions to progress with anti-HBV gene therapy development.

Quantitative HBsAg versus HBV DNA in Predicting Significant Hepatitis Activity of HBeAg-Positive Chronic HBV Infection

(1) Background: As specialparameters in predicting significant hepatitis activity of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B virus (HBV) infection, the quantitative standard of HBV DNA has not been agreed and that of hepatitis B surface antigen(HBsAg) has not been formed. Our objective is to evaluate the validity of HBsAg and HBV DNA in predicting the significant hepatitis activity of HBeAg-positive patients. (2) Methods: A population of 516 patients with HBeAg-positive chronic HBV infection was enrolled. Serum ALT was measured using an Abbott Architect c16000 autoanalyzer; diagnoses of liver pathological grade and stage referred to the Scheuer standard. Three levels of significant hepatitis activity were preset, which were successively “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1”, “ALT ≥ 30 IU/L or Grade > G1 or Stage > S1” and “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1”. (3) Results: A subpopulation of 288 patients with possible high HBV replication was selected based on locally weighted scatterplot smoothing regression curves between ALT and HBsAg, HBeAg and HBV DNA. In the subpopulation with possible high HBV replication, areas under receiver operating characteristic curves of HBsAg for predicting the three levels of significant hepatitis activity were successively 0.868, 0.839 and 0.789, which were all significantly greater than those of HBV DNA, as those were successively 0.553, 0.550 and 0.574 (p = 0.0002, p < 0.0001 and p < 0.0001). With the standard of HBsAg ≤ 4.699 log10 IU/mL, the sensitivity and specificity of HBsAg for predicting the three levels of significant hepatitis activity were successively 75.81% and 81.82%, 79.23% and 78.57% and 80.82% and 67.44%. (4) Conclusion: Quantitative HBsAg instead of HBV DNA is valuable in predicting significant hepatitis activity of HBeAg-positive chronic HBV infection.