IL24

original article

© The American Society of Gene Therapy

Systemic Cancer Gene Therapy Using Adeno-associated Virus Type 1 Vector Expressing MDA-7/IL24 Ichiro Tahara1,2, Koichi Miyake1, Hideki Hanawa1, Toshiyuki Kurai1, Yukihiko Hirai1, Masamichi Ishizaki3, Eiji Uchida2, Takashi Tajiri2 and Takashi Shimada1 Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research, Center for Advanced Medical Technology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan; 2Surgery for Organ Function and Biological Regulation, Nippon Medical School Graduate School of Medicine, Tokyo, Japan; 3Department of Analytic Human Pathology, Nippon Medical School Graduate School of Medicine, Tokyo, Japan 1

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vectormediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7transduced C2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 × 1011 viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cellspecific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-­mediated systemic delivery of MDA-7/ IL24 represents a potentially important new approach to anticancer therapy. Received 21 January 2007; accepted 2 May 2007; published online 5 June 2007. doi:10.1038/sj.mt.6300225

Introduction Melanoma differentiation associated gene-7 (mda-7; approved gene symbol IL24) was identified and cloned using the differentiation induction subtraction hybridization approach after treating the HO-1 human melanoma cell line with interferon-β and mezerein, which resulted in growth arrest and ­ terminal

­ ifferentiation.1 Expression of MDA-7/interleukin-24 (IL24) prod tein is normally restricted to cells of the immune system and to melanocytes,2,3 and reports of the loss of MDA-7/IL24 ­expression during the pathological progression of melanomas, and of the significant correlation between this loss and tumor invasion, suggests that mda-7/IL24 may function as a tumor suppressor gene in melanoma.1,2,4,5 Indeed, the results of in vitro ­studies, in vivo animal studies, and a phase I clinical trial, all indicate that MDA7/IL-24 has the ability to selectively induce apoptosis in cancer cells without harming normal cells.6–8 Intriguingly, MDA-7/ IL-24 not only induces apoptosis, but also has immunomodulatory and antiangiogenic properties (MDA-7/IL24 ­bioactivity was 20-fold to 50-fold more potent than endostatin or angiostatin), as well as potent antitumor bystander effects, making it an ideal candidate for anticancer gene therapy.8–10 Inhibition of angiogenesis is a promising anticancer strategy, as the neovasculature is essential for tumor growth and metastasis.11 The potential of antiangiogenic gene therapy in cancer is currently being evaluated using both viral and nonviral vectors.12,13 Achievement of therapeutic levels of antiangiogenic factors may be accomplished by targeting nontumor cells for gene transfer, thereby using normal tissues to provide a stable platform for transgene expression and production of secretory proteins. This approach requires vectors capable of sustaining expression for the-long term, without vector-associated toxicity or immunity. In that regard, adeno-associated virus (AAV)based vectors are nonpathogenic and less immunogenic than other vectors used for gene therapy. Epidemiological studies indicate that 90% of the human population has been exposed to AAV with no known associated pathologies.14 In addition to its nonpathogenic nature, AAV mediates persistent gene expression through integration into cellular chromosomes or by conversion into double-stranded episomal forms.14,15 There are at least 12 known AAV serotypes.16 Although at present AAV serotype 2-based vectors (AAV2 vectors) are most often employed in animal experiments and clinical trials, we found that AAV1 vectors mediate a higher level of long-lasting gene expression in muscle and liver than AAV2 vectors (data not shown).

Correspondence: Koichi Miyake, Department of Biochemistry and Molecular Biology, Nippon Medical School Graduate School of Medicine, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. E-mail: [email protected] Molecular Therapy vol. 15 no. 10, 1805–1811 oct. 2007

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Figure 1  MDA-7/IL24 expression with AAV-mda7 (serotype 1 and 2) in C2C12 cells. (a) MDA-7/IL24 expression in C2C12 cells. Cell lysates were collected from C2C12 cells treated with PBS (lane 1) and after transduction with AAV1-GFP (lane 2), AAV-mda7 serotype 2 (lane 3) or AAV-mda7 serotype 1 (lane 4). Samples were run on 10% SDS-PAGE, transferred to a nitrocellulose membrane and stained with goat anti-mda7/IL-24 antibody. (b) C2C12 cells were treated with PBS (bar 1) or transduced for 72 hours with AAV1-GFP (bar 2), AAV-mda7 serotype 2 (bar 3) or AAV-mda7 serotype 1 (bar 4), after which the supernatant was assessed by enzyme-linked immunosorbent assay. *P < 0.01, **P < 0.001. AAV, adeno-associated virus; GFP, green fluorescent protein; IL, interleukin; MDA, melanoma differentiation-associated gene.

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Bystander activity of MDA-7/IL24 in vitro To evaluate the bystander effects of MDA-7/IL24, we used terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays to assess the incidence of apoptosis among tumor cells (Ehrlich ascites tumor cell, PC3, PGHAM1) (­Figure 2). We found that the tumor cells incubated in the medium conditioned by AAV1-mda7-transduced C2C12 cells showed significantly higher incidences of apoptosis than the cells incubated in the medium conditioned by phosphate-­buffered saline (PBS)or AAV1-green fluorescence protein (GFP)-­transduced cells (­Figure 2a). Figure 2b shows that whereas only a small fraction (1.9 ± 1.0 and 2.1 ± 0.9% of the total cell population, respectively) of Ehrlich ascites tumor cells incubated in the medium conditioned by PBS- or AAV1-GFP-treated cells were TUNEL-positive, a markedly higher incidence of TUNEL-­positive cells (16.8 ± 2.8% and 27.1 ± 4.1%) was seen among Ehrlich ascites tumor cells incubated in the medium conditioned by AAV1-mda7-treated cells, and similar results were obtained with PGHAM1 and PC3 cells. Thus, medium conditioned by AAV1-mda7-treated cells induces significant apoptosis among tumor cells. Assays of in vitro tube formation by human umbilical vein endothelial cells (HUVECs), cocultured with fibroblasts, ­confirmed the antiangiogenic effect of AAV1-based, vector-mediated expression of MDA-7/IL24. HUVECs, incubated in medium conditioned by AAV1-mda7-treated C2C12 cells, showed significant and MDA-7 (10 ng/ml)

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After generating an AAV vector containing the gene encoding the secretable form of human MDA-7/IL24, expression of which was driven by the CAG promoter (AAV-mda7), we initially transduced C2C12 cells using AAV-mda7 serotype 1 or 2. Then after

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Results AAV vector-mediated expression of MDA-7/IL24 in vitro

72 hours, we used Western blotting and an enzyme-linked immunosorbent assay (ELISA) to assess the capacity of the vectors to mediate expression and secretion of MDA-7/IL24. ­Following centrifugation, intracellular MDA-7/IL24 (MW: 23 kd) was detected in the cell pellets (Figure 1a), while the ­corresponding secreted MDA-7/IL24 was detected in the cultured medium (­Figure 1b). Although the viral titers of AAV-mda7 serotype 1 or 2 were equivalent (7.5 × 109 viral genomes), higher levels of intracellular and secreted MDA-7/IL24 were detected with AAV-mda7 serotype 1-treated cells than those treated with serotype 2.

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The multifunctional tumor-specific cytotoxic effects of human MDA-7/IL24 make this molecule a promising gene-based therapeutic agent for the treatment of cancer.7,17 In this study, therefore, we used an AAV1 vector for systemic delivery of mda-7/IL24 to cultured cells and animal models and investigated the antitumor activity of secreted human MDA-7/IL24 against mouse and hamster tumor cells. Our aim was to evaluate the feasibility of AAV1-mediated systemic gene therapy using mda-7/IL24.

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Figure 2  Induction of apoptosis in tumor cells. (a) Tumor cells were cultured in the medium conditioned by AAV1-mda7-treated cells, after which apoptosis was assessed using TUNEL. Parallel cultures were treated with medium conditioned by PBS- and AAV1-GFP-treated cells (control medium). (b) Numbers of apoptotic cells in the various treatment groups were counted at least 1,000 cells per sample under a light microscope; the apoptosis index was calculated as a percentage of the total number of cells scored: 1, PBS; 2, control medium; 3, MDA-7/IL24 (10 ng/ml); 4, MDA-7/IL24 (30 ng/ml). *P < 0.05, **P < 0.01, ***P < 0.001. AAV, adeno-associated virus; GFP, green fluorescent protein; IL, interleukin; MDA, melanoma ­differentiation-associated gene; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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Figure 3  Antiangiogenic activity of AAV1-mda7 in tube formation assays. (a) Photomicrographs of HUVECs cocultured with human fibroblasts were incubated in medium conditioned by C2C12 cells treated with PBS, AAV1-GFP or AAV1-mda7. Where indicated, anti-MDA-7/IL24 antibody was added to the medium collected from AAV1-mda7-treated C2C12 cells. (b) The area and length of tube-like structures were measured using an image analyzer. *P < 0.001. AAV, adeno-associated virus; Ab, antibody; GFP, green fluorescent protein; IL, interleukin; MDA, melanoma differentiation-associated gene.

dose-dependent inhibition of tube formation, as compared to cells incubated in medium conditioned by PBS- or AAV1-GFP-treated C2C12 cells. This inhibitory effect was not detected when a neutralizing antibody against MDA-7/IL24 was added to the conditioned medium (Figure 3a), indicating that the inhibition of tube formation was a specific effect of the secreted MDA-7/IL24. Statistical analysis of tube area and length (Figure 3b) confirmed that the secreted MDA-7/IL24 significantly (P < 0.001) inhibits in vitro angiogenesis.

Up-regulation of BAX and p21 and activation of STAT3 It was previously reported that MDA-7/IL24-expressing adenovirus transduced human tumor cells accumulate p21 and Bax proteins.18 Consistent with that finding, our Western blot analysis showed accumulation of both Bax and p21 in human, mouse, and hamster tumor cells (Figure 4a). It was also reported that secreted MDA7/IL24 binds to the IL-20 and IL-22 receptors and activates signal transducer and activation of transcription-3 (STAT3) or STAT1.18,19 With that in mind, w e carried out a Western blot analysis to assess the capacity of secreted human MDA-7/IL24 to activate the Janus kinase-STAT pathway in mouse and hamster tumor cells. We found that MDA-7/IL24 induces phosphorylation (i.e., activation) of STAT3 in both mouse and hamster tumor cells (Figure 4b). Generation of a mouse subcutaneous cancer model We generated a subcutaneous cancer model by injecting Ehrlich ascites tumor cells into the dorsum of mice. In all cases, the injected cells proliferated rapidly and formed a mass within approximately 1 week after transplantation. Most of the animals receiving transplants died within approximately 9 weeks after transplantation as a result of the advanced tumor growth. Serum ELISA for circulating MDA-7/IL24 levels To determine circulating MDA-7/IL24 levels, we developed a sandwich ELISA as described in the Materials and Methods. Plasma samples were then obtained from all animals before and Molecular Therapy vol. 15 no. 10 oct. 2007

Figure 4  Western blot analysis showing the up-regulation of Bax and p21 and activation of signal transducer and activation of transcription-3 (STAT3) in lysates from AAV1-mda7-treated cells. (a) Up-regulation of Bax and p21 in the indicated cell lines. Lysates were collected from cells incubated in the medium conditioned by AAV1-mda7-treated C2C12 cells. Samples were run on 10% SDS-PAGE, transferred to a nitrocellulose membrane and stained with anti-Bax or anti-p21 antibody. Parallel cultures were treated with the medium conditioned by PBS- and AAV1-GFPtreated cells. 1, PBS; 2, control medium; 3, MDA-7/IL24 (10 ng/ml); 4, MDA-7/IL24 (30 ng/ml). (b) STAT3 activation. Lysates were collected from tumor cells (PC3, Ehrlich ascites and PGHAM1) after treatment with the medium lacking MDA-7/IL-24 (–) or with the medium containing MDA7/IL-24 (30 ng/ml) for 10 minutes, 1 and 4 hours. Western blot analysis was carried out as previously described using anti-phospho-STAT3 and anti-STAT3 antibodies. pSTAT refers to phosphorylated STAT3; tSTAT refers to total STAT3; 1: no treatment; 2: 10 minutes; 3: 1 hour; 4: 4 hours after treatment. AAV, adeno-associated virus; GFP, green fluorescent protein; IL, interleukin; MDA, melanoma differentiation-associated gene.

7, 10, 14, 21, 35, 49, and 56 days after Ehrlich ascites tumor cell implantation and were subjected to ELISA. We found that following intravenous administration of AAV1-mda7, the concentration of MDA-7/IL24 in plasma gradually increased, reaching a plateau (200 ng/ml) 2 weeks after vector injection. Thereafter, plasma MDA-7/IL24 levels remained stable for up to 3 months during the observation period (Figure 5a).

Inhibition of tumor growth after administration of AAV1-mda7 The growth kinetics of Ehrlich ascites tumors and survival rates among mice are shown in Figure 5b and c, respectively. We found that injection of AAV1-mda7 exerted a significant protective effect not seen with injection PBS or AAV1-GFP. The mean tumor volumes were significantly smaller in animals that received mda-7/IL24 than in those that received either PBS or AAV1-GFP (P < 0.001). In addition, polymerase chain reaction (PCR) analysis using mda-7/IL24-specific primers revealed mda-7/IL24 to be present in the liver, kidneys, and spleen following intravenous injection, but not in the tumor itself, indicating that suppression of tumor growth was caused by an antitumor bystander effect (Figure 5d). Administration of AAV1-mda7 enhances induction of apoptosis in vivo To assess the capacity of MDA-7/IL24 to mediate tumor cell apoptosis in vivo, we carried out TUNEL analysis of Ehrlich ascites tumors harvested from animals 8 weeks after their treatment with 1807

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Figure 6  Administration of AAV1-mda7 enhances apoptosis among tumor cells and reduces tumor vascularization. (a) Photomicrographs of Ehrlich ascites tumors harvested from mice 7 weeks after intravenous administration of PBS, AAV1-GFP, or AAV1-mda7 and analyzed for apoptosis using TUNEL (original magnification, ×400). (b) Numbers of apoptotic cells in the indicated treatment groups were determined by counting at least 1,000 cells per sample under a ×200 light microscope; the apoptosis index was calculated as a percentage of total number of cells scored. *P < 0.001. (c) Photomicrographs of subcutaneous Ehrlich ascites tumors harvested from mice 7 weeks after intravenous administration of PBS, AAV1-GFP or AAV1-mda7 and analyzed for platelet/endothelial cell adhesion molecule 1 (PECAM1) expression. Arrows indicate PECAM1-positive cells (original ­magnification, ×200). (d) Microvessel density. The density of microvessels staining positive was determined by counting under a light microscope. *P < 0.001. AAV, adeno-associated virus; GFP, green fluorescent protein; IL, interleukin; MDA, Melanoma differentiation-associated gene; TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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Figure 5 Time course of plasma MDA-7/IL24 concentrations, tumor volumes, and survival percentage, and vector distribution after intravenous injection of AAV1-mda7. Mice first received a subcutaneous injection of 3 × 107 Ehrlich ascites tumor cells (six mice per group) and then an intravenous injection of PBS or 2 × 1011 viral genomes (vg) of AAV1-GFP or AAV1-mda7 1 week later. (a) Plasma levels of human MDA-7/IL24 measured by enzyme-linked immunosorbent assay before tumor cell implantation and 7, 10, 14, 35, 49 and 56 days after injection. (b) Average tumor volume. *P < 0.001 versus PBS and AAV1-GFP control groups. (c) Survival rate. (d) ­Vector distribution after intravenous injection of AAV1-mda7. Genomic DNA was extracted from organs and analyzed by PCR using primers specific for the mda-7/IL24 gene or β-actin, which served as a control. AAV, adeno­associated virus; bp, base pair; GFP, green fluorescent protein; IL, ­interleukin; MDA, melanoma differentiation-associated gene.

AAV1-mda7, PBS, or AAV1-GFP. We found TUNEL-positive cells to be scattered throughout the histological sections, especially in specimens from the AAV1-mda7-treated mice (Figure 6a). Moreover, Figure 6b shows that the incidence of apoptosis was markedly higher (P < 0.001) among tumor cells in mice administered AAV1-mda7 (31.9 ± 5.5%) than among those administered PBS (4.5 ± 1.9%) or AAV1-GFP (3.2 ± 1.1%).

Administration of AAV1-mda7 suppresses tumor microvessel density An earlier report suggested that Ad-mda7 suppresses tumor growth by inhibiting angiogenesis detected on the basis of 1808

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­platelet/endothelial cell adhesion molecule 1 (PECAM1) staining.20 We therefore evaluated the antiangiogenic effect of AAV1-mda7 by harvesting Ehrlich ascites tumors 8 weeks after implantation and staining deparaffinized sections for PECAM1 to assess tumor microvessel density (Figure 6c). The average microvessel counts per ×400 microscope field in the PBS- and AAV1-GFP-treated tumors were 23.8 ± 4.3 and 27.3 ± 3.6 respectively. By contrast, tumor microvessel density was significantly (P < 0.001) diminished in tumors from mice administered AAV1-mda7, which showed an average microvessel count of only 8.8 ± 3.6 (Figure 6d).

Discussion MDA-7/IL24 has a number of attributes that would make it an effective therapeutic agent for the treatment of cancer, including the abilities to discriminate between normal and cancer cells, to induce apoptosis in diverse tumor cell types, to promote antitumor bystander effects, to inhibit tumor growth and angiogenesis, to act synergistically with radiation, and to modulate immune responses.7,17,21 Adenoviral vector-mediated expression of MDA7/IL24 (Ad-mda7) was previously reported to suppress tumor growth via direct transduction of tumor cells.8,10,22 More recently, however, it also was demonstrated that secreted MDA-7/IL24 exerts antitumor bystander effects after administration of Admda7.18,23 In this study, we focused on the antitumor bystander effects of secreted MDA-7/IL24, given its potential utility in development of a systemic anticancer strategy. Here, we showed that systemic administration of AAV-mda7 significantly and selectively inhibits tumor growth and induces apoptosis, indicating that AAV-mediated expression of secreted MDA-7/IL-24 also www.moleculartherapy.org vol. 15 no. 10 oct. 2007

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possesses potent bystander antitumor activity. Thus, systemic administration of AAV-mda7 is potentially useful for suppressing not only primary tumors but also metastatic ones. We chose an AAV1 vector to mediate expression of secreted MDA-7/IL24 in part, because AAV vectors are nonpathogenic and less immunogenic than adenoviral vectors. In addition, AAV vectors more efficiently transduce to both liver and muscle cells, two major targets for systemic cancer gene therapy, than other viral and nonviral delivery vehicles. To be clinically applicable, viral vectors should be expressed for a prolonged period and, of course, they must be safe. It has been reported that approximately 90% of the human population has been exposed to AAV2 and would likely have developed antibodies against it, which would be expected to diminish its capacity for sustained transduction. By contrast, because AAV1 was isolated from rhesus monkeys,24 and primate viral vectors are generally not recognized by prevailing antibodies in humans, it is less likely to cause adverse immunological side effects. Site-specific mutagenesis induced by integration of the viral vector into the cellular chromosome is potentially problematic. However, although AAV vector genomes have the capacity to integrate into the cellular chromosome, the earliest in vivo transgene expression is derived from episomal vectors,25 and at least in nondividing cells, integration is not required for transgene expression or for stability of the vector; indeed ≥90% of persisting vector DNA in the liver is reportedly not integrated.26,27 We therefore think the potential for AAV1 to induce mutagenesis is very low, as compared to retroviral vectors. Another risk associated with systemic administration of AAV vectors is germline transmission. Schuettrumpf et al. detected a dose-dependent increase in PCR-positive vector sequences in semen samples.28 Thus, germline transmission will need to be carefully evaluated before clinical usage. Using PCR, we detected the AAV vector genome in the liver, kidneys, and spleen following intravenous injection, but not in the tumor itself. Wu et al. recently suggested that α2,3-linked or α2,6linked sialic acid, which is different from the receptors of other serotype of AAV vectors (e.g., AAV2), may be the receptor mediating AAV1 cellular transduction.29 We suggest that the availability of these receptors play a significant role in the transduction profile of liver and kidney cells by AAV1. Antiangiogenic therapy is a promising approach to the control of solid tumor growth and metastasis. Antiangiogenic gene therapy offers the advantages of maximized cost effectiveness and the sustained therapeutic levels of antiangiogenic factors necessary for antitumor efficacy.30 Regulation of tumor angiogenesis occurs through a balance of angiogenic (e.g., vascular endothelial growth factor and β fibroblast growth factor) and antiangiogenic (e.g., angiostatin) factors produced by the tumor.31 MDA-7/IL24 can affect angiogenesis by inhibiting endothelial cell differentiation and by blocking the activities of VEGF and transforming growth factor-β via inhibition of src activity within tumor cells.21 The antiangiogenic activity of secreted MDA-7/IL24 may be mediated via the heterodimeric IL-22R1/IL-20R1 receptor complex20 and has proven to be 20-fold to 50-fold more potent than that of endostatin or angiostatin.18,20 We reported previously that AAV-mediated systemic expression of endostatin suppresses primary pancreatic tumors as well as metastatic liver tumors in Molecular Therapy vol. 15 no. 10 oct. 2007

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a ­ Hamster pancreatic cancer model.13 That MDA-7/IL24 shows stronger antiangiogenic activity than endostatin, suggests AAVmda7-­mediated systemic cancer gene therapy may represent a powerful new tool with which to treat cancer. In this study, we observed dose-dependent increases in the incidence of TUNEL-positivity among human PC3 tumor cells upon exposure to secreted human MDA-7/IL24. Apoptotic cells also were observed among both mouse Ehrlich ascites tumor cells and hamster PGHAM1 cells. Ad-mda7 induces a change in the ratio of proapoptotic to antiapoptotic members of the Bcl-2-gene family, including Bax and Bcl-2, resulting in diminished survival among multiple tumor cell types.5,22 Our Western blot analysis showed that both Bax and p21 accumulated in human, mouse, and hamster tumor cells. Thus, secreted human MDA-7/IL24 also appears to exert a significant proapoptotic effect. Many classical cytokines act by binding to receptors and activating the Janus kinase-STAT pathway. The signal transduction pathway(s), via which MDA-7/IL-24 exerts its bystander antitumor effects, remain unclear.20,23 Some studies have shown it to bind to a heterodimeric receptor complex comprised of IL-20R1 and IL-20R2,32,33 but it remains unknown whether both subunits are required for MDA-7/IL-24-mediated signaling events to occur. In any event, recent findings show that the binding of MDA-7/IL-24 to its receptor leads to activation of STAT3 and, to a lesser extent, STAT1.34 In this study, we found that human MDA-7/IL24 induces phosphorylation (i.e., activation) of STAT3 in human, mouse, and hamster tumor cells. We do not yet know, which receptor the secreted human MDA-7/IL24 binds to, but we suggest, it binds to and activates a component of the Janus kinase-STAT pathway. Additional studies have demonstrated that mda-7/IL24 can kill cancer cells in a Janus kinase/STAT-independent manner,35 and it can also kill cancer cells by an intracellular mechanism that is receptor-independent.36,37 These findings suggest an added level of complexity relative to the ability of mda-7/IL24 to induce cancerspecific cell apoptosis.21 In summary, we have shown here that AAV1-mediated expression of secretable human MDA-7/IL24 inhibits tumor growth and angiogenesis and induces apoptosis. Recently, Miyahara et al. reported that human MDA-7/IL24 induces antitumor immunity in a syngeneic murine model.38 Thus, MDA-7/IL24 has a number of attributes that would make it an effective therapeutic agent for the treatment of cancer, and our findings suggest AAV vector– mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to cancer gene therapy.

Materials and Methods Plasmid construction and vector production. Human MDA-7/IL24

com­plementary DNA (cDNA) (Accession BE788472) contained within Integrated Molecular Analysis of Genomes and Expression (IMAGE). cDNA Clone was purchased from Invitrogen Japan (Clone No. 3878467, Tokyo, Japan). To construct an AAV expression vector plasmid, human MDA-7/IL24 cDNA was amplified from I.M.A.G.E. by PCR using the oligonucleotide primers 5′-TTTCAATTGACCATGAATTTTCAACA­ GAGGCTGC-3′, and 5′-TTTCAATTGTCAGAGCTTGTAGAATTT­ CTGC-3′, which contain a MunI site (EcoRI compatible cohensive ends). After MunI digestion, the MDA-7/IL24 cDNA fragment was used to replace the EcoRI–EcoRI fragment, containing human α-galactosidase A cDNA within the AAV plasmid pCAaGBE,39 which contains the

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CAG promoter and the GFP gene driven by the B19 promoter. We used pAAV-GFP ­containing the GFP gene driven by the CAG promoter and the neomycin resistance gene driven by the herpes simplex virus thimidine kinase promoter as a control.13 The AAV vector-stocks were generated using an adenovirus-free triple transfection method. Briefly, the cis AAV vector plasmid (with AAV inverted terminal repeats), the trans plasmid (with the AAV type 2 Rep gene and type 1 Cap gene; p5E18RXC1; a gift from J. Wilson), and a helper plasmid (with an essential region from the adenovirus genome; pHelper, Stratagene, La Jolla, CA) were cotransfected into human embryonic kidney 293 cells at a ratio of 1:1:1 using calcium phosphate precipitation. After 6 hours of transfection, the medium was replaced with fresh culture medium, and the cells were incubated additional 50–60 hours at 37 °C under a 5% CO2 atmosphere. The cells were then scraped from the culture dishes, pelleted by centrifugation, resuspended in PBS (Sigma-Aldrich, St. Louis, MO), and subjected to three cycles of freezing and thawing. The resultant cell debris was centrifuged at 3000g for 20 minutes at 4 °C, after which the AAV vectors were purified by ammonium sulfate precipitation and iodixanol continuous gradient centrifugation.40 The genome titer of the AAV vector was determined by slot blot hybridization using a human mda-7/IL24 probe. Cell culture. Human embryonic kidney 293 and C2C12 mouse myo-

blast cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO). The PC3 human prostate cancer cell line and the mouse Ehrlich ascites tumor cell line were purchased from Japan Health Science Foundation (Tokyo, Japan) and cultured as previously described.41 The PGHAM1 cell line derived from hamster pancreatic cancer cells induced with N-nitrosobis (2-oxopropyl) amine42 were grown as previously described.20 Western blot analysis. Expression of MDA-7/IL24, Bax and p21, and

activation of STAT3 were analyzed by Western blotting. The cells were washed once and resuspended in PBS, after which they were boiled for 5 minutes in an equal volume of 2% sodium dodecyl sulfate (SDS) and sonicated on ice. The proteins in the samples were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked for 60 minutes with blocking buffer Tris-buffered saline (10 mmol/l Tris-Cl, 150 mmol/l NaCl, pH 8.0) supplemented with 0.1% Tween 20 (TBST) and 5% nonfat milk and then incubated with the primary antibodies against MDA7/IL-24, p21 (Santa Cruz Biotechnology, Santa Cruz, CA), Bax (Upstate, Charlottesville, VA), phospho-STAT3 (PY705), total-STAT3 or β-actin (BD Bioscience Pharmingen, Rockville, MD) in blocking buffer. Thereafter, the membranes were washed three times in TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, the membranes were incubated with ECL reagent (GE Healthcare BioSciences Corp., Piscataway, NJ) for 5 minutes at room temperature to develop the blots. Detection of apoptotic cells and antiangiogenesis. Apoptotic cell death

by the effect of medium conditioned by AAV1-mda7-treated cells was assessed by TUNEL assay43 using a DeadEnd Colorimetric TUNEL system (Promega, Madison, WI) according to the manufacturer’s instructions. The tumor cells were seeded into 6-well plates (3 × 105 cells/well) and incubated for 24 hours in standard normal culture medium. The culture medium was then replaced with medium conditioned by C2C12 cells that were treated with PBS, AAV1-GFP, or AAV1-mda7, after which the cells were ­incubated for an additional 3 days, and subjected to TUNEL assay. To evaluate the antiangiogenic activity of AAV1-mda7, in vitro tube formation assays were carried out using an angiogenesis kit (Kurabo, Okayama, Japan) as described previously.44 Briefly, HUVECs cocultured with human fibroblasts were treated with medium conditioned by PBS-, AAV1-GFP-, or AAV1-mda7-treated C2C12 cells. In some cases,

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anti-MDA-7/IL24 antibody (500 ng/ml) (R&D Systems, Minneapolis, MN) was added to the medium conditioned by AAV1-mda7-treated C2C12 cells (30 ng/ml). After 10 days, the cells were fixed and then incubated with mouse antihuman CD31 antibody (Kurabo, Okayama, Japan) and stained with metal-enhanced 3,3′-diaminobenzidine tetrahydrochloride. Total area and tube length were quantified using an angiogenesis image analyzer (Kurabo, Okayama, Japan). ELISA. The ELISA reaction used to detect human MDA-7/IL-24 was

run in 96-well plates using standard techniques and an antibody pair selected for sensitivity. Briefly, plates were coated with a polyclonal antibody (I) against MDA-7/IL-24 (R&D Systems, Minneapolis, MN) overnight at 4 °C in a standard sodium carbonate coating buffer, after which they were blocked for 2 hours at room temperature in blocking buffer (PBS containing 1% bovine serum albu­min and 1% thimerosal). Samples or recombinant MDA-7/IL-24 (R&D Systems, Minneapolis, MN) were diluted in diluent buffer (blocking buffer with 1% Tween 20), added to the plates and incubated for 2 hours at room temperature in ­diluent containing 2% nonfat dry milk. After extensive washing with 0.1% Tween 20 in PBS, a biotinylated polyclonal antibody (II) against MDA-7/IL-24 (R&D Systems, Minneapolis, MN) was added to the plate and incubated for 1 hour at room temperature. After washing, horseradish peroxidase-­streptavidin (Amersham Life Science, Piscataway, NJ) was added to the plate for 30 minutes at room temperature. The reaction was developed by addition of TMB microwell peroxidase substrate (Moss, Belfast, ME) and stopped with 1 N H2SO4 after 10 minutes. The OD450 was then recorded using a microtiter plate reader (Dynatech, Chantilly, VA). Animal experiments. Ehrlich ascites tumor cells (3 × 107 cells/mouse) in

100 µl of PBS were subcutaneously injected into the dorsum of male DDY mice (4 weeks old). One week later, the mice were administered AAV vectors in PBS. In one group, the AAV1-mda7 vector (2.0 × 1011 viral genomes) suspended in 100 µl of PBS was injected into the tail vein after anesthetization with diethyl ether. Control mice received intravenous injection of PBS or AAV1-GFP. After implantation of the tumor cells, tumor size was measured each week using a caliper; tumor volume was calculated using the formula 1/2 × larger diameter × (smaller diameter)2. At the end of the experiment (8 weeks after implantation), the animals were killed and the tumors were removed for histological examination. Additional set of the experiment was performed to evaluate survival of the mice after tumor implantation and administration of AAV vectors or PBS. All animal experiments were performed according to the regulations established by the Ethical Committee of Nippon Medical School. PCR. To evaluate the distribution of AAV1-mda7 following intravenous

injection, genomic DNA were extracted from selected organs 2 weeks after mice were administered PBS, AAV1-GFP, or AAV1-mda7. The DNA samples were then analyzed by PCR using primers that target a 329-base pair region derived from the human mda-7/IL24 gene (5′-CGGATGCTGAGA GCTGTTAC-3′ (sense) and 5′-TCAGAGCTTGTAGAATTTCTGC-3′ (antisense)). The PCR protocol entailed 1 cycle of 94 °C for 5 minutes; 30 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds and 72 °C for 1 ­minute; and 1 cycle of 72 °C for 5 minutes. The integrity of the DNA was deter­ mined by amplifying a 232-base pair region of the mouse β-actin gene using the appropriate primers (β-actin-1, 5′-CATTGTGATGGACTCCGG AGACGG-3′ and β-actin-2, 5′-CATCTCCTGCTCGAAGTCTAGAGC-3′).45 Histological and immunohistochemical examination. To assess apop-

tosis and microvessel density in vivo, we carried out TUNEL assays and PECAM1 staining on mda-7/IL24-treated or untreated tumors 8 weeks after implantation of Ehrlich ascites tumor cells. The materials and methods used for tumor implantation and TUNEL staining are described above. PECAM1 staining of endothelial cells was carried out using a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. Briefly, deparaffinized sections (4 µm) were www.moleculartherapy.org vol. 15 no. 10 oct. 2007

© The American Society of Gene Therapy

rinsed with ­methanol-hydrogen peroxide and then placed in citric buffer and irra­diated with microwaves. The sections were then incubated first with primary anti-PECAM1 antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and then with biotinylated secondary antibody at room temperature. For visualization, the sections were incubated with avidin-biotin complex for 30 minutes at room temperature, after which 3,3′-diaminobenzidine ­ tetrahydrochloride was added as the chromogen. Finally, the sections were counterstained with hematoxylin. Microvessels were detected through substrate reaction with diaminobenzidine.46 At least three microscope fields were counted per animal, and the average was taken as the microvessel density of each Ehrlich ascites tumor.47 Statistical analysis. Student’s t test was used to evaluate differences bet­

ween groups with respect to numbers of apoptotic cells, tumor volume, and microvessel density.

Acknowledgments This work was supported in part by grants from the Ministry of Health, Labour, and Welfare of Japan, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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