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 1129-1136 (June 2007) DOI: 10.1038/sj.mt.6300165 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

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 2007 15, 1129-1136DOI: (10.1038/sj.mt.6300165) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions