Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes

Abstract

e Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/ cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection. Humans are the only natural hosts of hepatitis B virus (HBV), and hepatocytes are the only recognized cells that support productive HBV infection in vivo (1). Viral gene transcription and replication in hepatic cells have been extensively studied (1), and high levels of infectious HBV virions can be produced in cultured cells with relative ease (2). The extremely restricted host and hepatocyte tropism of HBV infection, as well as the possibility of obtaining large amounts of virus in vitro, makes HBV an ideal candidate for the development of hepatocyte-targeting delivery vectors. HBV-based vectors are also invaluable for the study of HBV infection mechanisms.The highly compact HBV genome contains four overlapping open reading frames (ORFs) (preC/C, P, preS1/preS2/S, and X) (Fig. 1A). Multiple essential cis elements overlap these ORFs and function at the DNA or RNA level during different stages of the viral life cycle. Mature virions contain partially double-stranded, relaxed circular DNA (rcDNA) genomes. Upon infection of hepatocytes, rcDNA genomes are converted into covalently closed circular DNA (cccDNA), which serves as a transcription template for viral RNA species. Viral pregenomic RNA (pgRNA), which also functions as mRNA for polymerase, is bound by newly translated polymerase, preferentially in cis. The pgRNA-polymerase complex is then packaged by viral core proteins, also translated using pgRNA as mRNA. Reverse transcription and synthesis of rcDNA take place within the capsids. Mature capsids are subsequently enveloped by membranes containing viral large/middle/small (L/ M/S) surface proteins, encoded by the preS1/preS2/S ORF, to produce progeny virions that bud into the endoplasmic reticulum (ER) lumen to be secreted (1).The compact nature of HBV genome ...

Adenoviral Delivery of Recombinant Hepatitis B Virus Expressing Foreign Antigenic Epitopes for Immunotherapy of Persistent Viral Infection

“…Newly synthesized HBsAg was detected in culture supernatants 8 to 10 days later (Fig. 3A) (18). In line with this finding, immunofluorescent staining on day 10 postinfection revealed the foreign-antigenic rHBc, as well as HBsAg, in the cytoplasm.…”

“…Huh-7 cells constitutively expressing the HBV core antigen were generated by lentiviral vector transduction. Primary tupaia hepatocytes (PTHs) were isolated by a twostep perfusion protocol (17,18). For viral infection, 1 ϫ 10 6 freshly isolated PTHs were seeded in collagen-coated 6-well plates (BD Biosciences, USA); 1 day after seeding, the PTHs were inoculated with wtHBV isolated from the infectious sera of HBV-infected patients or rHBV virions concentrated from culture supernatants by polyethylene glycol 8000.…”

Establishment of an inducible HBV stable cell line that expresses cccDNA-dependent epitope-tagged HBeAg for screening of cccDNA modulators

“…However, due to the lack of specific and robust assays for direct measurement of low copy number cccDNA in HBV replicating cells, a cccDNA reporter system is thus needed for identification of cccDNA inhibitors in high throughput screening (Guo and Guo, 2015). Although recombinant HBV reporter viruses have been reported to support cccDNA-dependent infection, replacement or insertion of reporter gene in HBV genome will affect certain step(s) of virus replication and normally require transcomplementation of one or more viral proteins to make the infectious reporter viruses, resulting in decreases in the efficiency of replication or virion formation (Hong et al, 2013; Protzer et al, 1999; Untergasser and Protzer, 2004). In addition, infectious HBV reporter virus systems reproduce only the initial round of infection, and the current HBV in vitro infection systems require large amount of virus inoculum, but still exhibit relatively low infectivity and high variability (Yan et al, 2015a).…”

Intracellular and Organic miRNA In Situ Detection

“…The nonviral vectors show less efficiency compared to viral vector in delivering the gene probe and preventing it from degradation of the cell autoimmunity, but good biocompatibility and large-scale production of nonviral vectors make it promising in gene therapy research [4]. Various nonviral gene vectors such as liposomes, [5] cationic polymers, [6] dendritic polymers, [7] polymeric peptides [8], and nanoparticles [9][10][11] have been continuously explored for gene therapy.…”