[A minicircle DNA vector-mediated siRNA to stably suppress hepatitis B virus replication and expression]

We used a minicircle DNA vector system to express small interfering RNA (siRNA) and studied the inhibition of hepatitis B virus (HBV) replication and gene expression in vitro.

siRNA targeting HBV S gene (siHBS) was designed , synthesized and cloned into a minicircle DNA vector pMC. BESPX-MCS2. After sequencing, we transformed the recombinant pMC-H1-siHBS-U6 into E. coli ZYCY10P3S2T, and induced the degradation of its bacterial backbone by adding L-arabinose into the bacterial growth medium. As expected, a minicircle RNA interference (RNAi) vector pmc-H1-siHBS-U6 was generated only consisting of gene expression cassette. Then pmc-H1-siHBS-U6 was co-transfected into Huh-7 cells with HBV expression vector pHBV1.3. ELISA and Real-time PCR were performed to evaluate the inhibition effect of the secretion of HBsAg and HBeAg and the levels of HBV DNA and mRNA in Huh-7 cells.

We Successfully established the minicircle-based RNAi vector pmc-H1-siHBS-U6, which can significantly inhibit the secretion of HBsAg and HBeAg in Huh-7 cells for two to three weeks. Real-time PCR results show that HBV DNA and mRNA levels were also down-regulated about 71% and 80%.

The minicircle DNA-based RNAi vector pmc-H1-siHBS-U6 can suppress HBV replication and gene expression specifically, efficiently and steadily. Thus, this study provided us a new siRNA delivery system and a new gene therapy strategy of HBV infection.