An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas

An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas

© The American Society of Gene Therapy original article An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targ...

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© The American Society of Gene Therapy

original article

An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas Paula YP Lam1, Kian Chuan Sia1, Jenn H Khong2, Bart De Geest3, Kar S Lim4, Ivy AW Ho1, Grace Y Wang1, Lv Miao1, H Huynh1 and Kam M Hui1 Laboratory of Cancer Gene Therapy, Division of Cellular and Molecular Research, National Cancer Centre, Singapore; 2Institute of Molecular and Cellular Biology, Singapore; 3Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium; 4Genome Institute of Singapore, Singapore 1

Our previous studies have shown that transgene expression could be targeted to proliferating cells when cell cycle transcriptional regulatory elements were incorporated into herpes simplex virus type 1 (HSV-1) amplicon backbone vectors. In the study reported here, we further demonstrated the transcriptional activation of transgene expression in association with the onset of cellular proliferation using the mouse partial hepatectomy model. Moreover, transcriptional regulation could be rendered specific to human hepatocellular carcinoma (HCC) cells by inserting the chimeric gene Gal4/NF-YA under the regulation of the HCC-specific hybrid promoter. The hybrid promoter, which consists of four copies of the apolipoprotein E (ApoE) enhancer element inserted upstream of the human α1-antitrypsin (hAAT) promoter, induced an higher level of transcription than other liver-specific promoters such as alpha-­fetoprotein (AFP) and albumin (Alb) promoter. As a consequence, the enhancement of tissue-specific expression in the context of Gal4/NF-YA fusion proteins enabled the monitoring of transgene expression using a bioluminescence imaging system. Furthermore, these vectors have been shown to be non-toxic and exhibited potent infectivity for proliferating primary HCC cells and HCC cell lines. Together, these results demonstrated that the new hybrid vectors could provide options for the design of safe and efficient systemic gene therapeutic strategies for human HCC. Received 8 January 2007; accepted 6 March 2007; published online 10 April 2007. doi:10.1038/sj.mt.6300165

Introduction Proliferation-dependent transcriptional activation that is endogenously triggered could provide a means of improving the efficiency of existing transcriptional regulatory systems for cancer gene therapy. The strategy is based on the interaction of a transcriptional repressor protein, cell cycle–dependent factor 1, which is specifically expressed during the G0/G1 phase,

with the two contiguous repressor elements known as the cell cycle–dependent element and the cell cycle homology region.1,2 In resting or non-dividing cells, the occupation by these repression proteins of the cell cycle–dependent element/cell cycle homology region binding sites located within the minimal cyclin A promoter will interfere with the cascade of events leading to gene transcription via upstream factors such as the CCAATTbox binding factor and nuclear transcription factor Y, alpha (NF-YA).3 On the other hand, transactivation of the minimal cyclin A promoter could proceed in the absence of cell cycle– dependent factor 1 repressor protein in dividing cells (as illustrated in Figure 1a). The major limitation encountered in this system is the difficulty in achieving an optimal operational molar ratio of the various functional modules for cell cycle–dependent transcriptional regulation. Herpes simplex virus type 1 (HSV-1) amplicon viral vectors are efficient gene transfer vehicles for liver tumors, as 10–1,000 times fewer virions are usually required to achieve similar transduction efficiencies to other viral vectors.4 In addition, treatment of established hepatic tumors with HSV carrying interleukin-12 significantly reduced the growth of residual microscopic cancer at the time of resection of the parental tumor, suggesting that HSV could confer a hepatic-specific memory response.5 The hepatic tumor recurrence protection effect could be further enhanced by the combination of herpes amplicon vectors carrying transgenes for regulated upon activation, normal T-cell expressed, and secreted B7.1 and granulocyte–macrophage colony stimulating factor.6 HSV-based vectors could also be engineered to confer extended transgene expression in hepatocytes in vivo by incorporating desirable elements of the adeno-associated virus (AAV)7 or Epstein-Barr virus.8 Thus, recombinant HSV could serve as a potential vector for targeting human hepatocellular carcinomas (HCCs). We have previously reported that HSV-1 amplicon vectors could mediate cell cycle–dependent transgene expression through the incorporation of cell cycle transcriptional regulatory elements.9,10 In the study reported here, we demonstrated that a hybrid promoter consisting of four copies of the apolipoprotein E (ApoE) enhancer element upstream of the liver-specific

Correspondence: Paula YP Lam, Laboratory of Cancer Gene Therapy, Division of Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 169610. E-mail: [email protected] Molecular Therapy vol. 15 no. 6, 1129–1136 june 2007

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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 (ApoE enh) inserted upstream of the human α1antitrypsin promoter (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.

human α1-antitrypsin (hAAT) promoter induced a higher level of transcription than the conventional liver-specific promoters. This promoter was subsequently employed for the transcription of the chimeric transcription factor Gal4/NF-YA in the pApoE/ hAAT-cc-Luc viral vector. We demonstrate the transcriptional activation of transgene expression in association with the onset of cellular proliferation using a mouse partial hepatectomy model. Furthermore, the transcriptional targeting viral activities were also successfully monitored using a bioluminescence imaging system. This is of importance because reports of the efficacy of viral therapy such as HSV1716 (ref. 11) and G207 (ref. 12) in human clinical trials have been contradictory owing either to patient selection or to the lack of biological correlation between the treated patient and the status of the viral vectors after administration. Taken together, our results demonstrate the promise of using transcriptionally regulated HSV amplicon vectors as a means of targeting proliferating HCC cells. 1130

Results Construction of HCC-specific HSV-1 amplicon viral vectors Three basic HSV-1 amplicon vectors containing liver- or HCC disease–selective eukaryotic promoters have been constructed, as shown in Figure 1b–d. Luciferase reporter activities mediated by pApoE/hAAT-Luc were observed to be highest in human HCC cell lines (PLC/PRF/5 and HuH-7), followed by pSV40/Alb-Luc and pAFP/AFP-Luc amplicon plasmids (Figure 2a). In contrast, non-HCC cell lines (human lung carcinoma cell line A549, undifferentiated human nasopharyngeal carcinoma cell line CNE2, human glioma cell line Gli36, human cervix epithelial adenocarcinoma cell line HeLa) and human immortalized normal liver cell line (THLE-3) exhibited low levels of luciferase reporter activity. Recently, the establishment of primary human HCC xenografts has been reported by Huynh et al.13 These xenografts have been characterized extensively and were demonstrated to retain most www.moleculartherapy.org vol. 15 no. 6 june 2007

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of their normal architecture, function, and tumorigenicity.13 Thus, they may resemble the in vivo situation more accurately than cancer cell lines that have been passaged for many years under tissue culture conditions. To examine the transduction efficiency of pApoE/hAAT-Luc amplicon vectors, primary tumor cells from one of the HCC xenografts, line 26-1004, were prepared. At a multiplicity of infection of 0.3, 22% ± 3% of the proliferating primary HCC cells were transduced by pApoE/hAAT-Luc amplicon viral vectors (Figure 2b). High levels of luciferase activity were also measured in these infected cells (Figure 2c). Taken together, these results demonstrated that the hybrid promoter ApoE/hAAT is capable of conferring high transcriptional activities in both primary HCC cells and HCC cell lines.

Regulatable amplicon vectors that confer transgene expression in a cell cycle and disease-specific manner On the basis of these results, the hybrid ApoE/hAAT promoter was employed to regulate the transcriptional activities of the Gal4/NF-YA fusion gene in an attempt to target the transcriptional ­activities to HCC cells specifically. The new recombinant vector was denoted pApoE/hAAT-cc-Luc (Figure 1g). Luciferase reporter activities in proliferating HCC cells (HuH-7 and PLC/PRF/5) were subsequently observed to be significantly higher than in the corresponding G1-arrested cells (Figure 3a). In contrast, background levels were observed in non-HCC cells (Gli36 and HeLa). ­Immunoblotting performed on the representative HuH-7 cells showed a significantly higher level of endogenous cyclin A protein expression in proliferating cells than in the corresponding G1-arrested cells (Figure 3b), providing

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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 antihuman 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.

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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.

additional support for the cell cycle status of these two cell ­populations. To confirm that the pApoE/hAAT-cc-Luc amplicon virus could confer luciferase expression in a cell cycle– ­dependent manner at the single-cell level, confocal microscopy 1131

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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.

studies were performed. HuH-7 cells infected by pApoE/hAATcc-Luc amplicon viral vectors (multiplicity of infection = 1.0) for 6 hours were fed in either standard tissue culture medium or in the presence of lovastatin to induce G1 arrest. Similar transduction efficiency in both the proliferating and G1-arrested cell populations (~20%) was observed by fluorescence-activated cell sorting analysis (Figure 3c). The proliferation status was also confirmed by immunohistochemistry staining against endogenous cyclin A proteins. Confocal microscopy studies demonstrated that the pApoE/hAAT-cc-Luc amplicon– transduced HuH-7 cells, as shown by the presence of exogenous enhanced green fluorescent protein expression, were positively stained for luciferase (blue). In contrast, luciferase expression was minimal or not detectable in G1-arrested cell populations. Thus, these results demonstrated that the pApoE/hAAT-cc-Luc amplicon vector confer transcriptional activities in a cell cycle– dependent and cell type–specific manner in vitro.

Gene transfer efficiency of primary human HCC tumor cells by hybrid vectors Next, we examined the transduction efficiency of pApoE/hAATcc-Luc viral vectors in primary human HCC cells derived from line 26-1004 xenografts. These HCC cells were of histological grade 3 tumor types with a rapid proliferation rate in vivo.13 Immunohistochemistry showed that these cells over-expressed the hAAT cellular protein, a 52-kd serine proteinase inhibitor that is normally secreted from hepatocytes and circulates in the plasma (Figure 4a). The cells were also observed to be highly infectable by the pApoE/hAAT-cc-Luc amplicon virus (­Figure 4a). At a multiplicity of infection of 1, 47% ± 2.8% and 1132

66% ± 2.2% green fluorescent protein–positive cells were scored at 6 and 24 hours, respectively, after infection (Figure 4b). Thus, these amplicon vectors exhibit broad host range and are efficient in infecting primary HCC tumor cells.

Transcriptional regulation of hybrid vectors in a cell cycle–dependent manner in vivo The liver is an excellent tissue for the study of growth regulation because of its potent regeneration capacity, by a process of ­compensatory growth, after surgical resection or toxic injury. Thus, we have chosen normal liver regeneration after partial hepatectomy (PHx) as an animal model to study cell cycle–dependent regulation of transgene expression conferred by pApoE/hAAT-ccLuc amplicon viral vectors in vivo. Female BALB/c mice were separated into two experimental groups: a group that underwent PHx and a sham-operated group. Following PHx, the resected liver lobes were weighed ­immediately. The kinetics of liver regeneration was monitored closely, and the peak of the regeneration process was noted to occur at day 2 after PHx (Figure 5a). Flow cytometry analysis confirmed that the percentage of S-phase cells among actively proliferating cells was markedly higher in the PHx group (36.42%) than in the sham-operated group (12.25%) (Figure 5b). On day 2 after PHx, both the PHx and shamoperated mice (n = 5 per group) were injected, via the tail vein, with 1 × 106 transducing units (TU) of either pApoE/hAAT-ccLuc or pIH8GalLuc viral vectors. After 24 hours, the mice were killed, and luciferase activity in the liver tissues was assayed. In mice injected with pApoE/hAAT-cc-Luc, luciferase activities was approximately 11-fold higher in the PHx liver than in the sham-operated liver (P = 0.019) (Figure 5c). In contrast, control viral vectors (pIH8GalLuc) lacking the cell cycle regulatory elements failed to induce a significant level of luciferase activity in PHx mice compared with the sham-operated group. To confirm further that the observed increase in transcriptional activities is restricted to actively dividing liver cells and not other proliferating cells, the biodistribution of luciferase activity was analyzed in other organs. Our results showed that the ratio of cell cycle regulation, as measured by luciferase activity in PHx compared with sham-operated mice, was highest in the livers of mice injected with pApoE/hAAT-cc-Luc. In contrast, other organs exhibited a twofold or lower ratio of cell cycle regulation, which was consistent with the result observed for the negative controls in vitro. These data demonstrated that luciferase expression conferred by the viral vector pApoE/hAAT-cc-Luc is targeted to actively proliferating hepatocytes during liver regeneration. Cytotoxicity and specificity induced by pApoE/hAAT-cc-Luc amplicon viral vectors in vivo To examine whether administration of vectors may induce cytotoxicity, the levels of transaminases (alanine aminotransferase and aspartate aminotransferase) were analyzed in immunocompetent BALB/c mice (n = 5 per group) that received 1 × 106 TU of pApoE/hAAT-cc-Luc amplicon vectors. Mice injected with either Hank’s balanced salt solution or hrR3 (replication competent HSV-1) were used as negative and positive controls, respectively. No significantly elevated www.moleculartherapy.org vol. 15 no. 6 june 2007

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Figure 5  Cell cycle–controlled expression mediated by pApoE/hAATcc-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 (liverspecific). 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.

levels of transaminases were observed in mice injected with pApoE/hAAT-cc-Luc amplicon vectors compared with mice injected with Hank’s balanced salt solution (Figure 6a and b). In contrast, elevated hepatic transaminase activity was noted in mice injected with the hrR3 helper virus at day 1 after injection, which returned to near-normal levels over the following days. These data suggest that the regulatory elements employed in the context of the HSV-1 amplicon vector do not cause significant liver toxicity. To monitor the cell type–specific luciferase activities conferred by pApoE/hAAT-cc-Luc amplicon vectors in vivo, we used a model of human HCC based on Molecular Therapy vol. 15 no. 6 june 2007

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Figure 6  Transcriptional activities mediated by the pApoE/hAAT-ccLuc 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.

subcutaneous implantation of HuH-7 cells in severe combined immunodeficient mice. On the other flank of the same mouse, HeLa cells were inoculated. When tumors reached similar size, the animals were injected intra-tumorally with 1 × 106 TU of pApoE/hAAT-cc-Luc ­ amplicon vectors. As shown in Figure 6c, HuH-7 tumors injected with pApoE/hAAT-cc-Luc amplicon vectors had the strongest luciferase signals. Luciferase activity, measured in photons per second, was approximately 13-fold higher in HuH-7 tumor than in HeLa tumor. To confirm that the differential transgene expression was not a result of tissue depth variation between the reporter gene activities and the charge-coupled device detector, the tumors were removed and lysed to measure luciferase activity. Results obtained showed that the luciferase reporter activity was consistent, i.e., 13-fold higher in HuH-7 tumors than in HeLa tumors in mice injected with pApoE/hAAT-cc-Luc amplicon vectors (Figure 6d). Taken together, these data confirmed that the transgene expression conferred by pApoE/hAAT-cc-Luc amplicon vectors is HCC cell type–specific and that the viral vector itself is non-cytotoxic when administered systemically.

Discussion The liver is an organ that is maintained in a quiescent state in adults but has remarkable proliferative capacity after acute or chronic damage. Thus, precise control over hepatocyte proliferation is critical for the suppression of tumorigenesis in the liver, as chronic liver damage and the corresponding cycle of regeneration are known to fuel the development of hepatocellular 1133

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carcinoma.14 Specific transgene expression in liver-targeted gene therapy has been conventionally accomplished using tumor­specific promoters. However, high levels of gene expression are usually hard to achieve with these relatively weak promoters.15–17 The alpha-fetoprotein (AFP) promoter and its enhancer have been chosen for liver-targeted gene delivery because AFP is expressed in a high proportion of primary liver tumors but not in adult normal liver.18,19 In the present study, we demonstrated that the level of transgene expression and specificity in HCC cells was greatly enhanced in HCC cells transfected with the hybrid promoter construct (pApoE/hAAT-Luc) in comparison to the disease-specific AFP promoter (by 9–15-fold) or the liver-specific human albumin (Alb) promoter (by two to sixfold). In agreement with our findings, Condiotti et al. have compared five constitutive promoters, namely hAAT, human phosphoglycerate kinase, murine phosphoglycerate kinase, Rous sarcoma virus, and cytomegalovirus promoters in four HCC cell lines. Among these promoters, hAAT leads to the highest number of cells expressing the transgene,20 whereas cytomegalovirus is the weakest, probably owing to the transcriptional silencing in liver tissues.21,22 In a different context, this combination of hAAT promoter and human ApoE enhancer has also been reported to induce elevated and stable expression of human apolipoprotein A-I protein.23 Thus, the overall enhanced specificity of targeting proliferating HCC cells could be achieved through the employment of the hybrid promoter and the cell cycle regulatory elements. These findings suggest the feasibility of employing disease condition as a means of regulating transgene expression. In support of this, Pin et al. reported that when soluble fetal liver kinase-1 was expressed under the regulation of multimerized hypoxia-responsive enhancers in the context of HSV-1 amplicon vectors, a substantial reduction of vessel formation and hepatoma growth was observed under hypoxic conditions.24 The concept of targeting rapidly dividing tumor cells is an interesting one for cancer gene therapy. However, most of the current viral vectors are unable to accommodate the relatively large transcriptional regulatory cassettes. For example, the limited transgene packaging capacity of 4.7 kilobases of recombinant AAV–derived vectors can be increased by exploiting the fact that recombinant AAV genomes concatemerize after ­transduction.25 Thus, two recombinant AAV vectors independently encoding the regulatory and transgene cassette are required to reconstitute a functional gene.26 In an attempt to achieve a higher transgene expression level, a recombinant AAV vector encoding the regulatory elements was administered at a higher concentration than the latter vector carrying the therapeutic gene. Unfortunately, this led to a greater potential for adverse immune response. Recently, successful packaging and intact delivery of genomic inserts of more than 100 kilobases with efficiencies of up to 100% has been demonstrated in the HSV-1 amplicon vector platform.27–29 In addition, these amplicon vectors are relatively non-toxic. Using alanine ­aminotransferase/aspartate aminotransferase assays, we have demonstrated that the newly constructed pApoE/hAAT-ccLuc amplicon viral vectors exhibit enhanced safety compared with the replication-conditional hrR3 viruses, which have been demonstrated to exhibit potential therapeutic benefits in 1134

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the destruction of HCC.30,31 It may be worth noting that differential transaminase activities between the two viral vectors were recorded only at day 1. This difference may escalate with increased viral dose and could potentially induce a significant immune response when systemically administered to advanced cancer patients with multifocal HCC. Thus, we have shown that the regulatable gene expression system is relatively safe when mediated systemically by pApoE/hAAT-cc-Luc. Liver regeneration after PHx has been used as a model to study transcriptional activation of transgene expression in association with the onset of cellular proliferation. Almost immediately after PHx, quiescent hepatocytes are stimulated to proliferate.32 The expression of cyclin A increased from 12 hours until day 7 after PHx, whereas quiescent (G0 phase) liver cells of sham-operated animals were unable to re-enter the cell cycle and did not expressed cyclin A.33 To the best of our knowledge, this is the first demonstration of a functional correlation between the percentage of proliferating cells and the extent of luciferase reporter activity mediated by a regulatable gene expression system engineered into the HSV-1 amplicon viral vector. In addition, the enhancement of tissue-specific expression in the context of Gal4/NF-YA fusion proteins has enabled the real-time visualization of the luciferase reporter activity mediated by these viral vectors. The photon emission data acquired from the region of tumor masses were also consistent with the luciferase activity of the tumor biopsies measured using the luciferase assay. In conclusion, we have demonstrated that transcriptional regulation mediated by pApoE/hAAT-cc-Luc amplicon viral vectors is cell cycle dependent and cell type specific. The hybrid promoters in these vectors induced a high level of transcriptional activity compared with AFP and Alb promoters. They are also relatively non-toxic when administered systemically and exhibit high infectabilities in primary HCC cells. Overall, the newly generated vector construct may find application as a gene delivery vector to proliferating liver cells.

Materials and Methods Primary HCC cell isolation and cell lines. PLC/PRF/5, HeLa, A549,

A-431, and THLE-3 were obtained from American Type Culture Collection (Manassas, VA). HuH-7 was obtained from Japanese Collection of Research Bioresources (JCRB cell bank, Osaka, Japan). Gli36 and CNE2 cells were kindly provided by A.T. Campagnoni (UCLA School of ­Medicine, Los Angeles, CA) and H.M. Wang (Cancer Institute, Guangzhou, PRC), respectively. African green monkey kidney 2-2 cells were from Vero cells that constitutively expressed the HSV-1 ICP27 proteins (kindly provided by Sandri-Goldin, University of California, Los Angeles, CA)34 and were cultivated in the presence of 500 µg/ml Geneticin (Invitrogen, Grand Island, NY). All cells were grown under standard tissue culture conditions in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT). All HCC-bearing xenograft mice were maintained according to guidelines approved by the National Cancer Centre Animal Care and Use Committee. In brief, the tumors from these mice were removed and gently disrupted with fine forceps in the presence of 0.5 mg/ml collagenase (type IV, pH 7.5; Sigma, St. Louis, MO) as described by Pichard et al.35 Molecular cloning. pApoE/hAAT-Luc (Figure 1b) was constructed by

HindIII digestion of pApoE/hAAT-cc-Luc (see Figure 1g) to release the

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cassette from the Gal4 DNA binding domain to the minimal CycA promoter. The digested product was blunt-ended and self-ligated to generate pApoE/hAAT-Luc with four copies of the ApoE enhancer (650 base pairs, bp) and hAAT promoter (1,551 bp) that drive the expression of the luciferase gene. pAFP/AFP-Luc (Figure 1c) was generated by releasing the human AFP enhancer (1,854 bp) and human AFP promoter region (287 bp) derived from pDRIVE-AFP-hAFP (Invivogen, Carlsbad, CA) using the PstI and NotI restriction enzyme sites. This fragment was bluntended and ligated into the promoterless pHGX-Luc fragment to generate the recombinant construct. A similar strategy was used to construct pSV40/Alb-Luc (Figure 1d) containing the SV40 enhancer (235 bp) and human Alb promoter (213 bp) from pDRIVE-SV40-hAlb (Invivogen, Carlsbad, CA). To construct pHGCX-Luc (positive control), the firefly luciferase gene (Luc) was excised from GalCycA3 using HindIII and XbaI and subcloned into the parental pHGCX (kindly provided by Y. Saeki, Ohio State University Medical Centre, Columbus, OH). Subsequently, the cytomegalovirus promoter from pHGCX-Luc was removed using BglII and NheI, blunt-ended, and self-ligated to generate pHGX-Luc (Figure 1e). The construction of pIH8GalLuc plasmids (Figure 1f) has been described elsewhere.9 To construct the pApoE/hAAT-cc-Luc plasmid, the hAAT promoter (1,551 bp) was removed using the KpnI site from pBS-hAAT-bpA23,36 and subcloned into the pHGX-9 plasmid, using the same restriction enzyme site, to generate pHGX-hAAT. Plasmid pHGX-9 was similar to its parental vector, pHGCX,9 with the exception that the cytomegalovirus promoter was excised. Four copies of the human ApoE enhancer (650 bp), derived from the previously reported parental pUC19 construct,37 were excised with EcoRI and HindIII. The fragment was bluntended and ligated into pHGX-hAAT to generate pHGX-ApoE/hAAT. Using PmeI, the DNA fragment consisting of the Gal4/NF-YA and the 8GalLuc cassette was removed from the pC8-36 amplicon plasmid9 and subcloned into pHGX-ApoE/hAAT to generate the pApoE/hAAT-cc-Luc plasmid (Figure 1g). All recombinant plasmids were verified by restriction enzyme mapping followed by DNA sequencing (Applied Biosystems, Foster City, CA). Helper virus–free HSV-1 amplicon viral vectors. Packaging of HSV-1

amplicon virus was performed as described previously.9,38 In brief, 2-2 cells (3 × 106 cells) were transfected with 1.8 µg of amplicon DNA and 3.0 µg fHSV∆pac∆27 0+ using 45 µl LipofectAmine (Invitrogen, Carlsbad, CA) and 15 µl of Plus reagent (Invitrogen, Carlsbad, CA). The virus was purified through a 25% sucrose gradient and stored at −80 °C until use. To determine vector titer TU per ml, 2-2 cells were infected and 24 hours later green fluorescent cells were counted using a fluorescence microscope (Nikon, Tokyo, Japan). DNA transfections. Cells (3 × 105) were transfected in six-well plates

(Nunc, Roskilde, Denmark) using LipofectAmine (Invitrogen) according to the manufacturer’s instructions. Different ratios of plasmid DNA to LipofectAmine were employed for each of the cell lines studied to optimize the transfection efficiencies. The transfection efficiency was also normalized to the percentage of green fluorescent protein cells obtained from flow cytometry (Becton Dickinson, San Jose, CA). Synchronization of cells and cell cycle analysis. Depending on the cell

lines, fresh culture medium supplemented with 0.2% fetal bovine serum (Hyclone Laboratories, Logan, UT) and 50–70 µmol/l lovastatin (Merck, Singapore) were added to induce G1 arrest for 48 hours. After ethanol fixation at 4 °C overnight, flow cytometric analysis of cell cycles was performed as described previously.9 Luciferase reporter gene assay. Cells were harvested 48 hours after transfection and lysed in 120 µl lysis buffer (50 mmol/l Tris–Cl, 150 mmol NaCl, 1% Triton X-100). Luciferase activities and protein concentration were measured as described previously.9 Molecular Therapy vol. 15 no. 6 june 2007

HSV-1 Vector that Targets Proliferating HCC Cells

Assay for hepatic transaminase activities. The alanine aminotransferase and aspartate aminotransferase assay was performed as described previously.39 In brief, blood was drawn from the tail vein of mice at defined time points after intravenous injection of viruses. The serum was separated by centrifugation at 4,000 rpm at room temperature for 30 minutes and 10 µl of serum was used to assay alanine aminotransferase and aspartate aminotransferase activity. Immunofluorescent staining. HuH-7 cells (3 × 105) were infected with

pApoE/hAAT-cc-Luc amplicon viral vectors for 6 hours at a multiplicity of infection of approximately 1.0. Cells were cultured in either standard tissue culture medium or culture medium with lovastatin to induce G1 arrest for 24 hours. After fixation and permeabilization of the cells, which has been described in detail previously,9 the cells were incubated with primary rabbit anti-luciferase polyclonal antibody (2 µg/ml; Sigma-Aldrich, St. Louis, MO) overnight at 4 °C. They were then rinsed three times at 5-minute intervals and incubated with Alexa Fluor 350 goat anti-rabbit antibody (2 µg/ml; Molecular Probes, Eugene, OR) for an hour before mounting and examination using the LSM 510 Meta confocal microscope (Carl Zeiss Microscopy, Göttingen, Germany). Immunohistochemical staining. The procedure for fixing and permea-

bilization of cells was similar to that described for immunofluorescence studies. Primary HCC cells were incubated with either mouse anti-cyclin A monoclonal antibody (4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) or ready-to-use polyclonal rabbit anti-hAAT (DakoCytomation, Glostrup, Denmark) overnight at 4 °C. Cells were subsequently stained with either goat anti-mouse or anti-rabbit polymer, followed by detection using 3,3′-diaminobenzidine substrate solution (Dakocytomation, Glostrup, Denmark). Cells were counterstained with either hematoxylin or methyl green before mounting on microscope slides. Immunoblot analysis. Total HuH-7 cellular lysates (50 µg) were har-

vested for detection of endogenous cyclin A expression by immunoblot analysis with mouse anti-cyclin A monoclonal antibody (2 µg/ml; Becton Dickinson, San Jose, CA). The blot was re-probed with anti-β tubulin antibody (2 µg/ml; Becton Dickinson, San Jose, CA), and chemiluminescence detection was performed using SuperSignal West Pico Substrate (Pierce, Rockford, IL). Partial hepatectomy. Female BALB/c mice (6–8 weeks old) were pur-

chased from the Laboratory Animal Unit of the National University of Singapore. Mice were anesthetized with an intra-peritoneal injection of ketamine/xylazine (70/10 mg/kg), followed by the removal of 70% of the liver tissue as described by Higgins and Anderson.40 Forty-eight hours after PHx, 1 × 106 TU of viruses was administered via the tail vein. Regenerated liver tissues, as well as normal liver tissues from shamoperated groups, were subsequently removed and assayed for luciferase activities. Establishment of HCC xenograft and study of in vivo gene transfer efficiency. HuH-7 and HeLa cells (5 × 106) were injected subcutaneously into

4–6-week-old severe combined immunodeficient mice (Animal Resource Centre, Canningvale, Western Australia). When tumor nodules reached 40 mm3 in diameter approximately 3 weeks after inoculation, 1 × 106 TU of viruses (pApoE/hAAT-cc-Luc) was injected intra-tumorally (n = 3, experiments were performed twice). The following day, mice were anesthesized with 6 ml/kg body weight fentanyl/fluanison (Hypnorm; VetaPharma, Leeds, UK) and midazolam (Dormicum; Roche, Basel, Switzerland) in distilled water in a 1:1:2 (vol/vol/vol) (Hypnorm:Dormicum:water) ratio. Bioluminescence was measured non-invasively using a cryogenically cooled high-efficiency charge-coupled device camera system, the VersArray: 512B (Roper Scientific, Trenton, NJ). Images were taken 20 minutes after intraperitoneal injection of d-luciferin in phosphate-buffered saline (3 mg/18 g

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body weight; Invitrogen, Carlsbad, CA) at 300 seconds of exposure time, binning 1 × 1, and readout speed at 50 kHz. Image analysis and bioluminescence quantification was performed using Metavue software (Universal Imaging, Downingtown, PA). Statistical analysis. Data are presented throughout this study as mean ±

SEM. Statistical significance was determined using an unpaired t-test, and P < 0.05 was considered significant.

Acknowledgments We would like to express our thanks to Kon Oi Lian and Adrian Khoo (National Cancer Centre, Singapore) for their advice and technical assistance in performing partial hepatectomies. Special thanks go to Yoshinaga Saeki (Ohio State University Medical Centre, Columbus, OH) and Rolf Müller (Institute of Molecular Biology and Tumor ­ Research, Marburg, Germany) for providing the ICP27-deleted helper bacterial artificial chromosome and the reporter construct GalCycA, respectively. Technical support for non-invasive ­bioimaging was kindly provided by MicroLAMBDA Pte Ltd. (Singapore). This research was supported by grants from the Singapore National Medical ­Research Council.

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