Inhibition of Hepatitis B Virus Replication by Helper Dependent Adenoviral Vectors Expressing Artificial Anti-HBV Pri-miRs from a Liver-Specific Promoter
Figure 3
Liver specificity of MTTR promoter, RNA processed from anti-HBV pri-miR cassettes, and effect on HBsAg concentration in supernatants of cultured Huh7 cells. (a) Liver-derived Huh7 or kidney-derived HEK293 cells were transfected with 100 ng of pMTTR-FLuc or pCMV-FLuc, which contained the Firefly luciferase reporter gene under control of MTTR or CMV promoters, respectively. In addition the phRL-CMV (Promega) plasmid, constitutively expressing Renilla luciferase, was cotransfected to normalize the Firefly luciferase measurements. Forty-eight hours after transfection cells were harvested and activity of the two reporter genes was determined independently. Mean Firefly to Renilla luciferase activity is presented and error bars indicate the standard error of the mean (). (b) Northern blot analyses of 30 μg RNA isolated from Huh 7 cells infected with HD Ads at a multiplicity of infection (MOI) of 100 infectious viral particles per cell. The probes used were complementary to the predicted 5, 8, or 9 guide sequences derived from the panel of vectors expressing antiviral pri-miRs from the MTTR promoter. Equal loading of the lanes was verified by stripping and reprobing the blot with a labelled oligonucleotide complementary to U6 small nuclear RNA. Putative guide (G) and precursor (P) bands are indicated. (c) Inhibition of HBV replication in vitro was determined by measuring HBsAg levels using ELISA. Cell culture supernatants were obtained from Huh7 cells that had been transfected with the pCH-9/3091 replication-competent plasmid and then infected with the indicated HD Ads at MOIs of 100, 250, 500, or 1000. Data is expressed as SEM from three replicates. The statistical significance was calculated using a pair-wise comparison according to the Student t-test. values less than 0.05 () or less than 0.01 () were considered statistically significant.
