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A Novel Conditionally Replicative Adenovirus Vector Targeting Telomerase-Positive Tumor Cells

¹ Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina; ² No.1 People’s Hospital, Shanghai Jiao Tong University, Shanghai, People’s Republic of China; and ³ Huaxi Medical School, Sichuan University, Chengdu, Sichuan, People’s Republic of China

	ABSTRACT

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Purpose: To develop a novel conditionally replicative adenovirus vector that targets telomerase-positive cancer cells.

Experimental Design: A telomerase gene-derived promoter was used to control the expression of the E1a gene so that the E1a gene is only expressed in telomerase-positive tumor cells. In addition, a reporter gene was also engineered into the vector so that its infection and replication can be monitored easily.

Results: A novel recombinant adenovirus vector that could selectively replicate in telomerase-positive cancer cells was made successfully. This vector showed active replication in a panel of cancer cells and minimal replication in normal human fibroblast or epithelial cells. The recombinant vector could effectively lyse various cultured tumor cells even at very low multiplicity of infection. The replication efficiency in tumor cells is over 10³-fold more than normal fibroblast and epithelial cells. In s.c. tumor models, the newly developed telomerase-selective adenovirus vectors exhibited significantly more virus replication and reporter gene expression.

Conclusions: The telomerase-targeted adenovirus vector has significant potential as an oncolytic virus as well as a tumor-specific therapeutic gene delivery vehicle.

	INTRODUCTION

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The most promising feature of the cancer gene therapy approach is the potential to exploit the genetic differences between normal and tumor cells so that effective killing of tumor cells is achieved without harming normal cells in the body. With this goal in mind, many efforts are under way to develop recombinant replication-competent virus vectors that can selectively replicate in tumor cells (1, 2, 3) . The eradication of tumor cells is achieved by virus-mediated cell lysis and subsequent infection and killing of neighboring cells. These recombinant viruses are termed "oncolytic" virus vectors (4, 5, 6, 7) .

One of the most popular approaches to achieve tumor-specific virus replication is the use of tumor-specific gene promoters to control the expression of essential virus genes. In this approach, the higher the specificity of the promoter, the more specific the engineered vector will be. One of the most universal tumor-specific genes identified thus far is the telomere reverse-transcriptase (TERT) gene. TERT is a ribonucleoprotein enzyme that is responsible for the maintenance of the normal length of telomeres (8) . In the overwhelming majority of somatic human cells, TERT is inactive (9) . Therefore, these cells have a finite life span, dictated by the length of their telomeres. Only a few normal cell types, which included the embryonic cells, germ cells, stem cells, and hematopoietic cells, have active TERT activity to enable them to divide constantly (10) . Cancer cells, on the other hand, have regained the ability to maintain a stable telomere length. This ability has been shown to be a result of the reactivation of the TERT transcription. Indeed, it was demonstrated that late stage tumors had high levels of telomerase activity. Several studies indicate that over 85% of all human tumors possess active TERT activities (11) .

The TERT promoter has been shown to mediate tumor-specific expression of a variety of cytotoxic genes. In this study, we report the successful use of the human (h)TERT (telomerase) promoter (12 , 13) to control adenovirus vector replication. The newly engineered vectors showed strong tumor-specific replication in tissue-cultured cells. Effective lysis of the tumor cells was observed. In addition, strong intratumoral replication of the vectors was observed.

	MATERIALS AND METHODS

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Cell Lines
The 293 cells for virus production were obtained from the American Type Culture Collection (Manassas, VA). The other cell lines used include prostate cancer cell lines, LNCaP and Du145; breast cancer cell lines, MD-MB-231 and MCF-7; ovarian cancer cell lines, OVCR-3 and SKOV3; and colon cancer cell lines, HCT116 and HT29. These cell lines were obtained from the Cell Culture Facility of the Duke University Cancer Center and cultured in DMEM with supplementation of 10% fetal bovine serum. Two fibroblast cell strains from the Coriell Institute (Camden, NJ) were also used. They were also cultured in DMEM with 10% fetal bovine serum. In addition, prostate epithelial cells (PrECs) were used. They were purchased from Clonetics. Medium and supplements were obtained from the vendor to culture the cells. All of the tumor cell lines are known to possess telomerase activities (11) .

Engineering of Adenovirus Vectors
To engineer the telomerase-selective adenovirus, a 400-bp promoter for the hTERT gene was first cloned into the plasmid pTOPO-1 (Clontech, Palo Alto, CA) by PCR according to information published previously (12 , 13) . The primers used for the PCR reaction were as follows: forward primer, 5'-TGGCCCCTCCCTCGGGTTACCC-3' and reverse primer, 5'-CGCGGGGGTGGCCGGGGCCAG-3'.

The DNA fragment that contains adenovirus E1 gene (kindly provided by Dr. Chinghai Kao of Indiana University) was subsequently ligated 3' to the cloned hTERT promoter. The native E1a promoter was deleted so that the hTERT promoter now controls the expression of the E1a gene. The E1b gene remained under the control of its native promoter. The hTERT-E1 gene expression cassette was then transferred into a plasmid pE1sp1A (Microbix, Toronto, Canada), which contains the adenovirus left-side-inverted terminal repeat and virus-packaging signal. The dsRed2 gene, obtained from Clontech, was excised from the plasmid pDsRed2-N1 and transferred 5' to the hTERT-E1 and 3' to the packaging signal. The derived plasmid, pE1sp1A-hTERT-E1-dsRed2 was then cotransfected into 293 cells with pBHG10 (Microbix, Ontario, Canada; Ref. 14 ). The ligation mixture was then transfected into 293 cells (with the help of liposome) for virus packaging. Active recombinant adenovirus appeared in 7–10 days in the form of "comet"-like structures (15) .

A similar procedure was used to derive the nonreplicative adenovirus Ad-dsRed2, which encodes the dsRed2 protein under the control of the cytomegalovirus promoter.

Production of Adenovirus in 293 Cells
A procedure similar to those described by Graham and Prevec (16) was followed to produce the adenovirus in large scale. Briefly, 293 cells were infected at a multiplicity of infection (MOI) of 5–10 using a total 1 x 10⁹ 293 cells. After 3–4 days, the resultant viruses were purified by double CsCI banding. In general, about 3–10 x 10¹⁰ plaque-forming units (pfu) were yielded at the end of the amplification procedure by use of 293 cells cultured in 20 150-mm Petri dishes.

In Vitro Virus Infection Evaluation
To evaluate the capacity of the newly made adenovirus vectors to infect and replicate in various tumor and normal cells, the cells were infected at various MOIs. Virus infection and replication were then evaluated in the following ways:

DsRed2 Expression.
DsRed2 expression was followed by observing the cells under a fluorescence microscope (Axioscope; Zeiss). Infected cells were also subjected to flow cytometry analysis by use of the FACScan apparatus from Becton Dickinson. It is part of the Duke University Cancer Center Core Facility.

Virus Plaque Assays.
Virus plaque assays were carried by use of lysates from cells that have been infected with adenovirus vectors. The assays were carried out in 293 cells according to a published protocol (16) .

Evaluation of Oncolytic Activity of the Virus Vector
To evaluate the ability of AdTERT-E1a-dsRed2 to lyse tumor cells specifically, tumor and normal cells were infected with the virus vector at different MOI. At days 3 and 5 after viral infection, the number of live cells was quantified by use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which measures the activity of mitochondria enzymes in live cells. It was carried out by an established protocol (17) . 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was purchased from Sigma (St. Louis, MO). The catalogue number was M-5655.

In Vivo Evaluation of Virus Infection
To evaluate the ability of adenovirus vectors to infect and replicate in vivo, we established s.c. tumors by use of LNCaP prostate cancer cells (5 x 10⁶ cells in 50 µl) and athymic Balb/C-derived nude mice (Charles River, Raleigh, NC). Virus vectors were injected into tumors when they reached sizes of 10 mm. About 5 x 10⁷ pfu of virus vectors (in 50 µl of saline) were injected directly into the tumors by use of a 30-guage needle. At different time points after injection, the tumor-bearing animals were sacrificed and their tumors excised. Some tumors were sectioned and observed under a fluorescence microscope for dsRed2 expression although others were ground up in a tissue homogenizer. The homogenates were spun down, and the supernatants were evaluated for virus titer. Virus titer was determined using the approach of Graham and Prevec (16) . All animal procedures used in these experiments were approved by the Duke University Institutional Animal Use Committee.

	RESULTS

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Successful Construction of a Telomerase-Selective Adenovirus Vector Encoding Reporter Genes.
To make a conditionally replicative adenovirus vector, we adopted a design as shown in Fig. 1 . A hTERT was used to control the expression of the adenovirus E1 gene. In addition, a reporter gene, dsRed2 (a red fluorescent protein from Discosoma) was engineered into the vector. A cytomegalovirus promoter controls it. The vector, Ad5TERT-E1a-dsRed2, was successfully made. We were able to produce the vector with high titers (3–10 x 10¹⁰ pfu/ml).

View larger version (10K): [in this window] [in a new window]   Fig. 1. A schematic diagram of the telomerase-selective adenovirus vector. TERT-P, the TERT promoter; E1a and E1b, early E1 genes of the adenovirus; dsRed2, a gene encoding the red fluorescent protein; ITR, inverted repeats of the adenovirus genome; E3, deletion of the E3 region; CMV, cytomegalovirus.

An initial series of experiments were conducted to observe virus replication in cancer cells. Because our virus vector has a red fluorescent protein (dsRed2), virus infection and replication can be easily monitored by observation of dsRed2 expression under a fluorescent microscope.

Cells were infected with Ad5TERT-E1a-dsRed2 at a MOI of 0.05 pfu/cell, 0.1 pfu/cell, 0.5 pfu/cell, 1.0 pfu/cell, and 5 pfu/cell. As a control, they were also infected with Ad-dsRed2, which was a replication-deficient virus encoding the dsRed2 gene under the control of the cytomegalovirus promoter. The infected cells were then examined under a fluorescence microscope. A rhodamine filter was used. Active virus replication was seen in all tumor cell lines but was absent in all normal cell strains. The kinetics of virus replication was different among the tumor cell lines despite robust virus replication in all tumor lines. LNCaP, Du145, HCT116, and SKOV3 exhibited vary fast virus infection and replication kinetics whereas MCF-7 and MD-MB231 exhibited somewhat slower kinetics. The evidence for Du145 is shown in Fig. 2A , where the level of red fluorescent protein is reflective of the extent of virus replication. Among the normal fibroblast and epithelial cells, none showed the same level of virus replication as the tumor cells. Ad5TERT-E1a-dsRed showed roughly the same level of fluorescence as Ad-dsRed2 (data not shown), which indicated no significant virus replication in normal cells. Flow cytometry analysis was also conducted and the results confirmed the observations with fluorescence microscopy.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Selective replication of AdTERT-E1a-dsRed2 in tumor cells. A, infection of AdTERT-E1a-dsRed2 in Du145 and prostate epithelial cells (PrECs). Both cells were infected at a multiplicity of infection (MOI) of 2 at day 0. Photomicrographs of the cells were then taken on days 3 and 8. Top panels are cells illuminated by transmitted white light. Bottom panels are the corresponding cells illuminated by UV light with a rhodamine filter. Notice the high percentage of dsRed2 expression in Du145 cells versus the sporadic pattern of dsRed2 expression in the PrECs at days 8 or 7 after viral infection. The cytopathic effects are also apparent in Du145 cells although it is absent in PrECs. The size bar represents 100 µm. B, Western blot analysis of E1a gene expression. Cells were infected with AdTERT-E1a-dsRed2 at a MOI of 2. Cells were harvested 3 days later, and the lysates were analyzed by Western blot analysis. Top panel, E1A expression. Bottom panel, ß-actin expression, which serves as a loading control. C, active replication of AdTERT-E1a-dsRed in tumor cells and the absence of it in normal fibroblast cells GM8429 and PrEC. Each cell type was infected with AdTERT-E1a-dsRed2 and Ad-dsRed2 separately at an MOI of 0.5 plaque-forming units (pfu)/cell. Three days after infection, the cells were lysed and spun down in PBS. The supernatant was evaluated for virus titer in 293 cells. The bars represent the ratio of infectious virus particles in AdTERT-E1a-dsRed2-infected cells versus nonreplicative Ad-dsRed2-infected cells.

A third assay that was used to confirm tumor-specific virus replication by use of a virus plaque-forming assay. This assay was aimed at quantifying the amount of active virus replication in infected normal and tumor cells. A nonreplicative Ad-dsRed2, which encoded the dsRed2 gene under the control of a cytomegalovirus promoter, was used as a control. Seventy-two h after infection, the infected cells were harvested, and the amount of active viruses from the freeze-thawed cell lysates was evaluated by plaque-forming assays. The ratios of virus plaques in AdTERT-E1a-dsRed2-infected cells versus Ad-dsRed2-infected cells were then plotted. The results were shown in Fig. 2 . These results indicate that Ad5TERT-E1a-dsRed2 is at least three orders of magnitude more effective than Ad-dsRed2 in all of the tumor lines tested whereas the difference between the two is negligible in normal fibroblast cells and PrECs. These results essentially confirmed the observations made in the previous section.

A further assay was conducted to evaluate the tumor-specific oncolytic ability of Ad5TERT-E1a-dsRed2. After infection with Ad5TERT-E1a-dsRed2 at different MOI and incubation for different periods of time, the amount of viable cells were evaluated by staining with crystal violet or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Fig. 3 shows the results. It is clear that Ad5TERT-E1a-dsRed2 has very strong antitumor lytic capability and very weak activities in normal fibroblast or PrECs.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Cytolytic activities of AdTERT-E1a-dsRed2 in prostate cancer cells, fibroblast cells, and normal prostate epithelial cells (PrECs). A, cells were infected with different AdTERT-E1a-dsRed2 at multiplicity of infection (MOI; in plaque-forming units (pfu)/cell) at values indicated. They were fixed in methanol at day 7 postinfection and stained with crystal violet. The presence of staining indicates the presence of live cells. B, results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays carried out in cells that have been infected with AdTERT-E1a-dsRed2 at different MOI. GM8429, a fibroblast cell strain; PrEC, prostate epithelial cells; LNCaP and Du145, two prostate cancer cell lines.

View larger version (29K): [in this window] [in a new window]   Fig. 4. A, robust replication and virus spread of AdTERT-E1a-dsRed2 in a LNCap tumor versus the limited gene expression and virus spread for nonreplicative Ad-dsRed in the same tumor. Tumors were sectioned 5 days after initial viral injections. The sections were then observed under a fluorescence microscope equipped with a rhodamine filter. The scale bars represent 2 mm. B, virus titers in tumors after injection. About 5 x 107 plaque-forming units (pfu) of Ad-dsRed2 () and AdTERT-E1a-dsRed2 () were injected into established tumors at around 10 mm in diameter. Tumors were then sacrificed and lysed at days 1 and 3 after infection. The lysates were then evaluated for virus titer in 293 cells.

	DISCUSSION

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Specificity and efficiency of viral replication in target tumor cells are the two most important parameters used to evaluate the potential of any novel oncolytic vectors. In this study, we evaluated the merits of a novel oncolytic adenovirus strategy that can potentially be targeted to telomerase activities, which is present in over 90% of all tumor types. Our results, although limited in nature (more tumor lines need to be evaluated to test the ability of the virus to replicate in vivo), proved that this vector can indeed replicate efficiently in vitro and in vivo in tumor cells. More importantly, in normal cells where telomerase activities are absent, there was minimal virus replication. Therefore, our results demonstrated the great potential of the telomerase-targeted virus approach.

The prototype oncolytic adenovirus vector is dl1520, or Onyx-015. It is an adenovirus vector that can selectively replicate in any cells that have lost the p53 tumor suppressor gene (1 , 18) . This is based on the fact that adenovirus replication requires the inactivation of the p53 gene by the viral E1b protein. Therefore, Onyx-015, which has a mutation in the E1b gene that completely knocks out its p53 inactivation capability, can replicate in cells that have already lost p53 function but not in cells with wild-type p53. Because p53 is lost in over 50% of all tumors (70% for some tumors such as colon cancer), Onyx-015 can in theory be applied for the treatment of more than half of all tumors. To date, Onyx-015 has progressed into a phase III clinical trial (19) . Synergistic efficacy with chemotherapy has also been seen in patients in a phase I and a phase II trial (20) .

Another promising strategy involves the use of tumor or tissue-specific promoters to control the expression of genes required for viral replication. A typical example is the Calydon CN706, where the prostate-specific antigen gene promoter drives the expression of adenovirus E1a gene (21) . This virus was shown to have selective toxicity in prostate cancer cells. Even higher specificity was seen in another virus CN786, where the rat prostate-specific probasin promoter drives the expression of E1a although the prostate-specific antigen promoter drives the expression of E1b (22 , 23) . Synergistic interaction with radiotherapy has also been reported (13) . Similar promoter-driven approaches have been reported by many other groups aimed at targeting different tumor types (24, 25, 26, 27) .

The TERT promoter as tested in our study demonstrated high specificity for tumor cells and high efficiency in mediating tumor cell-specific virus replication. These two characteristics make a telomerase-selective adenovirus vector very potent and promising as an oncolytic/gene delivery vector for cancer treatment. Compared with other types of conditionally replicative adenovirus vectors, such as Onyx-015, which can selectively replicate in p53-negative tumor cells (1 , 18) , and Calydon CN706, which preferentially replicates in prostate-specific antigen-positive prostate cancer, the telomerase-based conditionally replicative adenovirus can, in theory, replicate in almost any tumor cells with telomerase activity. Because telomerase activity is by far the most common tumor marker (>90% in all tumor types), the telomerase-selective adenovirus vector can be adapted to almost all solid tumor types. Therefore, compared with other types of conditionally replicative adenovirus vectors, it has a much wider range for tumor therapy. For example, p53 defect is only detected in 65% of all tumors whereas the prostate-specific antigen expression is only specific for prostate cancer patients. In addition, telomerase activity is likely correlated with the malignancy of tumors (28 , 29) . It is also known that the myc oncogene is a powerful regulator of telomerase activities (30 , 31) . Therefore, a telomerase-selective gene therapy vector may have the potential of replicating in more malignant tumor types, such as those expressing higher levels of the myc oncogene, although this has to be proven experimentally.

Taken together, the telomerase-selective replicative adenovirus vector reported here may indeed warrant additional study as a novel treatment approach to achieve significant tumor cell killing with minimal normal tissue damage.

	ACKNOWLEDGMENTS

 
We thank Dr. Chinghai Kao of Indiana University for graciously providing a plasmid encoding the adenovirus E1 genes. We also thank Dr. Michael Cook of the Duke University Cancer Center Flow Cytometry Facility for expert help on the analysis of DsRed2 expression.

	FOOTNOTES

 
Grant support: Grant CA81512 from the United States National Cancer Institute, a grant from the Komen Foundation for Breast Cancer Research, and a grant from the United States Army Prostate Cancer Research Program (DAMD17-02-1-0052).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Chuan-Yuan Li, Box 3455, Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710. Phone: (919) 681-4897; Fax: (919) 684-8718; E-mail: cyli{at}radonc.duke.edu

Received 8/29/03; revised 11/ 7/03; accepted 11/13/03.

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