1. Volume 22, Issue 2, Pages 303-311 (February 2014)
An Efficient Antiviral Strategy for Targeting Hepatitis B Virus Genome Using Transcription Activator-Like Effector Nucleases 
Jieliang Chen, Wen Zhang, Junyu Lin, Fan Wang, Min Wu, Cuncun Chen, Ye Zheng, Xiuhua Peng, Jianhua Li, Zhenghong Yuan 
Molecular Therapy 
Volume 22, Issue 2, Pages (February 2014)
DOI: /mt
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
TAL effector nucleases designed for specific targeting HBV genome. (a) Schematic representation of the HBV genome and the target sites of the TALENs. (b) Schematic of a pair of TALENs (L2/R2) bound to the target HBV DNA. HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Expression of TALENs in cells. (a) Huh7 cells transfected with the plasmids encoding indicated Flag-tagged TALENs were lysed 48 hours post-transfection and the lysates were analyzed by immunoblotting with monoclonal antibody against Flag. (b) Huh7 cells were co-transfected with the plasmids encoding TALENs along with pEGFP. Immunofluorescence assay was performed 48 hours post-transfection. (c) The viability of the control cells and the cells expressing TALENs was measured by CCK8 assay every day for 4 days. TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
The effect of TALENs on the production of viral proteins and pgRNA. (a–d) Huh7 cells were co-transfected with the linear full-length HBV DNA (genotype A) and the plasmids encoding indicated TALENs. Forty-eight hours post-transfection, the supernatants were collected to detect HBeAg (a) and HBsAg (b) by ELISA and the cells were harvested for western blotting to detect the HBcAg expression (c) or for RT real-time PCR analysis to identify the level of pgRNA (d). ELISA, enzyme-linked immunosorbent assay; HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Suppression of HBeAg and HBsAg production from different HBV genotypes by TALENs. (a–f) Huh7 cells were co-transfected with the plasmids encoding indicated TALENs and pHBV1.3-AF genotype B (a,d), pHBV1.3-AF genotype C (b,e), or pCMV-HBV-ayw-genotype D (c,f). Forty-eight hours post-transfection, the supernatants were collected to detect HBeAg and HBsAg by ELISA. ELISA, enzyme-linked immunosorbent assay; HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
TALENs result in a decreased cccDNA level and misrepaired cccDNA. Huh7 cells were co-transfected with the linear full-length HBV DNA and the plasmids encoding indicated TALENs. (a,b) The levels of intracellular cccDNA 48 hours post-transfection were analyzed by PCR (a) and real-time PCR (b) using primers for cccDNA quantitation. (c) The cccDNA was extracted from the control and TALENs-expressing cells as described in methods. The DNA segments encompassing the recognition sites of TALENs-L2/R2 were PCR-amplified, and the DNA amplicons were melted and annealed. The annealed DNA was treated with five units of the mismatch-sensitive T7 endonuclease 1 (T7E1) for 15 minutes at 37 °C and then assessed by agarose gel electrophoresis. (d) Huh7 cells were co-transfected with the linear full-length HBV DNA and indicated plasmids. Forty-eight hours post-transfection, the supernatants were collected to detect HBeAg by ELISA. ELISA, enzyme-linked immunosorbent assay; HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Evaluation of the anti-HBV activity of TALENs in a hydrodynamic-based mouse model. The mice were hydrodynamically-injected with the linear full-length HBV DNA and the control vector (n = 4) or the plasmids encoding TALENs-L2 and -R2 (n = 4). (a,b) The levels of serum HBeAg (a) and HBsAg (b) of the mice were determined by ELISA at 3 and 6 days post-transfection. (c,d) The levels of serum HBV DNA (c) and intrahepatic pgRNA (d) were measured by quantitative real-time PCR. (e) Lysates of the mice liver samples were analyzed by western blotting to detect the expression of Flag-TALENs and the HBcAg. HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Enhanced antiviral effect with combinations of TALENs and IFN treatment. (a–b) Huh7 cells were co-transfected with the linear full-length HBV DNA and the plasmids encoding indicated TALENs. 1,000 IU/ml IFN-α was added to the cells at 36 hours post-transfection. At 72 hours post-transfection, the cells were extracted for pgRNA quantitation (a), or the cells from indicated group were lysed for cccDNA extraction followed by real-time PCR using primers for cccDNA quantitation (b). (c–d) Huh7 cells were co-transfected with the linear full-length HBV DNA and the plasmids encoding L2/R2 or the control vectors. 1,000 IU/ml IFN-α was added to the cells at 36 hours post-transfection. At 72 hours post-transfection, the cells were fixed to perform immunofluorescence staining (c) and the supernatants of the cells were collected to detect HBeAg by ELISA (d). (e) Huh7 cells co-transfected with pISRE-Luc and the indicated plasmids were treated with 1,000 IU/ml IFN-α- or mock-treated for 12 hours at 48 hours post-transfection and then harvested for the luciferase assay using the Dual-Luciferase Assay Kit (Promega). ELISA, enzyme-linked immunosorbent assay; HBV, hepatitis B virus; TALEN, transcription activator-like effector nucleases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions