Volume 13, Issue 2, Pages 411-421 (February 2006) Effective Inhibition of HBV Replication in Vivo by Anti-HBx Short Hairpin RNAs  Sergio Carmona, Abdullah Ely, Carol Crowther, Naazneen Moolla, Felix H. Salazar, Patricia L. Marion, Nicolas Ferry, Marc S. Weinberg, Patrick Arbuthnot  Molecular Therapy  Volume 13, Issue 2, Pages 411-421 (February 2006) DOI: 10.1016/j.ymthe.2005.10.013 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 HBV target sites and shRNA-encoding vectors. (A) Organization of the hepatitis B virus genome showing sites targeted by shRNA sequences. Coordinates of the genome are given relative to the single EcoRI restriction site. Partially double-stranded HBV DNA comprises + and − strands with cohesive complementary 5′ ends. The cis elements that regulate HBV transcription are represented by the circular and rectangular symbols. Immediately surrounding arrows indicate the viral open reading frames (with initiation codons) that encompass the entire genome. Four outer arrows indicate the HBV transcripts, which have common 3′ ends that include HBx. (B) (Top) Schematic illustration of anti-HBV shRNA indicating mismatches in the sense strand, miRNA 23 loop, and sequence of two U residues that are derived from the transcription termination signal. The DNA cassette with U6 promoter, sense, miRNA 23 loop (L), and antisense-encoding sequences is indicated below. Molecular Therapy 2006 13, 411-421DOI: (10.1016/j.ymthe.2005.10.013) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 shRNA-mediated inhibition of HBsAg secretion and HBV–eGFP fusion marker protein expression in transfected cells. (A) Measurement of HBsAg secretion from Huh7 cells cotransfected with indicated shRNA-encoding plasmids together with HBV target plasmid. HBsAg measurements from quantitative ELISA are given as a normalized mean relative to the mock-treated cells. Results are from four independent transfections and the bars indicate the standard error of the mean (SEM). (B) Schematic illustration of plasmid construct pCH-eGFP showing open reading frames, respective transcripts, and sites targeted by shRNAs. The disrupted polymerase ORF is not indicated. Representative fluorescence microscopy fields of Huh7 cells transfected with pCH-eGFP and indicated shRNA-expressing construct are also shown. (C) Quantitative comparison of the percentage of eGFP-positive Huh7 cells detected using flow cytometry after transfection with indicated shRNA-encoding expression vectors. Number of eGFP-positive cells is given as a normalized mean relative to the mock-treated cells, which represents approximately 45% eGFP-positive cells in the total population. Results are depicted as means from four independent transfections (each counting 100,000 events) with the SEM indicated. Molecular Therapy 2006 13, 411-421DOI: (10.1016/j.ymthe.2005.10.013) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 RNA analysis. (A) Huh7 hepatocytes were transfected with the indicated shRNA-encoding plasmids. Two days after transfection, total RNA was extracted from the cells and analyzed by Northern blot hybridization. The control lane represents analysis of RNA extracted from cells that had been transfected with a plasmid encoding shRNA 5 sequences under transcriptional regulation of the Pol II CMV immediate early promoter/enhancer. Blots were probed for HBV RNA and also GAPDH as a loading control. (B and C) Either radiolabeled HBV− or radiolabeled HBV+ oligonucleotides targeting the relevant sequences of shRNA 5 or shRNA 10 were hybridized to total RNA extracted from transfected Huh7 cells and then subjected to primer extension analysis using reverse transcriptase. Sizes of extended fragments were then detected using autoradiography after resolution with denaturing PAGE. The amount of template cellular RNA subjected to reverse transcription is indicated above the lanes and RNA molecular weight marker sizes (nt) are indicated on the left of the autoradiographs. Molecular Therapy 2006 13, 411-421DOI: (10.1016/j.ymthe.2005.10.013) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Effects of shRNA sequences on HBV antigen production in the hydrodynamic injection model of HBV replication. (A) Time course of relative serum HBsAg concentrations, measured using quantitative ELISA, in mice treated with hydrodynamic injection. Graphical depictions of individual measurements of the viral antigen are given on a log scale for four mock- and four U6 shRNA 5-treated animals. (B) Mice subjected to the hydrodynamic injection procedure were sacrificed after 4 days and the livers analyzed using immunohistochemistry to detect the HBcAg. Representative low- and high-power fields are shown for livers from mock- and U6 shRNA 5-treated animals. Frozen sections from the same animals were stained for LacZ activity to confirm similar delivery of DNA to hepatocytes. (C) Serum HBsAg concentrations and (D) viral loads on day 4 after injection of mice using the hydrodynamic injection procedure. Mice were injected with the indicated shRNA-encoding plasmids or synthetic RNA duplex equivalent to shRNA 5 (siRNA 5) together with pCH/9-3091 HBV target DNA. Mock-treated animals received backbone plasmid (pGEM-T Easy) without shRNA-encoding sequences. Viral loads were determined using real-time quantitative PCR and included EuroHep standards to calibrate the virion copy numbers. Groups comprised six to eight animals and the graphs indicate the mean and SEM for each group. Molecular Therapy 2006 13, 411-421DOI: (10.1016/j.ymthe.2005.10.013) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Effects of recombinant adenovirus-mediated shRNA delivery on viral replication markers in the HBV transgenic mouse model. (A) Time course of relative mouse serum HBsAg concentrations, measured using quantitative ELISA, after tail vein injection of indicated recombinant adenovirus vectors. Relative serum HBsAg concentrations over a period of 12 days are shown. Comparison of (B) serum HBeAg and (C) viral loads at day 12 after injection of animals with saline or indicated recombinant adenovirus. Molecular Therapy 2006 13, 411-421DOI: (10.1016/j.ymthe.2005.10.013) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions