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[Cancer Research 66, 372-377, January 1, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

In vivo Imaging of Adenovirus Transduction and Enhanced Therapeutic Efficacy of Combination Therapy with Conditionally Replicating Adenovirus and Adenovirus-p27

Choon-Taek Lee1,3, Yoon-Jin Lee1,3, Sung-Youn Kwon1,3, Jaeho Lee1,3, Kwang Il Kim2, Kyung-Ho Park4, Joo Hyun Kang2, Chul-Gyu Yoo1, Young Whan Kim1, Sung Koo Han1, June-Key Chung2, Young-Soo Shim1, David T. Curiel5 and David P. Carbone4

1 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Lung Institute of Medical Research Center, and 2 Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul, Korea; 3 Department of Medicine and Respiratory Center, Seoul National University Bundang Hospital, Seongnam, Korea; 4 Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee; and 5 Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama

Requests for reprints: Choon-Taek Lee, Department of Medicine, Seoul National University Bundang Hospital, 300 Gumi-Dong, Seongnam 463-707, Korea. Phone: 82-31-787-7002; Fax: 82-31-787-4052; E-mail: ctlee{at}snu.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Gene therapy is hampered by poor gene transfer to the tumor mass. We previously proposed a combination adenoviral gene therapy containing a conditionally replicating adenovirus (CRAD) expressing mutant E1 ({Delta}24RGD) and a replication-defective E1-deleted adenovirus to enhance the efficiency of gene transfer. Mutant E1 expressed by {Delta}24RGD enables the replication of replication-defective adenoviruses in tumors when cancer cells are co-infected with both viruses. In this study, gene transfer rates in xenografts tumors were monitored by bioluminescence in cells infected with the replication-defective adenovirus-luciferase (ad-luc). Tumor masses treated with CRAD + ad-luc showed dramatically stronger and more prolonged luciferase expression than ad-luc-treated tumors and this expression spread through the entire tumor mass without significant systemic spread. Transduction with CRAD + replication-defective adenovirus-p27 increased the expression of p27 by 24-fold versus transduction with ad-p27 alone. Treatment of a lung cancer cell line and of established lung cancer xenografts with CRAD + adenovirus-p27 also induced stronger growth suppression than treatment with either virus alone. These findings confirm the selective replication of E1-deleted adenovirus containing a therapeutic gene due to the presence of mutant E1 produced by {Delta}24RGD in tumors. Moreover, this replication increased the therapeutic gene transfer rate and enhanced its antitumor effects. (Cancer Res 2006; 66(1): 372-7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Low gene transfer rates in human cancers present a major obstacle to the efficacy of gene therapy. In human tumor masses, it is difficult to transfer therapeutic genes to tumor cells given the gene transfer vectors currently available. The intratumoral dispersion of replication-defective viral vectors is usually confined to the injection site and has produced impractical and ineffective clinical trial results.

A conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells has been developed to overcome this problem (1–3). This antitumor effect of CRADs depends on the oncolysis effect of adenovirus replication in tumor cells and the dissemination of adenovirus through the tumor mass. However, CRAD-based clinical studies have failed to meet expectations although some positive responses have been recorded (4–6). It seems that the majority of these viruses cannot carry a therapeutic gene due to size constraints. Furthermore, the genetic heterogeneity of tumors presents a problem as typically CRAD can only replicate in p53-mutated (7) or pRb/p16 pathway–inactivated cells or in cells expressing a specific protein [i.e., CRAD containing cancer-specific promoters such as telomerase or cyclooxygenase 2 (COX2) promoter].

The new CRAD strategy described here involves combining CRAD and E1-deleted, replication-defective adenovirus containing a therapeutic gene. We have previously shown that a conventional replication-defective adenovirus containing an E1 deletion can become replication competent when cotransduced with a CRAD capable of supplying E1 in trans. The resulting selective production of large numbers of the therapeutic adenovirus in situ within a tumor mass could transduce neighboring tumor cells and increase overall transduction efficiency (8).

This basic concept of trans-complementation was first reported with the demonstration that transduction with an E1-deleted adenovirus and transfection of plasmid containing E1 (pE1) in a cell induced the production of E1-deleted adenovirus and amplified viral transgene expression (9, 10).

Trans-complementation of two replication-defective adenoviral vectors with missing different genes was reported. Alemany et al. (11) showed that two replication-defective adenoviruses generated by splitting adenoviral genome complemented each other and replicated in certain tumor cells. Cotransduction of GT5610, an adenovirus containing minimal adenoviral genome for replication with E1, and AdHß, an adenovirus containing most adenoviral genome except E1, induced the coreplication of both and leaded to cell lysis. Wolkersdorfer et al. (12) combined AV.C2.TK, an E1-deleted nonreplicating adenovirus expressing herpes simplex thymidine kinase, and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus in malignant melanoma. Both E1-deleted virus and E4-deleted virus replicated in cancer cells by trans-complementing adenoviral gene products.

Habib et al. (13) showed that transduction with a replication competent adenovirus + replication-defective adenovirus containing reporter genes induced the replication of reporter viruses. In our previous study (8), we used a CRAD designated {Delta}24RGD, which produces a mutant E1 protein without the ability to bind Rb but which retains viral replication competence. Theoretically, this E1 can permit viral replication in cancer cells with a defective pRb/p16 pathway. To produce a replication-defective therapeutic adenovirus with an E1 deletion, we combined adenoviruses expressing a dominant negative insulin-like growth factor I receptor (IGF-IR; ad-IGF-IR/dn) with a stop codon at 950 or 482 to block IGF-I expression (14) and found that combined transduction with {Delta}24RGD and a dominant negative IGF-IR adenovirus increased transduction efficiency and therapeutic efficacy versus either approach in isolation in lung cancer xenografts (8). The theoretical advantage of this combination is that E1-deleted adenovirus with any therapeutic gene can be applied with a CRAD.

In this study, we combined an adenovirus expressing p27 [a known cell cycle–dependent kinase (CDK) inhibitor] with {Delta}24RGD in a lung cancer xenografts model. In addition, we visualized the distribution of transduced gene expression by noninvasive bioluminescent optical imaging techniques in living animals (15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant adenoviruses. {Delta}24RGD, kindly provided by David T. Curiel (Gene Therapy Center at the University of Alabama, Birmingham, AL), contains a 24-bp deletion in the CR2 region of E1A and an RGD-4C modification of the fiber gene of E3. The CR2 domain is responsible for binding pRb, which allows adenovirus-infected cells to enter the S phase. Therefore, adenoviruses with a deletion in this region can replicate only in the cells with defects in the pRb/p16 pathway where this binding is not necessary. Furthermore, the RGD-4C motif in the E3 region enables this virus to infect cells without binding to CAR (16).

We previously described the construction of adenoviruses expressing mutant p27 (ad-p27; ref. 17). Briefly, the cDNA of human p27 containing a mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) was subcloned into the KpnI and BamH1 sites of the polylinker pAC CMV pLpA (kindly provided by Robert Gerard, University of Texas Southwestern Medical Center, Dallas, TX). Adenovirus-p27 was generated by cotransfecting pAC CMV-p27mt and pJM17 into 293 cells. This ad-p27 was driven by CMVie promoter and was replication-defective due to E1 deletion. Adenovirus-luciferase (ad-luc) and adenovirus without the therapeutic gene (ad-null) were also driven by CMVie promoter and replication-defective due to E1 deletion.

Changes in the in vitro expression of luciferase by cotransduction with {Delta}24RGD. Human lung cancer cells (NCI H460) were seeded into a 24-well plate (3 x 105 per well). After incubation for 18 hours, cells were transduced with ad-luc [1 multiplicity of infection (m.o.i.)] from the first to the fifth lane. Cells from the second to fifth lane were cotransduced with 0.1, 0.5, 1, and 3 m.o.i. of {Delta}24RGD whereas cells in the first lane were transduced with ad-luc only and those in the sixth lane were transduced with {Delta}24RGD only. In vitro luciferase imaging was done by scanning (LAS-3000, 2-minute acquisition) 2 minutes after Bright Glo inoculation.

Changes in the transduction efficiency of ad-luc caused by {Delta}24RGD cotransduction by in vivo tumor imaging. Lung cancer xenografts were established by s.c. injecting human lung cancer cells (NCI H460, 1.0 x 106 per site) into nude mice. Four tumor masses were established in each animal (right and left shoulders and right and left thighs). Two weeks after injection, when tumor masses had reached 10 mm in diameter, adenoviruses were injected. PBS, {Delta}24RGD [1.0 x 107 plaque-forming units (pfu)], and ad-luc (1 x 107 pfu) were injected into tumor masses on the left shoulder, right shoulder, and left thigh, respectively, and a combination of {Delta}24RGD (1 x 107 pfu) and ad-luc (1 x 107 pfu) was injected into right thigh mass. On days 2, 5, and 14, D-luciferin was injected into the peritoneum, and 10 minutes after injection, tumor imaging was done using a charged-coupled device camera (LAS-3000) for 10 minutes (10-minute acquisition). Ratios of the luciferase activities of {Delta}24RGD + ad-luc injected tumors and ad-luc injected tumors in individual mice were calculated.

Changes of p27 expression in ad-p27 transduced lung cancer cell due to {Delta}24RGD cotransduction. NCI H460 (human lung cancer cell line) was transduced with ad-p27 alone, {Delta}24RGD, or {Delta}24RGD + ad-p27. Twenty-four hours after transduction, we compared the production of p27 protein in cells by Western blotting.

Changes in cell growth after cotransduction with ad-p27 and {Delta}24RGD. NCI H460 cells (a human lung cancer cell line) were plated into six-well plates and transduced with ad-p27 (1 m.o.i.), {Delta}24RGD (0.1 m.o.i.), or {Delta}24RGD (0.1 m.o.i.) + ad-p27 (1 m.o.i.). Cell numbers were counted daily using a hemocytometer and their survivals were compared against untransduced controls.

Combined treatment with {Delta}24RGD and ad-p27 in animal tumor models. Finally, we examined the antitumor effect of {Delta}24RGD + ad-27 in lung cancer xenografts established by injecting NCI H460 (1 x 106 per mouse) into s.c. tissue. Seven days later, ad-null (1.0 x 107 pfu), {Delta}24RGD (1.0 x 107 pfu), ad-p27 (1.0 x 107 pfu), {Delta}24RGD (1.0 x 107 pfu) + ad-null (1.0 x 107 pfu), and {Delta}24RGD (1.0 x 107 pfu) + ad-p27 (1.0 x 107 pfu) were injected intratumorally and this was repeated daily for 5 days.

Tumor sizes were measured using the formula (0.5 x length x width2). Tumor growths were analyzed using the repeated ANOVA test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased expression of luciferase in ad-luc transduced lung cancer cells due to {Delta}24RGD cotransduction. Luciferase expression from cells transduced with a low dose of ad-luc (1 m.o.i.) was low (lane 1) and almost the same as that of {Delta}24RGD transduced cells (containing no ad-luc; lane 6). However, cotransduction with {Delta}24RGD increased luciferase expression in a dose-dependent manner in the range 0.1 to 3 m.o.i. (Fig. 1).



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  		Figure 1. Luciferase expression by ad-luc transduced lung cancer cells was enhanced by cotransduction with {Delta}24RGD. Luciferase expression in cells transduced with ad-luc at 1 m.o.i. was barely detectable (lane 1). Cotransduction with {Delta}24RGD from 0.1 to 3 m.o.i. increased luciferase expression in ad-luc transduced cells in a dose-dependent manner. Lane 1, ad-luc (1 m.o.i.) only; lane 2, ad-luc (1 m.o.i.) + {Delta}24RGD (0.1 m.o.i.); lane 3, ad-luc (1 m.o.i.) + {Delta}24RGD (0.5 m.o.i.); lane 4, ad-luc (1 m.o.i.) + {Delta}24RGD (1 m.o.i.); lane 5, ad-luc (1 m.o.i.) + {Delta}24RGD (3 m.o.i.); lane 6, 24RGD (1 m.o.i.).

 
Strong and prolonged expression of luciferase was observed in tumors coinjected with {Delta}24RGD and ad-luc. Figure 2 shows increased luciferase expression in vivo in tumor masses treated with {Delta}24RGD + ad-p27. No luciferase expression was observed in two tumor masses treated with PBS (mass on left shoulder) or {Delta}24RGD (mass on right shoulder). Injection with ad-luc caused very limited luciferase transfer into tumor masses (i.e., weak and focal signals). However, injection with ad-luc + {Delta}24RGD produced strong prolonged luciferase expression for up to 2 weeks. This expression was observed throughout the tumor masses but without evidence of systemic spread, a very important characteristic of this combination therapy with respect to potential clinical application. This was true up to 2 weeks after transduction when the animals needed to be sacrificed due to large tumor burdens.



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  		Figure 2. Bioluminescence tumor imaging showed enhanced long-term luciferase expression throughout the tumor mass without systemic spread after {Delta}24RGD + ad-luc treatment. Four tumor masses established by injecting NCI H460 cells (a human lung cancer cell line) were treated with PBS (left shoulder), CRAD ({Delta}24RGD: 1.0 x 107 pfu; right shoulder), ad-luc (1.0 x 107 pfu; left thigh), and CRAD + ad-luc (right thigh). On days 2, 5, and 14, tumor luminescence was recorded using a charged-coupled device camera (LAS-3000) 10 minutes after the i.p. injection of D-luciferin. Transduction with ad-luc only induced weak focal signals (left thigh); however, strong long-term luciferase expression was observed at 2 weeks after treating with ad-luc + {Delta}24RGD (right thigh) and luciferase expression extended throughout the whole tumor mass without systemic spread. The ratios of the luciferase activities of CRAD + ad-luc injected and ad-luc injected tumors in the same mouse were 20.5 (day 2), 21.8 (day 5), and 58.7 (day 14; figures represent mean values for three mice).

 
Increased p27 expression in ad-p27 + {Delta}24RGD transduced cells. Western blots for p27 showed that cotransduction with {Delta}24RGD + ad-p27 markedly increased p27 production. The amount of p27 in {Delta}24RGD + ad-p27 cotransduced cells was 24-fold that of ad-27 transduced cells at the same ad-p27 dose (Fig. 3).



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  		Figure 3. Increased expression of p27 due to transduction with ad-p27 and CRAD ({Delta}24RGD). Twenty-four hours after transducing NCI H460 cells with ad-p27 (10 m.o.i.) + CRAD (1 m.o.i.), we measured p27 expression levels by Western blotting and found that this combined treatment increased p27 expression 24-fold versus transduction with ad-p27 alone.

 
Enhanced growth suppression of lung cancer cells by ad-p27 + {Delta}24RGD cotransduction in vitro. A single transduction with ad-p27 (1 m.o.i.) failed to show significant growth suppression versus the control. A single transduction with {Delta}24RGD (0.1 m.o.i.) showed moderate growth suppression initially but induced complete oncolysis at day 7. Transduction with ad-p27 + {Delta}24RGD produced more rapid and stronger growth suppression than {Delta}24RGD alone and induced complete oncolysis at day 5 (Fig. 4).



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  		Figure 4. Enhanced in vitro growth suppression of the lung cancer cell line (NCI H460) by transduction with CRAD ({Delta}24RGD: 0.1 m.o.i.) + ad-p27 (1 m.o.i.). Cell numbers were counted using a hemocytometer and compared with the untransduced control (relative survival). Combined transduction induced more rapid and stronger oncolysis than CRAD alone, especially from days 1 to 5 (P < 0.05).

 
{Delta}24RGD + ad-p27 had a more potent antitumor effect on established lung cancer xenografts in vivo. {Delta}24RGD injected intratumorally induced weak growth suppression. The ad-p27-treated group showed moderate growth suppression, which was similar to that shown by the {Delta}24RGD + ad-null–treated group. However, {Delta}24RGD + ad-p27 treatment induced stronger growth suppression than ad-p27 alone (P < 0.05) or {Delta}24RGD + ad-null (P < 0.05). Forty percent of {Delta}24RGD + ad-p27–treated mice showed complete regression of established tumors (Fig. 5).



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  		Figure 5. Treatment with ad-p27 + CRAD ({Delta}24RGD) more strongly suppressed the growths of established lung cancer xenografts. Intratumoral injections of ad-null (1 x107 pfu), CRAD (1 x 107 pfu), ad-p27 (1 x 107 pfu), CRAD (1 x 107 pfu) + ad-null (1 x 107 pfu), or CRAD (1 x 107 pfu) + ad-p27 (1 x 107 pfu) were made daily for 5 days into tumor masses established by injecting NCI H460 cells s.c. into nude mice (five mice per group). Ad-null showed no significant growth suppression. CRAD alone and ad-p27 alone did cause significant growth suppression versus the control (P < 0.05). Ad-null + CRAD seemed to have a greater antitumor effect than CRAD alone but this was not statistically significant. Treatment with CRAD + ad-p27 had the strongest growth-suppressing effect (P < 0.01, versus CRAD; P < 0.05, versus ad-p27; P < 0.05, versus CRAD + ad-null).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Disappointing gene therapy outcomes are primarily caused by poor gene transfer rates in human tumors. Vectors that are highly effective in culture result in minimal effects in tumors in vivo. CRAD was developed to selectively induce oncolysis and to induce adenoviral replication in tumors. However, although CRAD improves gene transfer in human tumors, its antitumor effects in human trials have not been impressive.

To potentiate the antitumor effect of CRAD, several modifications have been introduced. CRAD carrying therapeutic genes such as herpes simplex thymidine kinase (18, 19), uracil phosphoribosyltransferase (20), and p53 (21) in its E1 or E3 region showed substantially enhanced antitumor effects. CRADs that can replicate in cells expressing specific proteins have also been introduced. For example, CRAD containing a telomerase-derived promoter selectively replicated and destroyed telomerase-positive cancer cells (22, 23) and CRAD containing a COX2 promoter produced a promising result in in vivo tumor models (24, 25).

This study proposes a novel and straightforward way of increasing the therapeutic gene transfer of replication-defective adenovirus by combining with CRAD. As we previously showed, CRAD produces mutant E1 in pRb/p16–inactivated cancer cells enabling the replication of an E1-deleted adenovirus if both viruses were transduced in the same cancer with pRb inactivation. Both CRAD and the E1-deleted adenovirus expressing the therapeutic gene spread within the tumor mass and exerted their own antitumor effects. If both adenoviruses infect the same cancer cell, replication of the E1-deleted adenovirus will occur. Consequently, E1-deleted adenoviruses selectively replicate in tumor cells (Fig. 6).



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  		Figure 6. Strategy of combining CRAD ({Delta}24RGD) and an E1-deleted, replication-defective adenovirus containing therapeutic gene. When CRAD and E1-deleted adenovirus are cotransduced into same cancer cell in which pRb was inactivated, mutant E1 expressed by CRAD, and which functions normally in pRb/p16–inactivated cancer cells, helps replicate E1-deleted adenovirus. Both CRAD and the E1-deleted adenovirus expressing the therapeutic gene spread into the tumor mass and separate their antitumor effects. If both adenoviruses infect the same cancer cell again, E1-deleted adenovirus will be replicated. Consequently, E1-deleted adenovirus will selectively replicate in pRb/p16–inactivated cancer cells and spread through the tumor mass.

 
Gene transfer in vivo is not easily monitored but advances in molecular imaging may enable real-time noninvasive monitoring (15). Several gene products have been used for tumor imaging in vivo and firefly luciferase and green fluorescent protein are most commonly used for this purpose. Moreover, bioluminescence from cells transfected with luciferase can easily be monitored using a charged-coupled device camera after injecting D-luciferin, a luciferase substrate. Luciferase reacts with D-luciferin in the presence of ATP and oxygen to produce light that can be detected using a charged-coupled device camera (26). The in vivo distribution of adenovirus achieved by mediated gene therapy has been previously monitored by bioluminescence using luciferase gene transduced cells (27).

We adopted this technique to show the replication of ad-luc in tumor masses and to monitor the intratumoral and systemic viral transduction distributions and the duration of therapeutic gene expression. In Fig. 2, a single injection of ad-luc into the tumor mass showed weak, localized, and transient luciferase expression and, 14 days later, little luciferase activity remained. This finding partly explains why current gene therapies have failed to show a significant response in clinical trials. In contrast, tumors injected with ad-luc + {Delta}24RGD showed strong long-term activity for up to at least 2 weeks. Furthermore, luciferase activity was found throughout the tumor mass, showing ad-luc spread. Another important finding was the absence of luciferase activity in other organs in a mouse model. These findings support the value and safety of this gene therapy combination.

The p27 is a CDK inhibitor of the Cip/Kip family and participates at many points within the cell cycle (28). The main action of p27 arises from its binding with cyclin E/Cdk2 complex and its induction of the dephosphorylation of pRb (29, 30). Most non–small cell lung cancer cell lines contain inactivated p16 and intact pRb and are effectively killed by ad-p27 (17, 31) and by {Delta}24RGD.

The intracellular level of p27 is mainly regulated by its phosphorylation at Thr-187, which targets p27 for ubiquitination and proteosomal degradation (32, 33). We previously reported the development of an adenovirus expressing p27 with a mutation at Thr-187. This adenovirus expressing a stable mutant p27 has a more potent antitumor effects (via cell cycle arrest and apoptosis) than an adenovirus expressing wild-type p27 (17). This effect was later confirmed in lung cancer (17, 31), pancreatic cancer (34), and malignant glioblastoma (35). Thus, this adenovirus was used in the present study.

Transduction of ad-p27 + {Delta}24RGD into lung cancer cells was found to up-regulate p27 versus ad-p27-only transduced cells infected with identical doses of ad-p27. Moreover, treatment of established lung cancer xenografts produced dramatically improved therapeutic efficacy. Treatment with CRAD + ad-p27 suppressed tumor growth significantly more than CRAD, ad-p27, or CRAD + ad-null.

The lack of direct comparison of a CRAD + E1-deleted adenovirus with a therapeutic gene with a CRAD containing therapeutic gene in E1 or E3 region (armed CRAD) is the limitation of this study. Armed therapeutic adenovirus is limited with respect to the size of the inserted gene. In addition, Suzuki et al. (36) suggested that deletion of E3 for therapeutic gene insertion may decrease the oncolytic potency of CRAD. However, further study will be needed to directly compare the potencies of a CRAD + E1-deleted adenovirus with a therapeutic gene with armed CRAD.

In spite of this limitation, this study reconfirms the value of combination strategies based on CRAD and E1-deleted adenovirus containing a therapeutic gene by in vivo tumor imaging techniques in a lung cancer model and suggests that this strategy may be useful for therapies based on any E1-deleted recombinant adenovirus.


   Acknowledgments
 
Grant support: Korea Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea, grant 03-PJ1-PG3-20800-0048 and M 10534040003-05N3404-00310 by KOSEF (C-T. Lee), the Vanderbilt Specialized Program of Research Excellence in Lung Cancer grant CA90949 (D.P. Carbone), and NIH grants R01 CA083821 and R01 CA094084 (D.T. Curiel).

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.

Received 5/ 3/05. Revised 8/22/05. Accepted 10/12/05.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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