Abstract
BackgroundChronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. The major challenges for current therapies are the low efficacy of current drugs and the occurrence of drug resistant HBV mutations. RNA interference (RNAi) of virus-specific genes offers the possibility of developing a new anti-HBV therapy. Recent reports have shown that lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Herein, a lentivirus-based RNAi system was developed to drive expression and delivery of HBV-specific short hairpin RNA (shRNA) in a mouse model for HBV replication.MethodsHepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in the sera of the mice were analyzed by quantitative sandwich enzyme linked immunosorbent assay (ELISA) technique, hepatitis B core antigen (HBcAg) and HBsAg in the livers of the mice were detected by immunohistochemical assay, HBV DNA and HBV mRNA were measured by fluorogenic quantitative polymerase chain reaction (FQ-PCR) and quantitative real-time PCR respectively.ResultsCo-injection of HBV plasmids together with the lentivirus targeting HBV shRNA induced an RNAi response. Secreted HBsAg was reduced by 89% in mouse serum, and HBeAg was also significantly inhibited, immunohistochemical detection of HBcAg or HBsAg in the liver tissues also revealed substantial reduction. Lentiviral mediated shRNA caused a significant suppression in the levels of viral mRNA and DNA synthesis compared to the control group.ConclusionLentivirus-based RNAi can be used to suppress HBV replication in vivo, it might become a potential therapeutic strategy for treating HBV and other viral infections.
Highlights
Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma
Recent studies have shown that RNA interference (RNAi), which can be induced in mammalian cells by short hairpin RNAs, is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNAs by sequence-specific post-transcriptional silencing of homologous genes[8]
Selection of RNAi targets sites According to previous studies [27], two short hairpin RNA (shRNA) target sequences were chosen on the basis of their conservation among the major HBV genotypes adr, adw, ayr and ayw
Summary
Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma. Recent studies have shown that RNA interference (RNAi), which can be induced in mammalian cells by short hairpin RNAs (shRNAs), is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNAs (dsRNAs) by sequence-specific post-transcriptional silencing of homologous genes[8] It is most important for the processing of primary miRs, which have important functions for the regulation of gene expression, and inhibits the replication of HBV [9,10,11]. The majority of target cell types for gene therapy are non-dividing or slowly dividing, such as hepatocytes[25] These properties of RNAi vector systems based on HIV or other lentiviruses open up a possibility of efficiently controlling replication processes of infectious viruses such as HBV, and have the potential to become important tools in clinical gene therapy
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