O-3 DEVELOPMENT OF LENTIVIRAL VECTORS FOR INHIBITION OF HEPATITIS B VIRUS VIA SMALL INTERFERING RNA

Abstract

Introduction and Objectives

It is estimated that chronic hepatitis B virus (HBV) infection accounts for one million deaths/year due to cirrhosis and liver cancer. Currently, several drugs are used in the treatment of HBV; however, a complete cure is still controversial. The major challenge is the persistence of viral covalently closed circular DNA (cccDNA), as well as the ability of HBV to integrate into the host genome, which enables the infection's reactivation. Interfering RNA (RNAi) is a post-transcriptional mechanism of gene silencing and is a promising alternative for the treatment of chronic hepatitis B. We aimed to construct effective RNAi lentiviral vector to silencing HBV proteins (HBsAg, HBcAg, HBeAg) and pre-genomic RNA (pgRNA), via RNAi.

Materials and Methods

The silencing vector candidates targets overlapped Open Reading Frames (ORFs), allowing different viral proteins and the pgRNA to be silenced with a single RNAi. The efficiency of silencing by lentiviral vectors candidates used individually or in combination, have been assessed by quantification of HBV proteins by eletroquimioluminescence and quantification of HBV DNA during the post-transfection period by quantitative PCR.

Results

Three silencing vectors candidates were constructed and tested in silico to prevent off-target effects. Stability and secondary structures have also been tested. Huh7 cells were transfected with 1ug of purified HBV genome circular monomers (genotype A1) and 3 days later, infected with the first lentiviral candidate (siHBV-1), targeting S/Pol genes of HBV (108 TU/mL). From the third day post-infection, HBsAg became undetectable on cells infected by the lentiviral vectors, while untreated controls maintained viral protein expression (p<0.002). HBV DNA were also undetectable by PCR.

Conclusions

siHBV-1was able to silence HBV in vitro. This approach allows long- term, sustained knockdown of HBV replication and gene expression, which can effectively promote HBV clearance in chronic carriers.