Adenovirus-mediated Herpes Simplex Virus Thymidine Research Articles

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy for experimental prostate cancer

Objective To study the killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-TK/GCV) system mediated by adenovirus vector on the androgen-independent prostatic cancer (AIPC) cell line PC-3 in ivtro and explore the possibility of gene therapy for AIPC.Methods The plasmid pAdTK containing TK gene and pJMl7 with the main Adv gene fragments were co-infected into 293 cells by recombinant DNA technology.PC-3 cells were cultured with AdLacZ.MOI was 50,100,150,200 and 250.After 48 h,the transfection efficacy was calculated.The transfected cells with AdTK were treated with prodrug GCV ( 10 mg/L) in vitro.Killing effects were evaluated by methyl thiazol tetrazolium (MTT) assay.Electron microscopy and flow cytometry were utilized for assessing efficacy of AdTK/GCV system and related killing mechanisms were analyzed.Results The titer of prepared AdTK was 1 × 1011 pfu/ml.The outcomes indicated survival rate was decreased with the increase in AdTK MOI and GCV concentration.IC50 of GCV was 7.75 mg/L and 5.00 mg/L respectively when the AdTK MOI reached 150 and 250.There was significant difference in suicide effects of TK gene.Apoptosis mechanism was demonstrated to be involved in such suicide course as well as bystander effects.Conclusion AdTK/GCV gene therapy may play a key role in inhibition of growth of PC-3 cellsin vitro. Key words: Prpstate neoplasms; Adenovirus vector; Gene therapy

Adenovirus-mediated herpes simplex virus thymidine kinase gene transfer driver by KDR promoter in treatment of experimental human hepatocelLular carcinoma in nude mice

To investigate the therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transfer under the driving of KDR promoter (AdKDR-tk) in combination of ganciclovir (GCV) against human hepatocellular carcinoma in nude mice. HepG2 cell line was implanted subcutaneously into 32 nude mice, which were subsequently divided into 4 groups (n=8 each group): Ganciclovir group (I), Ad group (II), AdCMV-tk/GCV group (under the driving of CMV promoter) (III) and AdKDR-tk/GCV group (IV). Then intratumoral injection of recombinant adenovirus or Ad was performed in all nude mice, and repeated 24 h later. For the following 10 d GCV was given at a dose of 100 mg/(kg·d), ip. All the treated animals were killed to evaluate the tumor weight and the histopathological changes and the microvessel density of tumors after the treatment was determined. Compared with group I, the tumor inhibitory rate was 12.3% in group III and 24.5% in group IV; the inhibition rates were significantly different between group III and IV (P<0.05). The mean MVDs in group I, II, III and IV were 37.4±8.6, 30.6±7.8, 27.6±7.1, and 10.7±4.1 (microvessels/mm2), respectively. Significant differences were found between group III and II (P<0.05), IV and II (P<0.01), and IV and III (P<0.01). Intratumoral injection of AdKDR-tk results in marked inhibition of HCC growth through inhibition angiogenesis in nude mice. It may be a new treatment approach for human HCC.

Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase (HSV-tk) targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. Recombinant adenovirus containing kinase domain insert with receptor (KDR) or cytomegalovirus (CMV) promoter-controlled HSV-tk gene (AdKDR-tk and AdCMV-tk) was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells (HUVEC) and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir (GCV), the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups: ganciclovir group (I), Ad group (II), AdCMV-tk group (III) and AdKDR-tk group (IV). Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir (GCV) was given at a dose of 100 mg kg(-1) d(-1) (ip) started on the following day and lasted 10 d. Microvessel density (MVD) of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and after the last GCV dose. Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk-transfected HUVEC and HepG2 decreased from 100% to (28.94 +/- 5.67)% and (75.45 +/- 2.91)% at proper order, respectively (P < 0.01), while the survival rate of AdCMV-tk-transfected HUVEC and HepG2 declined from 100% to (17.56 +/- 2.48)% and (23.15 +/- 5.72)%, respectively (P > 0.05). Compared with group I, there was a decrease of tumor weight by 14.7% in group III and by 23.6% in group IV. And there was a distinct difference between group III and IV (P < 0.05). The median MVD for all groups was 37.4 +/- 8.6, 30.6 +/- 7.8, 27.6 +/- 7.1, and 10.7 +/- 4.1 (microvessels/mm(2)) in group I, II, III and IV, respectively. And there was a marked difference between group III and II (P < 0.05), IV and II (P < 0.01), and IV and III (P < 0.01). KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

569. Gene Therapy of Uterine Leiomyoma: Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase/Ganciclovir Treatment Inhibits Growth of Human and Rat Leiomyoma Cells In Vitro and in a Nude Mouse Model

569. Gene Therapy of Uterine Leiomyoma: Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase/Ganciclovir Treatment Inhibits Growth of Human and Rat Leiomyoma Cells In Vitro and in a Nude Mouse Model

259. Uterine Fibroids Gene Therapy: Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase/Ganciclovir Treatment Inhibits Growth of Human and Rat Leiomyoma Cells

Introduction: Adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) is one of the major gene therapy strategies to eradicate tumor cells. Leiomyoma is a benign uterine tumor that affects high percentage of women in their reproductive age. Unfortunately, there is no effective and safe medical treatment available. Leiomyom is developed as discrete well-defined tumor, easily accessible with imaging techniques which makes it a good candidate for localized gene therapy approaches.

Adenovirus-mediated herpes simplex virus thymidine kinase gene therapy in BT4C rat glioma model

Adenovirus (Adv)-mediated herpes simplex virus thymidine kinase (adv/tk) gene therapy combined with ganciclovir (GCV) medication is a promising approach for the treatment of malignant glioma. However, optimal administration and the effect of possible adjuvant treatments have not been fully examined. In the present study, we examined the efficacy of adv/tk/GCV gene therapy in a syngeneic BT4C rat malignant glioma model, either as a single administration or given as three injections during three consecutive days. The effect of combined adv-mediated macrophage colony-stimulating factor (MCSF) and adv/tk gene transfer was also studied. BT4C malignant glioma cells were injected into the right corpus callosum of BDIX rats (n=112). Before gene therapy, the presence of tumors was verified by MRI. The rats were divided into eight groups as follows: group I (n=20) received a single adv/tk gene transfer (total dose 4x10(8) pfu) and GCV treatment for 14 days; group II (n=5) received the same gene transfer without GCV; group III (n=28) received three adv/tk injections (total dose 4x10(8) pfu) on three consecutive days and GCV for 14 days; group IV (n=5) received three similar adv/tk injections without GCV medication; group V (n=13) received three adv/MCSF injections (total dose 2x10(8) pfu) on three consecutive days and GCV medication; group VI (n=12) received three adv/tk and adv/MCSF (total dose 6x10(8) pfu) injections on three consecutive days followed by GCV medication; and group VII (n=12) the same treatment without GCV. Group VIII (n=17) consisted of wild-type BT4C malignant glioma tumors without any treatment. Treatment effect and tissue responses were characterized by general histology, immunohistochemistry, MRI, and survival of the study groups. The best treatment effect and survival was found in rats treated with adv/tk gene transfer once a day for three consecutive days (P<.05). No improvement of the treatment effect was seen after the combined adv/tk and adv/MCSF gene transfer compared with the repeated adv/tk gene transfer. The results show that 20% of the rats can be cured (survival >6 months) after optimized adv/tk gene therapy. It is concluded that repeated intratumoral administration of adv/tk is a promising approach for the treatment of malignant glioma tumors in vivo.

The combination of HSV-tk and endostatin gene therapy eradicates orthotopic human renal cell carcinomas in nude mice

Gene therapy may offer a new tool for the treatment of renal cell carcinoma (RCC). We have tested a combination of cytotoxic and antiangiogenic gene therapy for wild-type orthotopic human RCC xenografts in nude mice using intratumoral adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) and endostatin (ES) gene therapy. In vivo magnetic resonance imaging, morphometry, immunocytochemistry, and survival were used to evaluate the treatment effect. Adenovirus-mediated marker gene transfers (GFP) were used as controls. In vivo transduction efficiency, measured using GFP gene transfer, was 27+/-7%. The combination gene therapy with HSV-tk and ES adenoviruses resulted in a significant antitumor effect (P<.01) compared to single HSV-tk (n.s.) or ES (n.s.). In the survival study, all tumors with single gene therapy using HSV-tk, ES, and marker gene adenoviruses showed progression in magnetic resonance imaging. In contrast, the majority of the tumors in the combination treatment group remained dormant or were eradicated (57%). Survival of these mice equaled healthy nude mice, and was significantly prolonged (P<.0001) compared to HSV-tk (P<.028) and ES (n.s.) groups. It is concluded that the inhibition of angiogenesis using ES gene transfer together with the cytotoxic HSV-tk gene therapy results in a significantly improved treatment effect in RCC compared to the single gene treatments.

Adenoviral herpes simplex virus thymidine kinase gene therapy in an orthotopic lung cancer model

Background. Although adenovirus-mediated herpes simplex virus thymidine kinase (HSV tk) gene therapy is a potential candidate for a novel effective therapy for lung cancer, previous studies have been performed only in a subcutaneous tumor model employing nude mice. We studied therapeutic potentials in correlation with accurate adenoviral gene transduction efficiency in a more clinically relevant orthotopic lung cancer model employing imunocompetent mice. Methods. To analyze the cytotoxicity of adenoviral HSV tk gene transduction and ganciclovir, a cell proliferation assay was performed in vitro. A survival study was carried out in immunocompetent mice with orthotopic lung cancer, which was generated by intrapulmonary inoculation with syngeneic murine lung cancer cells that had been infected beforehand with each adenoviral vector at the predetermined gene transduction efficiencies. Results. Tumor cells were efficiently killed by infection with adenovirus carrying the HSV tk gene with the addition of ganciclovir in vitro. In the in vivo experiment all control mice died of rapid growth of the primary lung cancer and of metastases to mediastinal lymph nodes within 26 days after tumor inoculation. In contrast 50% and 100% of mice survived more than 40 days after inoculation with adenovirally HSV tk-transfected tumor cells that moderately and highly expressed HSV tk, respectively, when followed by ganciclovir administration. Gene transduction efficiencies were 67%. Conclusions. Adenovirus-mediated HSV tk gene therapy may be therapeutic for lung cancer when gene transduction efficiencies and sufficient expression levels of HSV tk can be achieved. Moreover, the present findings underscore the importance of the mouse orthotopic lung cancer model for studies of gene therapy.

Adenovirus-Mediated Thymidine Kinase Gene Therapy for Recurrent Ovarian Cancer: Expression of Coxsackie-Adenovirus Receptor and Integrins αvβ3 and αvβ5

Ten patients with recurrent ovarian cancer received a combined treatment of optimal tumor debulking, adenovirus-mediated herpes simplex virus thymidine kinase gene therapy (GT), and systemic application of acyclovir or valacyclovir and topotecan. Biopsies were taken at the time of secondary debulking about 1 month after the application of GT and chemotherapy and were analyzed for expression of coxsackie-adenovirus receptor (CAR) and integrins alphavbeta3 and alphavbeta5 with respect to treatment response. Treatment modalities and study design have been described recently. Immunohistochemistry was used to visualize expression of CAR and integrins alphavbeta3 and alphavbeta5 in tumor samples taken before and after application of GT. Before GT six of ten patients presented with CAR-positive and four with CAR-negative tumors. After GT all tumors showed CAR expression. Integrin alphavbeta3 was found in all tumors before and after GT. Expression of integrin alphavbeta5 was seen in eight of ten tumor samples before GT and in all samples after GT. Despite the importance of CAR and integrin expression for successful adenovirus internalization, other cell surface receptors might be involved in this process. It is too early to decide whether expressions of CAR and integrin alphavbeta3/alphavbeta5 on tumor cells are appropriate additional inclusion criteria for the enrollment of patients in GT trials. Further research is necessary to evaluate the effect of GT plus chemotherapy on CAR and integrin expression.

Transduction efficacy, antitumoral effect, and toxicity of adenovirus-mediated herpes simplex virus thymidine kinase/ ganciclovir therapy of hepatocellular carcinoma: the woodchuck animal model.

Gene therapy for hepatocellular carcinoma (HCC) has shown some promise, but its evaluation requires relevant experimental models. With this aim, we present an evaluation of the interest of using the woodchuck model of HCC to assess in vivo gene transfer efficiency. We tested the transduction efficacy of the adenoviral vectors directing lacZ gene product expression under the control of the cytomegalovirus and alpha-fetoprotein (AFP) regulatory sequences. We have also investigated whether an adenoviral cytomegalovirus-thymidine kinase (Tk) vector might induce an antitumoral effect in this model. Our results demonstrate that with direct intratumoral and intrahepatic arterial injections, transduction of a significant proportion of tumor cells occurred even in large HCC nodules. Furthermore, due to intra-arterial anastomoses, direct intratumoral injection led to transduction of some noninjected HCC nodules. Moreover, direct intratumoral injection of a herpes simplex virus-1 Tk-encoding vector induced, on ganciclovir administration, a significant antitumoral effect in the two animals evaluated. However, in one animal, massive hepatic failure occurred due to Tk expression in nontumor cells. These results emphasize the need to target the expression of the Tk gene to tumor cells using a hepatoma-specific promoter such as AFP promoter. However, we showed that, in vivo, lacZ expression as driven by the AFP promoter was extremely low, thus emphasizing some potential pitfalls when using this approach. Altogether, our data stress the need to test gene therapy-based strategies in such in vivo animal models of HCC and evaluate gene transduction, expression, and biological activity, as well as its potential toxicity.

Differential chemosensitivity of breast cancer cells to ganciclovir treatment following adenovirus-mediated herpes simplex virus thymidine kinase gene transfer.

The development of resistance to radiation and chemotherapeutic agents that cause DNA damage is a major problem for the treatment of breast and other cancers. The p53 tumor suppressor gene plays a direct role in the signaling of cell cycle arrest and apoptosis in response to DNA damage, and p53 gene mutations have been correlated with increased resistance to DNA-damaging agents. Herpes simplex virus thymidine kinase (HSV-tk) gene transfer followed by ganciclovir (GCV) treatment is a novel tumor ablation strategy that has shown good success in a variety of experimental tumor models. However, GCV cytotoxicity is believed to be mediated by DNA damage-induced apoptosis, and the relationship between p53 gene status, p53-mediated apoptosis, and the sensitivity of human tumors to HSV-tk/GCV treatment has not been firmly established. To address this issue, we compared the therapeutic efficacy of adenovirus-mediated HSV-tk gene transfer and GCV treatment in two human breast cancer cell lines: MCF-7 cells, which express wild-type p53, and MDA-MB-468 cells, which express high levels of a mutant p53 (273 Arg-His). Treating MCF-7 cells with AdHSV-tk/GCV led to the predicted increase in endogenous p53 and p21WAF1/CIP1 protein levels, and apoptosis was observed in a significant proportion of the target cell population. However, treating MDA-MB-468 cells under the same conditions resulted in a much stronger apoptotic response in the absence of induction in p21WAF1/CIP1 protein levels. This latter result suggested that HSV-tk/GCV treatment can activate a strong p53-independent apoptotic response in tumor cells that lack functional p53. To confirm this observation, four additional human breast cancer cell lines expressing mutant p53 were examined. Although a significant degree of variability in GCV chemosensitivity was observed in these cell lines, all displayed a greater reduction in cell viability than MCF-7 or normal mammary cells treated under the same conditions. These results suggest that endogenous p53 status does not correlate with chemosensitivity to HSV-tk/GCV treatment. Furthermore, evidence for a p53-independent apoptotic response serves to extend the potential of this therapeutic strategy to tumors that express mutant p53 and that may have developed resistance to conventional genotoxic agents.

Cooperative therapeutic effects of androgen ablation and adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy in experimental prostate cancer.

Adenovirus-mediated transduction of the herpes simplex thymidine kinase gene (HSV-tk) in conjunction with ganciclovir (GCV) has been shown to result in significant growth suppression and to enhance survival in a model of mouse prostate cancer. However, this therapeutic activity is not sustained, because in most cases tumors eventually regrow and ultimately cause the death of the host. Androgen ablation, an inducer of apoptosis in prostate cells which is used widely as palliative therapy in patients with prostate cancer, was combined with HSV-tk plus GCV using an androgen-sensitive mouse prostate cancer cell line. The combination of castration and HSV-tk plus GCV led to markedly enhanced tumor growth suppression in both subcutaneous and orthotopic models compared with either treatment alone and resulted in an enhanced survival in which combination-treated animals lived twice as long as controls in the subcutaneous model and over 50% longer than controls in the orthotopic model. Further analysis of apoptotic activity demonstrated high levels of apoptosis only in combined androgen ablation and HSV-tk plus GCV-treated tumors after 14 days of growth in an androgen-depleted environment and 8 days after HSV-tk plus GCV therapy. At this time, the apoptotic index, but not the percent of necrotic tissue, was significantly higher for combination therapy-treated tumors relative to control-treated tumors or either treatment alone. These data indicate that the therapeutic effects of androgen ablation and HSV-tk plus GCV are cooperative and that increased apoptosis may, in part, underlie these activities.

Adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy leads to systemic activity against spontaneous and induced metastasis in an orthotopic mouse model of prostate cancer.

It is critical to develop new therapies, such as gene therapy, which can impact on both local and metastatic prostate cancer progression. We have developed an orthotopic mouse model of metastatic prostate cancer using a cell line (RM-1) derived from the mouse prostate reconstitution (MPR) model system. This mouse model closely simulates the anatomical and biological milieu of the prostate and allows for realistic testing of experimental gene therapy protocols. Adenovirus (ADV)-mediated transduction of the herpes simplex virus thymidine kinase (HSV-tk) gene in conjunction with ganciclovir (GCV) in this model led to significant suppression of growth and of spontaneous metastasis at 14 days post-tumor inoculation. Longer-term studies produced a significant survival advantage and a continued suppression of metastatic activity for treatment animals despite regrowth of the primary tumor. Challenge by injection of tumor cells into the tail vein following excision of treated and control s.c. primary tumors resulted in 40% reduction in lung colonization in the treatment group, indicating the possible production of systemic anti-metastatic activity following a single in situ treatment with ADV/HSV-tk + GCV in this model system.

Adenovirus-mediated gene transfer of herpes simplex virus thymidine kinase in an ascites model of human breast cancer.

In this study, the growth of locally disseminated breast cancer was modeled using a human breast cancer cell line, MDA-MB-435A, adapted to grow as an ascites tumor in athymic mice. Ex vivo infection of MDA-MB-435A cells with adenovirus containing the herpes simplex virus thymidine kinase gene (HSV-tk) were injected into the intraperitoneal cavity of athymic mice. Ganciclovir (GCV) treatment resulted in prolonged median survival (117 vs. 34 days, p < 0.001) compared to untreated or control animals. Adenovirus containing HSV-tk also demonstrated therapeutic activity after in vivo transduction resulting in prolongation of median survival after GCV treatment (32 vs. 25 days, p < 0.001). However, compared to ex vivo treatment, the effect was modest. In an attempt to increase survival, the viral dose was increased three-fold. Instead of prolonging survival, the increased dose resulted in more toxic deaths. Necropsy demonstrated that the most significant histologic abnormality was marked, diffuse, cytomegalic changes in the liver. Polymerase chain reaction (PCR) analysis of hepatic DNA demonstrated the presence of the virus in the affected tissue. Similar host toxicity and hepatic abnormalities were seen in non-tumor-bearing mice treated with ADV/RSV-tk plus GCV. In conclusion, adenoviral vectors can successfully transfer genes in vivo to cancer cells growing as ascites tumors. Transduction with HSV-tk followed by GCV treatment can prolong survival in this model system of disseminated disease, however toxicity can be substantial. Further refinement in targeting expression of HSV-tk will be required to enhance the therapeutic benefit.

Inhibition of Melanoma Growth by Adenoviral-Mediated HSV Thymidine Kinase Gene Transfer In Vivo

To assess the potential of an in vivo, adenovirus-mediated gene therapy approach for the treatment of malignant melanoma, the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-Ek) transfer and administration of ganciclovir (GCV) was investigated using a nude mouse model. Initially, B16 murine melanoma cells were efficiently transduced in vitro by a recombinant replication-defective adenovirus containing the HSV-tk gene (ADV/RSVtk), and rendered sensitive to cell killing by 10 micrograms/ml GCV. A significant "bystander effect" was observed at low multiplicity of infection in comparison of cell killing to control B16 transduction by adenovirus containing the beta-galactosidase gene (ADV/RSV-beta-gal). In vivo, melanomas established from subcutaneous injection of 4 x 10(5) B16 cells were injected after 14 d with 1 x 10(10) ADV/RSV-tk viral particles. Subsequent treatment for 6 d with GCV resulted in an inhibition of melanoma growth, with an approximately 40-50% reduction in melanoma volume in comparison to controls in repeated experiments. These data demonstrate that adenovirus-mediated gene transfer can function as an efficient delivery system to reduce established tumor burden in vivo. This result may hold significant promise for the development of effective in situ gene therapy for melanoma in humans.

Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo.

The therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transduction of rat C6 glioma cells followed by ganciclovir (GCV) administration was studied in tumors generated in the brains of nude mice. C6 glioma cells were efficiently transduced in vitro by a replicative-defective recombinant adenovirus carrying the HSV-tk gene (ADV/RSV-tk) that rendered them sensitive to GCV in a dose-dependent manner. Tumors were generated by stereotaxic intracerebral injection of 1 x 10(4) C6 cells in nude mice. After 8 days of tumor growth, 3 x 10(8) ADV/RSV-tk viral particles were injected into the tumors and the mice subsequently were treated with GCV for 6 days. Tumor size in untreated and treated animals was compared 20 days after tumor implantation. The mean cross-sectional area of the tumors in the treated animals was 23-fold smaller than in control animals and the tumor volume was reduced by > 500-fold. These results demonstrate that the recombinant adenoviral vector can function as an efficient gene delivery vehicle for the treatment of gliomas by in vivo gene therapy.