1. Volume 15, Issue 6, Pages 1129-1136 (June 2007)
An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas 
Paula YP Lam, Kian Chuan Sia, Jenn H Khong, Bart De Geest, Kar S Lim, Ivy AW Ho, Grace Y Wang, Lv Miao, H Huynh, Kam M Hui 
Molecular Therapy 
Volume 15, Issue 6, Pages (June 2007)
DOI: /sj.mt
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Proposed mechanism for the recombinant transcriptional activator system and schematic representation of the herpes simplex virus type 1 (HSV-1) amplicon vector constructs. (a) In proliferating tumor cells, the activator module comprising the Gal4-DNA binding domain and the nuclear transcription factor Y, alpha (NF-YA) transcription activator domain will bind to Gal4-binding sites (Gal4 bs) upstream of a minimal cyclin A promoter harboring the cell cycle–dependent element (CDE)/cell cycle homology region (CHR) and drive the transcription of the luciferase reporter gene. In quiescent cells, transactivation of the minimal cyclin A promoter is prevented by the binding of cell cycle–dependent factor 1 (CDF-1) to the CDE/CHR element. (b) pApoE/hAAT-Luc contained four apolipoprotein E enhancers (ApoEenh) inserted upstream of the human α1-antitrypsinpromoter (hAATp). (c) pAFP/AFP-Luc contained the human alpha-fetoprotein (AFP) enhancer with the human AFP promoter. (d) pSV40/Alb-Luc contained the simian virus 40 enhancer inserted upstream of the human albumin (Alb) promoter. (e) pHGX-Luc was a promoterless backbone vector that served as the negative control. (f) The pIH8GalLuc construct lacked the activator module. (g) pApoE/hAAT-cc-Luc contained both the activator module (Gal4/NF-YA) and the reporter module (8GalLuc). ampR, ampicillin resistant gene; bGHp(A), bovine growth hormone poly-adenylation signal; eGFP, enhanced green fluorescent protein; Oris, HSV origin of replication; p(A), SV40 poly-adenylation signal; pac, HSV-1 packaging signal; pIE4/5, promoter of HSV-1 immediate early gene 4/5.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Liver-specific promoter activities exhibited in a range of different human cell lines. (a) Luciferase expression in hepatocellular carcinoma (HCC), non-HCC, and normal liver cell lines transfected with pHGX-Luc, pApoE/hAAT-Luc, pAFP/AFP-Luc, and pSV40/Alb-Luc was determined. (b) Immunohistochemistry of primary HCC cells with anti-human cyclin A showing that the HCC cells were in the proliferating stage (left). Primary HCC cells were infected with amplicon virus packaged with pApoE/hAAT-Luc carrying the green fluorescent protein (GFP) reporter gene (right). (c) Luciferase activities conferred by the pApoE/hAAT-Luc amplicon virus 24 hours after infection of the primary HCC cells. The control was derived from the cell lysate of primary HCC cells. RLU, relative light unit.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Transgene expression mediated by the pApoE/hAAT-cc-Luc amplicon vector was cell cycle dependent. (a) Cell cycle–regulated luciferase reporter activity mediated by the pApoE/hAAT-cc-Luc amplicon plasmid was analyzed in proliferating and G1-arrested cell populations in HuH-7, PLC/PRF/5, Gli36, and HeLa cells. (b) The cell cycle status of representative HuH-7 cells was determined using anti-human cyclin A antibodies. Cell-free extract from A-431 was used as a positive control. (c) Transgene expression mediated by pApoE/hAAT-cc-Luc amplicon viral vectors was compared in proliferating and G1-arrested HuH-7 cells. Transduction efficiencies in these two cell populations were examined by fluorescence-activated cell sorting analysis (row 1). The level of endogenous cyclin A was not detectable in G1-arrested cells after immunohistochemical staining with anti-cyclin A antibody. Actively proliferating cells expressing endogenous cyclin A were positively immunostained (row 2). Cells infected with pApoE/hAAT-cc-Luc amplicon viral vectors expressed the enhanced green fluorescent protein (eGFP) gene (row 3). Luciferase gene expression (immunostained blue) could be detected only in pApoE/hAAT-cc-Luc-infected proliferating cells (row 4). RLU, relative light unit.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Primary human hepatocellular carcinoma (HCC) cells were highly infectable by pApoE/hAAT-cc-Luc amplicon vectors. (a) Negative control for immunohistochemistry on the primary HCC tumor section (left). The primary HCC tumor section was immunostained using polyclonal rabbit anti-hAAT(middle). Infectability of primary HCC cells with pApoE/hAAT-cc-Luc amplicon virus (right). (b) Transduction efficiency of primary HCC cells estimated at 6 and 24 hours after pApoE/hAAT-cc-Luc viral incubation.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Cell cycle–controlled expression mediated by pApoE/hAAT-cc-Luc in the mouse liver regeneration model. (a) Liver regeneration kinetics in BALB/c mice. At each time point, the regenerated livers from mice undergoing PHx were removed and weighed. The regenerated liver mass was expressed as a percentage of the mass of livers obtained from a similar region of control mice killed at the same time point. (b) Flow cytometry analysis demonstrating the cell cycle profile of sham-operated and PHx mice. (c) In vivo luciferase activity in various organs mediated by pApoE/hAAT-cc-Luc amplicon viral vectors for PHx mice. Sham-operated and PHx mice were injected with similar viral doses (1 × 106 transducing units): pIH8GalLuc (negative control) and pApoE/hAAT-cc-Luc (liver-specific). Luciferase activity from various organs in all groups was analyzed 24 hours after viral injection. Data shown represent average + SEM for five mice. RLU, relative light unit.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Transcriptional activities mediated by the pApoE/hAAT-cc-Luc amplicon vector in hepatocellular carcinoma tumor–bearing mice can be visualized by bioimaging. Potential liver cytotoxicity induced by the pApoE/hAAT-cc-Luc amplicon vector was measured by (a) alanine aminotransferase (ALT) and (b) aspartate aminotransferase (AST) assays at days 1, 3, and 7 after viral injections. Hank's balanced salt solution (HBSS) was use as a negative control, and hrR3 was use as a positive control. (c) pApoE/hAAT-cc-Luc (1 × 106 transducing units) was intra-tumorally injected into the HeLa or HuH-7 tumor–bearing severe combined immunodeficient mice. Luciferase activity was measured using the non-invasive bioimaging system 24 hours after viral injections. (d) Luciferase assays were performed on harvested tumors. All data shown represent average + SEM for five mice.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2007 The American Society of Gene Therapy Terms and Conditions