1. Volume 15, Issue 1, Pages 131-138 (January 2007)
Development of a Conditionally Replicating Pseudorabies Virus for HER-2/neu- overexpressing Bladder Cancer Therapy 
Ai-Li Shiau, Yu-Ping Lin, Gia-Shing Shieh, Chih-Hau Su, Wen-Luan Wu, Yuh-Shyan Tsai, Chih-Wei Cheng, Ming-Derg Lai, Chao-Liang Wu 
Molecular Therapy 
Volume 15, Issue 1, Pages (January 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Construction and characterization of YP2. (a) Schematic presentation of the strategy for generating YP2 virus, a gE-defective PrV carrying gD and HSV-1 TK genes driven by the human HER-2/neu promoter. The CW1 virus is a gD/gE/TK-defective PrV mutant carrying the HSV-TK gene inserted in the place of the gD gene. The bicistronic expression vector pDgD/HER2pr-gD-IRES-TK contains the human HER-2/neu promoter and the coding regions of gD and HSV-TK linked by an IRES sequence, in addition to 5′- and 3′-flanking regions encompassing gG and gI, respectively, to provide homology regions for homologous recombination with the CW1 virus. MBT-2 cells were transfected with pDgD/HER2pr-gD-IRES-TK plasmid followed by infection with CW1 virus, yielding YP2 virus. Restriction enzyme cleavage sites shown are BamHI (B), HindIII (H), NotI (N), EcoRI (R), and XhoI (X). Thin and thick arrows represent the locations of primers used for PCR. (b) Detection of gD and HSV-TK sequences from YP2 by PCR. DNA from YP2 (lanes 1 and 2) and CW1 (lanes 3 and 4) served as the templates for PCR amplification using primer pairs specific for HSV-TK (lanes 1 and 3) and gD (lanes 2 and 4), producing the expected 1,131- and 262-bp amplicons, respectively. M, 100-bp DNA ladder. (c) Southern hybridization of YP2. DNA from CW1 (lane 1), TNL (lane 2), and YP2 (lane 3) were digested with BamHI. After electrophoresis and transfer, the replica membrane was hybridized with a labeled probe specific for gD. TNL and YP2 gave 7.4- and 10.5-kb bands, respectively, whereas no signal was produced on CW1.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Expression of HER-2/neu proteins and transcriptional activities of HER-2/neu and CMV promoters in various cells. (a) Expression of HER-2/neu proteins in various cancer cells and NIH3T3 fibroblasts examined by immunoblot analysis. Expression of β-actin served as the quantitative control. (b) HER-2/neu and CMV promoter activities were assessed by the luciferase reporter assay. Cells were transfected with pGL-HER-2-Luc or pGL-CMV-Luc and the transfection efficiency was standardized to the cotransfected plasmid pTCY-LacZ expressing β-gal driven by the β-actin promoter. Relative promoter activity is expressed as a ratio of relative luciferase activity driven by the HER-2/neu promoter divided by that driven by the CMV promoter in (c) MBT-2, HCDB-1, and NIH3T3 cells, and (d) TSGH-8301, J82, and TCC-SUP cells. Each value represents the mean±SD (n=3; P<0.05 for J82 vs TSGH-8301 and P<0.01 for TCC-SUP vs TSGH-8301 by Student's t-test).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
CPE in various cell lines after infection with YP2, TNL, or CW1. (a) CPE in MBT-2 cells infected with YP2 or TNL, but not CW1. MBT-2 cells were infected with 2 × 105 TCID50 of YP2, TNL, or CW1 and monitored for CPE by photomicrographic examination at 3 days post-infection (original magnification × 200). (b) CPE in MBT-2, NIH3T3, TSGH-8301, TCC-SUP, J82, and HCDB-1 cells after infection with TNL or YP2. Cells were infected with varying doses of TNL or YP2, and monitored for CPE by crystal violet staining at 3 days post-infection. Cell viability in (c) NIH3T3 and (d) MBT-2 cells after infection with 2 × 104 TCID50 of TNL, CW1, or YP2. The viable cell numbers were determined by Trypan blue exclusion at 4 days post-infection. Each value represents the mean±SD (n=4; ***P<0.001 compared with mock-infected cells by Student's t-test).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Modulation of HER-2/neu promoter activity affected YP2-induced cytolysis in MBT-2 cells. MBT-2 cells were transfected with pTCY-E1A (1.2, 0.8, 0.4, and 0 μg) and pGL-HER-2-Luc (0.5 μg). The total amount of plasmid DNA for transfection was kept constant by the addition of pTCY-LacZ. (a) Cell lysates were harvested 30 h after transfection and their luciferase activities were determined. (b) Alternatively, 24 h after transfection, cells were infected with various doses of YP2 and monitored for CPE by crystal violet staining at 3 days post-infection. (c) MBT-2 cells were transfected with pGL-HER-2-Luc (0.5 μg) and pTCY-LacZ (0.2 μg). After 24 h, cells were treated with TPA (10 nM) and retinoic acid (10 μM) for 6 h and their luciferase activities were determined. (d) Alternatively, MBT-2 cells were infected with various doses of YP2. After 5 h, cells were washed, fed with complete medium containing TPA and retinoic acid, and cultured for an additional 18 h. Cells were then provided fresh medium and CPE was monitored by crystal violet staining 3 days later. Each value represents the mean±SEM (n=3; ***P<0.001 by Student's t-test).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Antitumor effects of YP2 on immunocompetent mice bearing bladder tumors. Groups of eight or nine C3H/HeN mice that had been inoculated subcutaneously with 2 × 106 MBT-2 cells at day 0 were treated intratumorally with 2 × 104 TCID50 of YP2 or CW1, or with PBS daily from days 7 to 13. (a) The mean tumor volume is shown and the data are presented as mean±SD (P< for YP2 Vs CW1 or PBS by Student's t-test). (b) Kaplan–Meier survival curves at day 50 are shown (P= for YP2 vs CW1 and P= for YP2 vs PBS by the log-rank test). (c) Detection of PrV proteins within MBT-2 tumors in vivo. C3H/HeN mice bearing MBT-2 tumors were injected intratumorally with 2 × 106 TCID50 of YP2 or TNL, or PBS for 3 consecutive days. Immunofluorescent staining of cryostat sections of tumors with swine anti-PrV antiserum and fluorescein-conjugated goat anti-swine IgG was performed at 24 h for TNL- or PBS-treated animals and at 72 h for YP2-treated animals after last virus injection. The stained sections were examined under fluorescence and bright-field microscopes. Immunofluorescent images (left panel) and bright-field images (middle panel) in the same field are displayed (original magnification × 640). Formalin-fixed, paraffin-embedded sections were examined by H&E stain (right panel, original magnification × 400).
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions