Cloning of carrier cells infected with oncolytic adenovirus driven by midkine promoter and biosafety studies.

BACKGROUND: A549 carrier cells infected with oncolytic adenovirus can induce complete tumor reduction of subcutaneous ovarian tumors but not intraperitoneal disseminated ovarian tumors. This appears to be a result of the insufficient antitumor effect of A549 carrier cells. Therefore, in the present study, we cloned a novel carrier cell with the aim of improving the antitumor effects. METHODS: Carrier cells infected with oncolytic adenovirus AdE3-midkine with a midkine promoter were cloned by limiting dilution. We examined the antitumor effects of these cells on subcutaneous and intraperitoneal OVHM ovarian tumors in a syngeneic mouse model. Biosafety tests were conducted in beagle dogs and rabbits. RESULTS: We cloned EHMK-51-35 carrier cells with 10-fold higher antitumor effects compared to A549 carrier cells in vitro. EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-mGM-CSF induced a 100% complete tumor reduction in subcutaneous tumors and a 60% reduction of intraperitoneal disseminated tumors. Single-dose acute toxicity test on beagle dogs with EHMK-51-35 carrier cells co-infected with AdE3-midkine and Ad-cGM-CSF showed no serious side effects. Biologically active adenoviruses were not detected in the blood, saliva, feces, urine or whole organs. In a chronic toxicity test, VX2 tumors in rabbits were injected five times with EHMK-51-35 carrier cells infected with AdE3-midkine and these rabbits showed no serious side effects. CONCLUSIONS: Significant antitumor effects and safety of cloned EHMK-51-35 carrier cells were confirmed in intraperitoneal ovarian tumors and toxicity tests, respectively. These findings will be extended to preclinical efficacy studies using dogs and cats, with the aim of conducting human clinical trials on refractory solid tumors.

Enantioselective synthesis of ß-amino acid derivatives via nickel-promoted regioselective carboxylation of ynamides and rhodium-catalyzed asymmetric hydrogenation.

We succeeded in the development of a new method for enantioselective synthesis of α-substituted-ß-amino acid derivatives. Thus, nickel(0)-promoted carboxylation of ynamide gave the α-substituted-ß-aminoacrylate derivative in a highly regioselective manner. Then, rhodium-catalyzed asymmetric hydrogenation of the α-substituted ß-aminoacrylate produced the corresponding α-substituted ß-amino acid derivative as an optically active form.

Antitumor effect of chondroitin sulfate-coated ternary granulocyte macrophage-colony-stimulating factor plasmid complex for ovarian cancer.

BACKGROUND: Although replication-competent viruses have been developed for treating cancers, their cytotoxic effects are insufficient as a result of infection inhibited by the generation of neutralizing antibodies, and systemic administration is difficult as a result of the life-threatening serious side-effects of virus-induced cytokine surge. To overcome these critical problems, we devised a plasmid/polycation/polyanion complex and assessed the potential of ternary plasmid complexes coated with chondroitin sulfate in gene therapy for ovarian cancer. The antitumor effects of chondroitin sulfate-coated complex as an anionic component were compared with those of hyaluronic acid on ovarian cancer. METHODS: Plasmid harboring the gene of murine granulocyte macrophage-colony-stimulating factor (mGM-CSF) was complexed with polyethyleneimine (PEI) and hyaluronic acid or chondroitin sulfate. Murine ovarian cancer cells were injected into (C57BL/6 × C3H/He) F(1) mice to prepare a subcutaneous or intraperitoneal tumor model. RESULTS: DNA/PEI was charged positively and DNA/PEI/chondroitin sulfate or DNA/PEI/hyaluronic acid was charged negatively. Plasmid-green fluorescent protein (GFP)/PEI coated with 10-kilodalton (kDa) chondroitin sulfate increased transfection efficiency compared to coating with chondroitin sulfate of higher-molecular-weight or hyaluronic acid. The transfection efficiency of GFP/PEI/10-kDa chondroitin sulfate in ovarian cancer cells was six-fold higher than that in normal cells. Intraperitoneal injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate prolonged survival compared to that coated with hyaluronic acid. Intratumoral injection of mGM-CSF/PEI coated with 10-kDa chondroitin sulfate achieved mouse survival rates of 100%, although that with hyaluronic acid did not. CONCLUSIONS: These findings suggest that GM-CSF/PEI coated with 10-kDa chondroitin sulfate has the potential for use in gene therapy of ovarian cancer.

Intravenous injection of irradiated tumor cell vaccine carrying oncolytic adenovirus suppressed the growth of multiple lung tumors in a mouse squamous cell carcinoma model.

BACKGROUND: Although cancer therapy using replication-selective oncolytic adenoviruses has been available for many years, its anti-tumor efficacy is suboptimal as a result of low and nonspecific infectivity that depends on coxsackie adenovirus receptor expression of the target cancer and normal cells, and generation of an anti-adenovirus neutralizing antibody. In addition, concerns of triggering a severe innate immune response against the adenovirus limit the systemic administration. We developed the carrier cell-based oncolytic virus system (CBOVS) using irradiated tumor cells as carrier cells and concealing the adenovirus (Ad-IAI.3B) inside to improve the specific infectivity. We investigated the anti-tumor effect of CBOVS in a multiple lung tumor mouse model. METHODS: The ability of CBOVS to infect Ad-IAI.3B to the target cancer cells was examined in vitro in the presence of anti-adenovirus antibodies. To evaluate the systemic effect of CBOVS, we intravenously injected CBOVS into mice with lung tumors (KLN205 cell lines). RESULTS: CBOVS enhanced the infectivity of Ad-IAI.3B to tumor cells in the presence of anti-adenovirus antibodies in vitro. Intravenous injections of CBOVS produced an accumulation of the adenovirus in the lung-bearing tumors and produced a strong anti-tumor effect in vivo. Furthermore, lymphocytes collected from the CBOVS-treated mice induced an increase in cytokines related to the Th1 response (interferon-γ, interleukin-12) by pulsing with KLN205. CONCLUSIONS: These findings suggest that CBOVS could protect adenoviruses from neutralizing antibodies and systemically deliver them to lung tumors. Furthermore, CBOVS appears to have potential as a tumor cell vaccine that activates cytotoxic immunity against cancer cells.

Cancer Cell-specific Transfection of hCas9 Gene Using Ad5F35 Vector.

BACKGROUND: The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. MATERIALS AND METHODS: We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. RESULTS: hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. CONCLUSION: Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.

Microbial Antigen-Presenting Extracellular Vesicles Derived from Genetically Modified Tumor Cells Promote Antitumor Activity of Dendritic Cells.

Tumor-derived extracellular vesicles (EVs), as tumor vaccines, carry tumor-associated antigens (TAAs), and were expected to transfer TAAs to antigen-presenting cells. However, treatment with tumor-derived EVs exhibited no obvious antitumor effect on the established tumors, likely due to their immuno-suppressive functions, and also to the poor immunogenicity of TAAs. In order to improve the immune stimulating properties, EVs expressing a highly immunogenic bacterial antigen, 6 kDa early secretory antigenic target (ESAT-6), from Mycobacterium tuberculosis were prepared by genetically modifying the parent tumor cells with a plasmid coding for ESAT-6. Cultured B16 tumor cells were transfected with a ternary complex system consisting of pDNA, polyethylenimine (PEI), and chondroitin sulfate. The cells that were transfected with the ternary complex secreted EVs with a higher number of ESAT-6 epitopes than those transfected by a conventional DNA/PEI binary complex, due to the low cytotoxicity, and durable high expression efficiency of the ternary complex systems. The EVs presenting the ESAT-6 epitope (ESAT-EV) were collected and explored as immune modulatory agents. Dendritic cells (DCs) were differentiated from mouse bone marrow cells and incubated with ESAT-EV. After incubating with the EVs for one day, the DCs expressed a significantly higher level of DC maturation marker, CD86. The DCs treated with ESAT-EV showed a significantly improved antitumor activity in tumor-bearing mice.

Carrier cell-mediated cell lysis of squamous cell carcinoma cells by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus.

BACKGROUND: The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes: SCCA1 and SCCA2. SCCA1 gene is up-regulated in squamous cell carcinoma cells. We analyzed the proximal region of the SCCA1 promoter and the antitumor effect of oncolytic adenovirus driven by the SCCA1 promoter in squamous cell carcinoma cells. METHODS: The SCCA1 promoter was analyzed by dual luciferase assay and substituted with the E1A promoter to construct the oncolytic adenovirus to determine the squamous cell carcinoma-specific cell lysis. RESULTS: Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at -525 to -475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by four-fold. Oncolytic adenovirus driven by this SCCA1 enhancer-promoter complex specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of syngeneic squamous cell carcinoma cell tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. CONCLUSIONS: SCCA1 was up-regulated in squamous cell carcinoma cells and oncolytic adenovirus driven by SCCA1 promoter specifically killed these cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma.

Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer.

We evaluated the long-term safety and efficacy of Ad-OC-TK (recombinant adenoviral vector carrying an osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) gene therapy for hormone-refractory prostate cancer. Ad-OC-TK/VAL therapy is the first in vivo adenovirus-mediated gene therapy to be used to treat metastatic prostate cancer, including bone metastasis. Six patients were enrolled in this trial, and two doses of Ad-OC-TK (2.5 x 10(9) or 2.5 x 10(10) plaque-forming units) were injected into locally recurrent tumor or bone metastasis on day 1 and day 8. Patients were also given VAL (3 g/day) for 21 days. Safety and efficacy were evaluated for at least 8 months in each patient. All patients tolerated this therapy with no serious adverse events. One prostate-specific antigen (PSA) response (from 318.3 to 4.9 ng/ml) was observed with a time to PSA progression (TTP) of 12 months. Docetaxel (30 mg/m2 per week) and estramustine (560 mg/day) combination chemotherapy (DE) was given to three docetaxel-naive patients on PSA failure after gene therapy. All three patients had a PSA response to DE therapy with 21, 7, and 4 months of TTP. These results suggest that additional trials are warranted.

PTEN gene transfer suppresses the invasive potential of human malignant gliomas by regulating cell invasion-related molecules.

Loss of function of the tumor suppressor gene PTEN is more frequently encountered in high-grade malignant gliomas than in low-grade gliomas. High-grade gliomas are characterized by their extremely invasive behavior, suggesting that PTEN is one of the important regulators of cell motility and that alterations of its coding gene contribute to a much more invasive tumor cell phenotype. In order to clarify a role of PTEN in glioma invasion, we introduced the wild-type PTEN gene into human malignant glioma cell lines and investigated their motile and invasive activity in a brain slice model that presents circumstances analogous to normal brain conditions in vivo. In addition, we analyzed biochemical and molecular changes resulting from the transfer of PTEN in the glioma cells. Infection of recombinant replication-defective adenovirus vector containing the wild-type PTEN cDNA (Ad5CMV-PTEN) significantly inhibited the cell migration and invasion activities of PTEN-mutated glioma cell lines in in vitro migration and chemoinvasion assays. In an organotypic brain slice model, co-culture of glioma spheroids and rat brain slices demonstrated that Ad5CMV-PTEN transfected cells failed to invade surrounding normal brain tissues. Ad5CMV-PTEN transfer into the glioma cell lines lacking the wild-type gene product decreased the levels of matrix metalloproteinase (MMP)-2 mRNA and inhibited the enzymatic activities of MMP-2 and MMP-9. In contrast, mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-2 was upregulated by the PTEN gene transfer. Introduction of PTEN gene in glioma cell lines markedly reduced the levels of Rac-GTP and Cdc42-GTP, activated forms of these small GTP-binding proteins, and decreased the phosphorylation levels of focal adhesion kinase. These results suggest that PTEN inhibits glioma cell invasion in two ways: suppressing proteolysis of the extracellular matrix by MMPs and modulating the migratory activity of glioma cells to a less motile nature by inactivating two Rho-family GTP-binding proteins, Rac and Cdc42.

Identification of the human IAI.3B promoter element and its use in the construction of a replication-selective adenovirus for ovarian cancer therapy.

Little is known concerning promoters or gene therapy specific for ovarian cancer. To explore the potential use of IAI.3B isolated from ovarian cancer cells in gene therapy for ovarian cancer, we identified the promoter region of the IAI.3B gene and created a replication-selective adenovirus, AdE3-IAI.3B, driven by the promoter. Transient transfection experiments showed that the DNA segment located between -1816 and -1 bp resulted in preferential expression in ovarian cancer cells with negligible expression in squamous cell carcinoma and normal cells. The promoter activity of IAI.3B was almost the same as that of cytomegalovirus and an order of magnitude higher than those of midkine and cyclooxygenase-2 in ovarian cancer cells. AdE3-IAI.3B replicated as efficiently as the wild-type adenovirus and caused extensive cell killing in a panel of ovarian cancer cells in vitro. In contrast, squamous cell carcinoma and normal cells were not able to support AdE3-IAI.3B replication. In animal studies, AdE3-IAI.3B administered to flank and i.p. xenografts of ovarian cancer cells led to a significant therapeutic effect. These results demonstrate the usefulness of the IAI.3B promoter for generation of ovarian cancer-specific adenoviral vectors and provide a potential for the development of ovarian cancer-specific oncolytic viral therapies.

Immune activation during the implantation phase causes preeclampsia-like symptoms via the CD40-CD40 ligand pathway in pregnant mice.

The CD40 ligand (CD40L) is expressed by T cells and has a critical role in immune system regulation. Interventions targeting CD40L interactions following embryo implantation represent an approach to preventing preeclampsia (PE). To better understand the role of CD40L in PE, we developed a PE mouse model in which we examined how CD40L-induced immune activation affects embryo implantation. Blastocysts were incubated with CD40L-expressing adenovirus and then were transferred into the uterine horns of pseudopregnant ICR mice. Histology, biochemistry and flow cytometry experiments were performed to examine the characteristics of the mouse model. In early pregnancy, decidualization and spiral artery remodeling were reduced in CD40L-transfected mice (CD40L mice) compared with control mice. Hematoxylin-eosin (HE) staining revealed hemorrhaging and excess fibrin deposition at the labyrinth layer-junctional zone interface of the placenta, and PAS staining demonstrated prominent focal and segmental sclerosis with collapsed glomerular capillaries in the kidneys of the CD40L mice. Flow cytometry data showed that interferon-γ production derived from CD4(+) T cells was elevated in the splenic cells of CD40L mice. Blood pressure (measured by the tail-cuff method) and urine albumin concentrations were significantly increased in CD40L mice compared with control mice. Furthermore, the plasma concentrations of soluble Flt-1 and soluble endoglin were increased in CD40L mice, as occurs in human patients with PE. Thus, CD40L-induced T-helper cell type 1 differentiation during embryo implantation may have a critical role in the pathogenesis of a PE-like presentation in a novel mouse model of PE.

A novel, polymer-coated oncolytic measles virus overcomes immune suppression and induces robust antitumor activity.

Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future.

A cox-2 promoter-based replication-selective adenoviral vector to target the cox-2-expressing human bladder cancer cells.

PURPOSE: Cyclooxygenase-2 (Cox-2), an enzyme that catalyzes the synthesis of prostaglandins, is overexpressed in a variety of premalignant and malignant conditions, including urinary bladder cancer. In the present study, we examined the feasibility of using Cox-2 promoter-based replication-selective adenovirus for targeting bladder cancer cells that express Cox-2 transcriptional activity. EXPERIMENTAL DESIGN: A series of human cancer cell lines, including three bladder cancer cell lines (KK47, T24, and 5637), were evaluated for their Cox-2 and CAR (the Coxsackievirus and adenovirus receptor) mRNA expression levels by quantitative real-time PCR. AdE3-cox2-327, a replication-selective adenovirus in which the expression of E1a is controlled by the Cox-2 promoter, was generated, and its tissue-specific activity was tested in vitro and in vivo. RESULTS: Three bladder cancer cell lines express higher levels of Cox-2 mRNA than does the human prostate cancer cell line PC3, the primary cultured human benign prostatic fibroblast, PF cells, and the human colon cancer cell line Colo320. Relatively higher expression of CAR mRNA was detected in the KK47, 5637, respectively, and Colo320 than in the T24, PC-3, and PF cells. In vitro assays revealed significant growth suppression of both Cox-2- and CAR-expressing bladder cancer cells KK47 and 5637 in comparison with the other cells that lack Cox-2 expression and/or CAR expression. CONCLUSIONS: The present study demonstrated both specificity and efficacy of AdE3-cox2-327, a selectively replicated adenovirus, toward the Cox-2-expressing bladder cancer cells in vitro and in vivo. We also found that CAR expression in the target cancer cells is an important factor for the efficacy of selectively replicated adenovirus-based gene therapy.

A replication-selective adenoviral vector for head and neck cancers.

OBJECTIVE: To test the oncolytic activity of cyclo-oxygenase 2 (COX-2) promoter-based conditional replication-selective adenovirus vector for squamous cell carcinoma cells of the head and neck. DESIGN: In vitro study. SUBJECTS: None. INTERVENTIONS: A conditional replication-selective adenovirus vector in which the expression of E1a, required for viral replication, is controlled by the COX-2 promoter, Ad-COX2-E1a, was generated. Its oncolytic activity according to the levels of COX-2 and of Coxsackie and adenovirus receptor expression was tested in a series of human head and neck squamous cell carcinoma cell lines. RESULTS: The respective COX-2 messenger RNA expression ratios of KB, H891, T891, T892, and L871 were 1.5, 60.0, 1.0, 14.6, and 1.3. The corresponding Coxsackie and adenovirus receptor messenger RNA expression ratios were 1, 1, 5, 3, and 1. In vitro assays showed significant growth suppression of cancer cell lines with strong expressions of COX-2. CONCLUSION: This study demonstrated the possibility of oncolytic therapy using the COX-2 promoter-based conditional replication-selective adenovirus for head and neck squamous cell carcinoma expressing COX-2.

Insertion of an exogenous promoter in the E1A regulatory region of adenovirus does not disturb viral replication despite reduced E1A transcription.

Insertion of an exogenous promoter into adenoviral regulatory regions may result in altered specificity and activity of the integrated promoter-mediated transcription in the context of recombinant adenoviruses (Ad). The alteration is due to the influence of the viral regulatory elements. Specificity of oncolytic Ad, in which the E1A expression is designed to be controlled by a tumor-specific promoter, could thus be modulated by the Ad E1A enhancer/promoters. We prepared recombinant Ad bearing a midkine (MK) promoter region in the 3'-side of the E1A promoter and investigated the relationship between transcriptional activity by the E1A promoter-fused MK fragment and the viral replication. Reporter assays revealed that the transcriptional activity of the fused E1A-MK fragment was significantly lower than that of respective E1A and the MK promoters. However, the replication of the Ad bearing the MK promoter was greater than or comparable to that of wild-type Ad. The present study suggests that the replication of oncolytic Ad in tumors is not directly correlated with the promoter activity to transcribe the E1A gene and that insertion of an exogenous promoter downstream to the E1A regulatory region may not always disturb Ad replication.

Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells.

Malignant gliomas are distinguished from low-grade gliomas by their intense angiogenesis. In gliomas, p53 is the most frequently altered gene and is involved in the early phase of glioma development. In contrast, homozygous p16 gene deletion is more common in high-grade gliomas. In order to understand the mechanism by which gliomas become more angiogenic during the malignant transformation, we examined the relationship between thrombospondin-1, a negative regulator in angiogenesis, and these tumor suppressor genes in malignant gliomas. Human glioma cell line U-251 MG, which has mutated p53 and deleted p16, was transduced with recombinant replication-defective adenovirus vectors containing the cDNA of wild-type p53, p16, and p21. Only the induction of wild-type p53 enhanced expression of thrombospondin-1 mRNA and the protein in U-251 MG cells. Furthermore, thrombospondin-1 that was secreted in the culture medium was significantly increased (3.8-fold) as compared with that of the viral control 36 h after infection with Ad5CMV-p53. In the presence of wild-type p53 plasmid DNA, the promoter activity was increased 7.4-fold as compared with an empty expression vector control. These studies may suggest that mutation of p53 gene endows gliomas with an angiogenic phenotype by reducing thrombospondin-1 production as well as enhancing the angiogenesis inducers in the early phase of malignant progression.

Introduction of p16INK4a inhibits telomerase activity through transcriptional suppression of human telomerase reverse transcriptase expression in human gliomas.

The p16 and p53 tumor suppressor proteins, which are frequently altered in malignant gliomas, have been noted as regulators of telomerase activity. However, the link between telomerase regulation and these suppressor proteins has not been adequately clarified. In the present study, we demonstrated that p16, as well as p53, suppress telomerase activity through transcriptional regulation of human telomerase reverse transcriptase (hTERT) in malignant glioma. To examine the effect of p16 and p53 on telomerase activity, we utilized wild-type p16 or p53 expression plasmid and three human glioma cell lines differing in their p53 and p16 status. Restoring p16 significantly reduced the level of telomerase activity of glioma cells. Furthermore, cotransfection of the p16 gene with 5'-deletion constructs of the hTERT promoter carrying Sp1 binding sites, repressed the transcriptional activity of hTERT promoter in p16-deleted cells. In addition, electrophoretic mobility shift assay revealed that p16 expression inhibited the binding of Sp1 to the consensus Sp1 responsive element, indicating that the recruitment of Sp1 to the hTERT proximal core promoter is inhibited by p16 protein. These results were similar to those from a p53 transfection study in p53-mutated cells. These findings implicate p16 in the transcriptional regulation of telomerase activity by inhibiting the function of Sp1 in human malignant gliomas.

Midkine promoter-based conditionally replicative adenovirus for malignant glioma therapy.

Little is known concerning promoters or gene therapy specific for malignant glioma. To explore the potential use of midkine promoter in gene therapy for malignant glioma, we constructed a midkine promoter-based conditionally replicating adenovirus (Ad-MK). Midkine was overexpressed in malignant glioma tissues but cyclooxygenase-2 was not. The midkine promoter activity of the 600-bp fragment was 2 orders of magnitude higher in midkine-positive glioma cells than in midkine-negative primary normal brain cells. Ad-MK showed strong oncolytic effects in midkine-positive glioma cells but did not exhibit cytotoxicity in midkine-negative primary normal brain cells. The cell-killing effect was evident in E3-intact Ad-MK more than in E3-deleted Ad-MK. In an animal experiment, Ad-MK completely eradicated midkine-positive glioma xenografts. These findings indicate that midkine promoter-based conditionally replicative adenovirus might be a promising new modality of gene therapy for malignant glioma.

Atypical carcinoid of the uterine cervix with aggressive clinical behavior: A case report.

•We herein report a case of a 44-year-old Japanese woman diagnosed with stage IB1 atypical carcinoid of the uterine cervix.•After radical hysterectomy, she developed recurrence with aggressive clinical behavior, resistance to CPT-11 + cisplatin and paclitaxel + CBDCA chemotherapy.

Biosafety studies of carrier cells infected with a replication-competent adenovirus introduced by IAI.3B promoter.

The use of carrier cells infected with oncolytic viruses in cancer gene therapy is an attractive method because it can overcome viral immunogenicity and induce tumor immunity and significant antitumor activity. To enable human clinical trials of this treatment, acute and chronic toxicity tests must first be performed to ensure safety. IAI.3B promoter, oncolytic adenovirus AdE3-IAI.3B introduced by IAI.3B promoter, and A549 carrier cells infected with AdE3-IAI.3B were highly active in cancer cells but not in normal cells. Freeze-thawing increased the antitumor effect of A549 carrier cells by promoting the translocation of oncolytic adenovirus particles from the nucleus to the cytoplasm following the rupture of the nuclear membranes. No deaths or abnormal blood test data resulted from acute toxicity tests conducted in nude mice after a single dose. In chronic toxicity tests in rabbits, there were no serious side effects after eight doses of 1.25 × 10(7) cells/kg or less for 4 weeks; a significant immune response is known to elicit increased numbers of antiadenovirus antibodies and enlarge the spleen. From these results, it could be concluded that cancer gene therapy of recurrent solid tumors using carrier cells can be safely trialed in humans.