Ganciclovir Gene Therapy Research Articles

790 SYSTEMIC T-CELL ACTIVATION FOLLOWING NEOADJUVANT IN SITU GENE THERAPY IN HIGH-RISK PROSTATE CANCER PATIENTS

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012790 SYSTEMIC T-CELL ACTIVATION FOLLOWING NEOADJUVANT IN SITU GENE THERAPY IN HIGH-RISK PROSTATE CANCER PATIENTS Takefumi Satoh, Makoto Kubo, Ken-ichi Tabata, Shinji Kurosaka, Kazumasa Matsumoto, Tetsuo Fujita, Fumiya Obata, Yasutomo Nasu, Hiromi Kumon, Dov Kadmon, Malcolm K. Brenner, Timothy C. Thompson, and Shiro Baba Takefumi SatohTakefumi Satoh Kanagawa, Japan More articles by this author , Makoto KuboMakoto Kubo Kanagawa, Japan More articles by this author , Ken-ichi TabataKen-ichi Tabata Kanagawa, Japan More articles by this author , Shinji KurosakaShinji Kurosaka Kanagawa, Japan More articles by this author , Kazumasa MatsumotoKazumasa Matsumoto Kanagawa, Japan More articles by this author , Tetsuo FujitaTetsuo Fujita Kanagawa, Japan More articles by this author , Fumiya ObataFumiya Obata Kanagawa, Japan More articles by this author , Yasutomo NasuYasutomo Nasu Okayama, Japan More articles by this author , Hiromi KumonHiromi Kumon Okayama, Japan More articles by this author , Dov KadmonDov Kadmon Houston, TX More articles by this author , Malcolm K. BrennerMalcolm K. Brenner Houston, TX More articles by this author , Timothy C. ThompsonTimothy C. Thompson Houston, TX More articles by this author , and Shiro BabaShiro Baba Kanagawa, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.878AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Neoadjuvant in situ cytotoxic gene therapy can potentially trigger a systemic immune response, which could impact occult micro-metastatic disease. We are currently conducting adenoviral vector mediated Herpes Simplex Virus-thymidine kinese (HSV-tk) gene plus ganciclovir (GCV) therapy as neoadjuvant intraprostatic injection for localized high-risk prostate cancer. This study evaluates the systemic T-cell response following gene therapy. METHODS We enrolled 5 men with clinically localized prostate cancer but high risk for recurrence (Kattan nomogram score > 115) in this Phase I–II trial. Intraprostatic viral injections (two) were followed by 2 weeks of GCV and prostatectomy 4 weeks later. Peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting (FACS). Samples were taken at the time of HSV-tk virus injection and at selected intervals afterwards. 100 μl of heparin-treated blood was incubated with dual color labeled antibody pairs: CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/HLA-DR, CD4/HLA-DR, CD3/HLA-DR, CD3/CD56+CD16 and CD4/CD25. Forward and side scatter was set to distinguish lymphocyte, macrophage, and granulocyte populations with an EPICS XL flow cytometer. RESULTS A reduction in serum PSA was observed immediately after vector injection and GCV therapy in all patients. The mean reduction was 31.1% and ranged from 24.8 to 38.9%. The pretreatment mean percentage of CD8+ T cells positive for the HLA-DR marker of activation was 10.6%. For day 2, day 7, day 14, day 16 and day 56 post treatment, the mean percent of CD8+ DR+ T cells increased by 14.0%, 12.3%, 19.7%, 25.4% and 14.9%, which were statistically significant (day 14; p = 0.0431, day 16; p = 0.0431). CONCLUSIONS We present evidence of systemic T-cell responses following HSV-tk + GCV gene therapy under clinical trial condition. There was an increase in activated CD8+ T cells in the peripheral blood following vector injection suggesting the potential for activation of components of cell-mediated immune response in this neoadjuvant setting. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e323 Peer Review Report Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takefumi Satoh Kanagawa, Japan More articles by this author Makoto Kubo Kanagawa, Japan More articles by this author Ken-ichi Tabata Kanagawa, Japan More articles by this author Shinji Kurosaka Kanagawa, Japan More articles by this author Kazumasa Matsumoto Kanagawa, Japan More articles by this author Tetsuo Fujita Kanagawa, Japan More articles by this author Fumiya Obata Kanagawa, Japan More articles by this author Yasutomo Nasu Okayama, Japan More articles by this author Hiromi Kumon Okayama, Japan More articles by this author Dov Kadmon Houston, TX More articles by this author Malcolm K. Brenner Houston, TX More articles by this author Timothy C. Thompson Houston, TX More articles by this author Shiro Baba Kanagawa, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Effective Ablation of Pancreatic Cancer Cells in SCID Mice Using Systemic Adenoviral RIP-TK/GCV Gene Therapy

Studies have demonstrated that adenovirus subtype 5 mediated rat insulin promoter directed thymidine kinase (A-5-RIP-TK)/ganciclovir (GCV) gene therapy resulted in significant enhanced cytotoxicity to both PANC-1 and MIA PaCa2 pancreatic cancer cells in vitro. However, little is known about the effect in vivo. In this study we examine the in vivo safety and efficacy of intravenous A-5-RIP-TK/GCV gene therapy. 1 x 10(6) Mia PaCa2 cells were injected intraperitoneally (i.p.) into SCID mice to create a mouse model of human pancreatic cancer. A-5-RIP-TK gene construct was administered intravenously (i.v.), followed by i.p. GCV administration. Intravenous injection of A-5-RIP-lacZ reporter gene constructs was used for evaluation of Ad-RIP-gene expression in tumors and other tissues. Optimal adenoviral and GCV doses and treatment duration were determined. Tumor volume, serum insulin, and glucose levels were measured. Immunohistochemical staining of pancreata and tumors were performed to assess morphology and hormone expression and apoptotic rates were determined. All A-5-RIP-TK/GCV-treated mice had reduced tumor volume compared with controls, but maximal tumor volume reduction was observed with 10(8) vp followed by GCV treatment for 4 wk. A-5-RIP-TK/GCV gene therapy contributed to significant survival advantage in MIA PaCa2 bearing mice, and the greatest survival benefit was observed with 10(8) vp and was not affected by length of treatment of GCV. A-5-RIP-TK/GCV therapy increased PDX-1 expression and tumor cells apoptosis, and altered islet morphology. However, A-5-RIP-TK/GCV gene therapy caused diabetes associated with islet cell apoptosis, increased delta-cells and reduced pancreatic polypeptide (PP)-cell numbers. Systemically administered A-5-RIP-TK/GCV is an effective treatment of pancreatic cancer. A-5-RIP-TK/GCV cytotoxicity to malignant cells varies with adenoviral dose and length of GCV treatment. However, A-5-RIP-TK/GCV is associated with islet cell toxicity and diabetogenesis. The type of diabetes observed is distinct from Types 1 and 2 and is associated with islet cell apoptosis and reduced delta- and PP-cells.

Dexamethasone inhibits the HSV-tk/ ganciclovir bystander effect in malignant glioma cells

BackgroundHSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed.MethodsStable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL.ResultsDexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade.ConclusionThe overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.

Non-small cell lung cancer as a target disease for herpes simplex type 1 thymidine kinase - ganciclovir gene therapy

Lung cancer is a group of diseases that are difficult to cure and new treatment modalities, like gene therapy are actively tested to find alternatives for currently used strategies. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) method is one of the most frequently utilized forms of gene therapy and it has been tested on lung cancer, but no systematic study with comparison of different lung cancer types has been published. In this study, we examined in vitro and in vivo how good targets non-small cell lung cancer (NSCLC) cell lines representing adenocarcinoma, squamous cell lung cancer and large cell lung cancer are for adenovirus-mediated HSV-TK/GCV gene therapy. By using an adenovirus vector carrying a fusion gene of HSV-TK and green fluorescent protein (GFP), we found that: a) adenoviruses were efficient gene transfer vehicles for all types of NSCLCs; b) all adenocarcinoma and large cell lung cancer cells were good targets for HSV-TK/GCV therapy, whereas one of the squamous cell carcinoma cell lines was not responsive to the treatment; c) bystander effect played a major role in the success of this gene therapy form; d) subcutaneous tumors representing all three NSCLC types were efficiently treated with adenovirus-mediated HSV-TK/GCV gene therapy. In summary, this form of gene therapy appeared to be efficient treatment for human NSCLC and these results warrant further studies with primary lung cancer cells and orthotopic lung tumor models.

Enhanced therapeutic effect of mutiple injections of HSV-TK + GCV gene therapy in combination with ionizing radiation in a mouse mammary tumor model

Purpose: Standard therapies for breast cancer lack tumor specificity and have significant risk for recurrence and toxicities. Herpes simplex virus-thymidine kinase (HSV-tk) gene therapy combined with radiation therapy (XRT) may be effective because of complementary mechanisms and distinct toxicity profiles. HSV-tk gene therapy followed by systemic administration of ganciclovir (GCV) enhances radiation-induced DNA damage by generating high local concentrations of phosphorylated nucleotide analogs that increase radiation-induced DNA breaks and interfere with DNA repair mechanisms. In addition, radiation-induced membrane damage enhances the “bystander effect” by facilitating transfer of nucleotide analogs to neighboring nontransduced cells and by promoting local and systemic immune responses. This study assesses the effect of single and multiple courses of HSV-tk gene therapy in combination with ionizing radiation in a mouse mammary cancer model.Methods and Materials: Mouse mammary TM40D tumors transplanted s.c. in syngeneic immunocompetent BALB-c mice were treated with either adenoviral-mediated HSV-tk gene therapy or local radiation or the combination of gene and radiation therapy. A vector consisting of a replication-deficient (E1-deleted) adenovirus type 5 was injected intratumorally to administer the HSV-tk gene, and GCV was initiated 24 h later for a total of 6 days. Radiation was given as a single dose of 5 Gy 48 h after the HSV-tk injection. A metastatic model was developed by tail vein injection of TM40D cells on the same day that the s.c. tumors were established. Systemic antitumor effect was evaluated by counting the number of lung nodules after treating only the primary tumors with gene therapy, radiation, or the combination of gene and radiation therapy. To assess the therapeutic efficacy of multiple courses of this combinatorial approach, one, two, and three courses of HSV-tk + GCV gene therapy, in combination with radiation, were compared to HSV-tk or XRT alone and to sham-treated animals. (Treatments were repeated at 7-day intervals from the HSV-tk injection.)Results:Both single-therapy modalities reduced tumor growth by 11% compared to controls, while the combined therapy resulted in a decrease of 29%. Median survival was 36 days in the combined therapy group, compared to 33 days in the monotherapy groups and 26 days in the control group. In the metastatic model, the number of lung nodules was reduced by 59.5% after HSV-tk gene therapy, whereas radiotherapy had no effect on metastatic growth. Combined therapy led to an additional 66.7% reduction in lung colonization. Compared to controls, local tumor growth was maximally suppressed by three courses of combined therapy (51.5%), followed by two courses of combined therapy (37.2%), and three sessions of XRT alone (35.6%). Median survival was also significantly prolonged to 58 days with the three courses of combined therapy, followed by two courses, to 45 days. All other treatment groups demonstrated median survival times between 26 and 35 days, while controls had a median survival of 24 days.Conclusions: These results indicate that multiple courses of HSV-tk therapy in combination with radiation improve the therapeutic efficacy of this approach and may provide therapeutic implications for the treatment of human breast cancer and other solid tumors.

Sequential histopathological changes in vivo after suicide gene therapy of gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy, such as with the herpes simplex virus type 1 thymidine kinase (HSV‐TK) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. We previously reported in situ gene transfer and gene therapy for gastric cancer induced by N‐ethyl‐.N′‐nitro‐.N‐nitrosoguanidine (ENNG) in dogs. Here, we describe the sequential histopathological changes after suicide gene therapy of N‐methyl‐.N′‐nitro‐.N nitrosoguanidine (MNNG)‐induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38/73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV‐TK gene driven by CAG promoter (Ad.CAGHSV‐TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). To observe the histopathological changes at various tunes after HSV‐TK/GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 days after gene transfer. Apoptosis in the gastric tumors was detected by the TUNEL method to assess the efficacy of HSV‐TK/GCV gene therapy, and it was marked in the 8‐ and 30‐day treatment groups compared to the sham operation controls (P<0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30‐day treatment group. The HSV‐TK gene was detectable in peripheral blood by PCR until 30 days after gene transfer. These results may be useful in devising a method of suicide gene therapy for humans.

Gene Therapy of Established Medullary Thyroid Carcinoma with Herpes Simplex Viral Thymidine Kinase in a Rat Tumor Model: Relationship of Bystander Effect and Antitumor Efficacy

Bystander effect (BSE) refers to killing of cells adjacent to a cell engineered to express a killing gene segment. BSE is considered an important aspect of suicide gene therapy with thymidine kinase. We evaluated the BSE of adenovirus expressing herpes simplex thymidine kinase (AdCMVtk) in rat medullary thyroid carcinomas (rMTC) and three rat thyroid epithelial cancer cell lines using an in vitro BSE assay. In the assay, different proportions of infected and uninfected cells are mixed. Only the proportion of directly infected cells was inhibited in the proliferation assay using rMTC cells. This indicates that there is little BSE in this cell line. One rat thyroid epithelial cancer cell line (RTC-R2) has a high BSE, with BSE index (BSEi) of 7. In the proliferation assay a greater proportion of cells was inhibited than those directly infected. BSE was also evaluated during in vivo tumor growth by subcutaneous injection of mixtures of AdCMVtk infected and uninfected cells. Ganciclovir (GCV) treatment of tumors developing from a 1:1 mixture of infected to uninfected rMTC cells failed to inhibit their growth. In contrast, GCV treatment of a 2:8 mixture of infected to uninfected RTC-R2 cells completely inhibit tumor development, indicating a high BSE. BSE is related to in vivo antitumor efficacy when replication-defective adenovirus AdCMVtk is directly injected into rMTC tumors. After treatment with 100 mg/kg per day of GCV, a growth-retardation effect was observed in small tumors (<100 mm3), but there was little antitumor activity in large tumors (>100 mm3). Our results indicate that there is a good correlation between this in vitro BSE assay and in vivo treatment efficacy. Not all kinds of tumors are suitable for thymidine kinase (TK)/GCV gene therapy because some lack BSE. Methods to improve BSE and/or transduction efficiency are needed in order to obtain an effective therapeutic result. It will be appropriate to test the BSE in human tumor cells before performing clinical trials with current adenoviral vectors expressing TK.

In situ gene transfer and suicide gene therapy of gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine in dogs.

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N‐ethyl‐N′‐nitro‐N‐nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV‐TK) gene was introduced in situ into cancer tissues in the stomach of three dog, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus‐producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus‐producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAG lacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV‐TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV‐TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAG lacZ gene transfer, but was found after high‐titer Ad.CAGHSV‐TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.

GENE THERAPY FOR MALIGNANT PLEURAL MESOTHELIOMA

Malignant mesothelioma (MM) is a fatal malignancy refractory to all forms of standard anticancer therapy. This article reports the results of a phase I clinical trial assessing the safety of intrapleural delivery and efficacy of intratumoral gene transfer of recombinant adenovirus (rAd) containing herpes simplex virus thymidine kinase (HSVtk) gene into the pleural space of patients with MM, followed by systematic treatment with the antiviral drug ganciclovir (GCV) for 14 days. AD.RSVtk/GCV gene therapy proved to be well tolerated, with evidence of significant gene transfer particularly at high vector doses and with elimination of preliminary biopsy. Ongoing gene therapy trials for mesothelioma at two other centers, focusing on immunostimulation and using suicide gene therapy as a tumor vaccine, are also reviewed in this article.