title: [A minicircle DNA vector-mediated siRNA to stably suppress hepatitis B virus replication and expression].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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%.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China. lxm1926@hotmail.com
authors.forename: Xiaoman
authors.initials: X
authors.lastname: Liu
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Yang
authors.forename: Tao
authors.initials: T
authors.lastname: Feng
mesh_terms: D001483:Base Sequence; D004270:DNA, Circular / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006509:Hepatitis B / Q000628:therapy*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000502:physiology*; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics*; D014779:Virus Replication
chemical_list: D004270:DNA, Circular; D006514:Hepatitis B Surface Antigens; D034741:RNA, Small Interfering