Mediated Suicide Gene Therapy Research Articles

Long-term treatment with valganciclovir improves lentiviral suicide gene therapy of glioblastoma.

Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

Nanoscale bubble ultrasound contrast agents-mediated suicide gene therapy system, Nanoscale bubble-LV5-YCD-TK/GCV/5-FC, effectively inhibits bladder cancer cell growth.

Bladder cancer is the 2nd most common reason for human genitourinary cancer-associated mortality. This study aimed to investigate the effects of Nanoscale bubble ultrasound contrast agents-mediated yeast-cytosine-deaminase-thymidine kinase/ganciclovir (YCD-TK/GCV) or YCD-TK/5-fluorocytosine (5-FC) suicide gene therapy system on BIU-87 cell growth. Targeted nanoscale bubble ultrasound contrast agents were prepared by utilizing thin-film hydration-sonication approach. Nanoscale bubble-LV5-YCD-TK/GCV(5-FC) was constructed and transfected to BIU-87 cells. Hematoxylin and eosin (HE) staining was used to evaluate inflammation. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was used to examine cell viability. Cell-cycle distribution was analyzed with cell cycle assay. Flow cytometry assay was utilized to test apoptosis of BIU-87 cells. YCD-TK expression was examined using Western blot and quantitative Real Time-PCR (qRT-PCR), respectively. YCD-TK highly expressed in Nanoscale bubble mediated suicide gene therapy system. Nanoscale bubble-mediated suicide gene therapy system significantly induced inflammatory response and apoptosis compared to that of Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly reduced cell viability compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble mediated suicide gene therapy system significantly inhibited cell cycle arrest compared to that of the Nanoscale bubble group (p<0.05). Nanoscale bubble-LV5-YCD-TK/GCV/5-FC therapy system significantly reduced BIU-87 cell viability compared to that of the Nanoscale bubble-associated groups (p<0.05). Nanoscale bubble-mediated suicide gene therapy system, bubble-LV5-YCD-TK/GCV/5-FC, acts as a novel therapeutic strategy for bladder cancer treatment.

Tumoricidal effect of human olfactory ensheathing cell mediated suicide gene therapy in human glioblastoma cells.

The potential of herpes simplex virus type 1 thymidine kinase (HSV-tk)-expressing olfactory ensheathing cells (OEC) treated with ganciclovir (GCV) to induce cell death in adjacent HSV-tk-negative cells (bystander effect) has been well demonstrated. Although it has been shown that bystander effect occurs through the delivery of phosphorylated GCV, the bystander effect mechanism and the role of gap junctions for human OECs mediated suicide gene therapy in primary astrocytes of human glioblastma remain obscure. In the present study, the efficacy of a new method for the transfer of phosphorylated GCV from OECs into primary astrocytes was evaluated. Surgical biopsy of glioblastoma was used to isolate primary astrocyte. Biopsy of olfactory mucosa was applied to isolate olfactory ensheathing cell. Expression of S100-beta antigen was confirmed immunocytochemically in astrocytes and OECs. OECs were transduced to lentiviral containing thymidine kinase gene (TK) and co-cultured with astrocytes. Fluorescent dye transfer and western blot analysis indicated the expression of connexin43 between olfactory ensheathing cells and astrocytes whereas, expression of the gap junction protein connexin43 was inhibited by the gap junction inhibitor 18α-glycyrrhethinic acid (AGA, 20µg/ml). Furthermore, co-culture of astrocytes with OEC-TK in the presence of concentration of 30µg/ml GCV led to a decrease in astrocytes survival rate. Also, apoptosis hallmarks, including DNA fragmentation in cell nuclear, expression increase of Bax to Bcl-2 ratio and increase of caspase3 activation were observed in this study. Our findings suggest that human olfactory ensheathing cells can deliver phosphorylated GCV into the glioblastoma derived astrocytes through gap junction communication for apoptosis induction.

4:10 PM Abstract No. 50 Intratumoral radiofrequency hyperthermia-enhanced herpes simplex virus thymidine kinase (HSV-TK) gene therapy of orthotopic lung cancer through the augmentation of apoptosis and heat shock protein (HSP70) expression pathway

To investigate the associated biomolecular mechanisms of intratumoral RFH- enhanced local HSV-TK/ ganciclovir (GCV)-mediated suicide gene therapy of rat orthotopic lung cancers. Human lung cancer cells (A549) transduced with Luciferase/mCherry/lentivirus and 24 nude rats with orthotopic lung cancers were divided into four study groups with treatments of (i) intratumoral HSV-TK/GCV gene therapy followed by RFH at 41-42 °C for 30 minutes; (ii) HSV-TK/GCV therapy alone; (iii) RFH alone; (iv) PBS as a control. Flow cytometry, bioluminescence assay and confocal microscopy were used to determine the apoptosis and viability of cells. Bioluminescence optical imaging was used to evaluate the changes of bioluminescent signals among the groups over 2 weeks. The expression of Bcl-2/Bax, Caspase-3 and HSP70 in both cells and tumor tissue were quantified by western blotting (WB) and immunohistochemistical (IHC) staining respectively. Compared with gene therapy alone, RFH alone or PBS, combination therapy induced the lowest cell viability (30.56 ± 9.12% VS 65.47 ± 8.02% VS 89.95 ± 6.33% VS 100% P<0.05), the highest cell apoptosis (20.05 ± 2.12% VS 9.87 ± 2.02% VS 4.31 ± 4.31% VS 3.12 ± 1.03%, P<0.05), and a remarkable decrease of relative bioluminescence signal (16.54 ± 0.12% VS 54.8 ± 0.10% VS 85.01 ± 0.08% VS 100%, P<0.01). Optical imaging showed a markedly decreased bioluminescence signal of the tumor in combination therapy (1.29 ± 0.18 VS 1.76 ± 0.13 VS 2.26 ± 0.17 VS 2.94 ± 0.15, P<0.05). Both WB analysis and IHC staining displayed the significantly decreased expression of Bcl-2 (1.03 ± 0.28 VS 2.15 ± 0.33 VS 3.48 ± 0.12 VS 3.87 ± 0.17×107 IOD, P<0.01), as well as increased expression of Bax (3.30 ± 0.0.36 VS 2.02 ± 0.20 VS 1.48 ± 0.31 VS 1.26 ± 0.07, 107 IOD, P<0.01), Caspase-3 (5.58 ± 0.52 VS 3.36 ± 0.29 VS 1.42 ± 0.34VS 1.21 ± 0.52, 107 IOD, P<0.05) and HSP70 (8.79 ± 0.62 VS 3.97 ± 0.31 VS 4.48 ± 0.42 VS 1.60 ± 0.23, 107 IOD, P<0.01) in cancer tissues of combination therapy group. Intratumoral RFH-enhanced suicide gene therapy on rat orthotopic lung cancers through the mechanisms of augmenting apoptosis and the expression of HSP-70 pathway, which may provide additional insights to further improving treatment of lung cancers.

Shape-controlled magnetic mesoporous silica nanoparticles for magnetically-mediated suicide gene therapy of hepatocellular carcinoma

Magnetic nanoparticles (NPs) have emerged as a promising tool for suicide gene therapy. However, the separate delivery of the suicide gene and prodrug in current systems limits their clinical translation. Therefore, improving magnetically mediated suicide gene therapy by exploring higher performance magnetic NP-based hybrid nanoplatforms is an important challenge. In the current study, shape-controlled magnetic mesoporous silica nanoparticles (M-MSNs) were prepared, and their performance in magnetic resonance imaging (MRI)-guided, magnetically targeted and hyperthermia-enhanced suicide gene therapy of hepatocellular carcinoma (HCC) was investigated. Compared with sphere-like MSNs, rod-like MSNs exhibited higher loading capacity, faster prodrug release behavior, stronger magnetically enhanced gene delivery and better magnetic hyperthermia properties. Utilizing the improved magnetic properties of the M-MSNs allowed us to demonstrate highly effective dual magnetically enhanced suicide gene therapy in vivo with decreased systematic toxicity and with the ability to monitor therapeutic outcome by MRI. Because of their magnetic targeting abilities, magnetic hyperthermia performance and MRI properties, these M-MSNs might prove to be a potentially superior candidate for suicide gene therapy of HCC.

Lentiviral HSV-Tk.007-mediated suicide gene therapy is not toxic for normal brain cells.

Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation. In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis. During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed. We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd.

475. Effects of HSV-Tk Mediated Suicide Gene Therapy on Normal Brain Cells

Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. For glioblastoma, the most malignant form of glioma, the median survival time is generally less than one year. Lentiviral vector mediated suicide gene therapy has been reported to be a potential therapeutic option for glioma and has been validated in clinically relevant animal models. At this end, specific targeting of tumor cells sparing normal brain tissue is a significant concern. Although several studies may suggest that HSV-Tk mediated suicide gene therapy is not likely to be toxic for normal brain cells, it has never been investigated which effect lentiviral vector mediated HSV-Tk suicide gene therapy exerts on the normal brain in a non-tumor setting. We addressed this important question by delivering the suicide gene HSV-Tk. 007 to the brain of healthy, immunocompetent rats using lentiviral vectors pseudotyped with VSV-G. First, we studied behavior patterns and assessed any potential neurological symptoms in treatment and the control groups, during prodrug treatment with Ganciclovir. We did not observe any significant changes in their physical and behavioral patterns. Then, we carried detailed histological/immunohistochemical analyses of the brains to analyze potential differences in the number of transduced brain cells between control and treatment groups. There was no significant difference in numbers of neurons, astrocytes and oligodendrocyte progenitor cells among the groups, indicating successful survival of these cells after pro-drug treatment. Furthermore, potential inflammation and apoptosis of brain cells were also investigated. No significant infiltration of immune cells was detected across the groups. We could not detect any sign of apoptosis or necrosis across all groups. In conclusion, our study which suggests that lentiviral suicide gene therapy mediated by HSV-Tk. 007 is not toxic for normal brain cells.View Large Image | Download PowerPoint Slide

Genetically engineered theranostic mesenchymal stem cells for the evaluation of the anticancer efficacy of enzyme/prodrug systems

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase:uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose–response relationship studies in animals. Considering the limitations imposed by the prodrugs' bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems.

Anti-tumor effect of suicide gene therapy using chimeric promoter plus radiotherapy on cancer cell lines

To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation. The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis. The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells. Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.

Positive selection of gene-modified cells increases the efficacy of pancreatic cancer suicide gene therapy

Thymidine kinase (TK)-mediated suicide gene therapy has been considered for the treatment of pancreatic cancer. However, despite a bystander effect, the proportion of transduced tumor cells has proven too low to result in efficacy. We propose the use of a drug-selectable marker (MDR1) to enrich TK-expressing cells using chemotherapy. This enrichment or positive selection phase may increase the efficacy of suicide gene therapy. To test this strategy, we generated stable NP18MDR/TK-GFP transfectants and showed docetaxel resistance in vivo. Mixed tumors of MDR/TK-expressing cells and parental NP18 cells were established and docetaxel was used to increase the proportion of TK-expressing cells. After this positive selection phase, suicide gene therapy with ganciclovir was applied. Upon positive selection, the proportion of TK-expressing cells increased from 4% to 22%. Subsequent suicide gene therapy was more effective compared with a control group without positive selection. Starting with 10% of TK-expressing cells the positive-negative selection strategy completely inhibited tumor growth. Taken together, these results suggest that a positive-negative selection strategy based on MDR and TK genes represents an efficient way to increase the proportion of TK-expressing cells in the tumor and the efficacy of TK-mediated suicide gene therapy.

HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus‐HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

Efficacy of suicide gene therapy in hypoxic rat 9L glioma cells

Viral vector mediated suicide gene therapy (SGT) involving thymidine kinase (TK) or cytosine deaminase (CD) have considerable promise in the treatment of malignant brain tumors. An unresolved issue is to what extent tumor hypoxia influences the outcome of SGT since brain tumors characterized by regions of hypoxia have potentially reduced cellular metabolism and SGT's cytotoxicity is manifest through cellular metabolism. We studied in vitro and in vivo, the effect of hypoxia on the cytotoxicity of SGT in rat 9L glioma cells. Neither acute nor chronic hypoxia affected the cell killing of SGT by TK or CD. In vivo confirmation that SGT efficacy was not adversely affected by tumor hypoxia using the hypoxic cell marker pimonidazole was shown by the absence of a change in tumor hypoxia by SGT. These studies support the use of SGT utilizing either TK or CD gene strategies even when tumors are characterized by a hypoxic microenvironment.

Apoptotic Induction with Bifunctional E.coli Cytosine Deaminase-Uracil Phosphoribosyltransferase Mediated Suicide Gene Therapy is Synergized by Curcumin Treatment In vitro

Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment.

Polyethylene Glycol (Molecular Weight 400 DA) Vehicle Improves Gene Expression of Adenovirus Mediated Gene Therapy

A significant limitation of adenoviral mediated suicide gene therapy is poor gene distribution in vivo. The choice of vehicle has been demonstrated to affect the level of adenoviral delivered gene transduction. We examined the hypotheses that 1) adenovirus suspended in PEG400 improves gene expression in the naïve canine prostate model, 2) improved transgene expression with PEG400 results in improved tumor control and 3) vehicle affects the initial adenoviral spread from a single intratumor injection. The magnitude and volume of gene expression were measured 24 hours following intraprostatic injection of adenovirus suspended in PEG400 (12.5% weight per volume) or saline as vehicle. Tumor growth delay was measured in mice bearing human tumor xenografts following the injection of adenovirus in PEG400 and saline. The initial spread of adenovirus was measured by confocal microscopy following a single injection of fluorescently labeled adenoviral particles in human tumor xenografts using each vehicle. Adenovirus suspended in PEG400 provided an average of twice the level of gene expression in the canine prostate and significantly better tumor control relative to saline in preclinical tumor models (p = 0.046 and 0.036, respectively). The initial spread of adenovirus with PEG400 was superior to that of adenovirus in saline and the latter was largely limited to the needle tract. Adenoviral gene therapy vectors suspended in PEG400 results in improved tumor control because of greater initial adenoviral spread, and the increased volume and magnitude of gene expression in vivo.

945. Combination Treatment of Adenovirus Mediated HSV-Thymidine Kinase Suicide Gene Therapy and Docetaxel in Bladder Cancer

Purpose: The objective of this study was to evaluate the combined effect of adenovirus vector encoding Herpes simplex thymidine kinase plus ganciclovir (GCV) suicide gene therapy and Docetaxel (DTX) in human bladder cancer cell lines.

306. A Novel Thymidine Kinase in Enhanced Gap Junction Mediated Suicide Gene Therapy

Top of pageAbstract Patients with malignant gliomas have a poor prognosis due to failure of conventional therapy, and new treatment paradigms are urgently needed. The extensive heterogeneity of glioblastoma cells calls for a treatment strategy with multiple (cellular) targets. Suicide gene therapy for malignant gliomas has showed promising results in experimental animal models, but has failed in clinical trials due to low transfection rate, insufficient expression of the transgene in tumor cells, and low bystander killing effect. Gap junction communication (GJC) - allowing intercellular transport of small water soluble molecules - is generally low in malignant cells, leading to insufficient bystander effect. We have previously shown that treatment of glioma cells with the histone deacetylase inhibitor 4-phenyl butyrate (PB) leads to upregulation of the gap junction protein connexin 43 expression in both its phosphorylated and non-phosphorylated forms 1. This correlates with enhanced intercellular gap junction communication and increased bystander killing effect in a herpes simplex thymidine kinase (HSVtk)/ganciclovir suicide gene therapy in vitro model 2. A novel plant thymidine kinase with high affinity for the nucleoside analogue AZT was employed in a suicide gene therapy in vitro model. We show that the plant-TK/AZT system is superior to the HSV-TK/GCV system (app. 450- vs 55-fold time of activation) in the glioblastoma cell line U-87 MG. In addition, U-87 MG cells recombinantly expressing this thymidine kinase, showed a synergistic sensitivity to an AZT/PB combination therapy. This effect was shown to be partially gap junction mediated. Similar to glioblastoma cells, we show that neural stem cells (NSC) are subject to enhanced gap junction communication by treatment with PB. This was shown by enhanced fluorescent dye transfer between NSCs and glioblastoma cells. Furthermore, the bystander killing of tumor cells was investigated using plant kinase expressing NSCs co-cultured with glioblastoma cells in the presence of an AZT/PB combination compared to AZT alone. The presence of PB also resulted in an upregulation of connexin 43 in NSCs, as was shown for glioma cells. The successful cell killing in vitro using the plant-TK/AZT system prompted us to test this in vivo. An intracranial glioblastoma xenograft model in immuno-suppressed rats was developed. Plant-TK expressing U-87 MG glioma cell xenografts were established. The sensitivity of the tumor cells to AZT was assessed by intracranial infusion by use of an osmotic minipump. Drug concentrations of 40, 80, and 160 mM, with a flow of 5 ml/hour were used. 80 mM AZT was well tolerated by the rats for a 2 week period. The anti-tumor effect was assessed by histological and immunohistochemical techniques after sacrifice of animals and sectioning of the brain. To explore the NSC migration in rat brains carrying glioma xenografts, BrdU labeled NSCs or NSCs recombinantly expressing green fluorescent protein have been injected at some distance from the tumor site and their migrational capacity is being studied.

926. FK228 Increases Adenovirus Transgene Expression and Enhances Adenovirus Mediated Suicide Gene Therapy in Human Bladder Cancer Cells

926. FK228 Increases Adenovirus Transgene Expression and Enhances Adenovirus Mediated Suicide Gene Therapy in Human Bladder Cancer Cells

In vitro thymidine kinase/ganciclovir-based suicide gene therapy using replication defective herpes simplex virus-1 against leukemic B-cell malignancies (MCL, HCL, B-CLL)

A replication defective herpes simplex virus-1 was evaluated as a therapeutic vector. Mantle cell lymphoma (MCL), hairy cell leukemia (HCL), and B-cell chronic lymphocytic leukemia (B-CLL) were chosen because leukemic cells were collectable from peripheral bloods in these diseases. Cells from six MCL, one HCL, and nine B-CLL were infected in vitro with T0Z.1 at 3 multiplicity of infection (MOI). Herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV)-mediated suicide gene therapy showed 14.7% of mean tumor killing against leukemic B-cell malignancies. The mean tumor-killing effects were 8.7 and 17.1% in MCL and B-CLL, respectively. The effect against HCL was 29%. The study indicates that herpes simplex virus (HSV)-based gene therapy might be an effective strategy.

Adenovirus Mediated Prostate Specific Enzyme Prodrug Gene Therapy Using Prostate Specific Antigen Promoter Enhanced by the Cre-loxP System

Tissue or tumor specific gene delivery is crucial for achieving successful results in suicide gene therapy. Prostate specific antigen (PSA) promoter is known to be highly specific in prostate tissue but its promoter activity is much weaker than that of constitutive viral promoters. In the current study we enhanced PSA promoter activity by combining it with the Cre-loxP system. We also applied this system to adenovirus mediated suicide gene therapy with the cytosine deaminase (CD) gene. The Cre-loxP DNA recombination system was used to enhance PSA promoter. A plasmid with the PSA promoter-enhancer combination was constructed to drive Cre recombinase. Another plasmid contained the cytomegalovirus promoter-loxP-flanked stop signal-luciferase gene. LNCaP human prostate cancer cells were co-transfected with these 2 plasmids and luciferase activity was measured to assess promoter activities. Adenoviral vectors with the CD suicide gene were constructed in similar fashion and tested in LNCaP cells in in vitro/in vivo prostate cancer models. Promoter activity of the combined PSA promoter/enhancer and Cre-loxP system was 3 times stronger than that of PSA promoter/enhancer alone. It was further enhanced 7-fold in the presence of testosterone. Application of this system to CD suicide gene therapy by adenoviral vectors inhibited subcutaneous LNCaP tumor growth in nude mice. Combining the Cre-loxP system with PSA promoter/enhancer amplified promoter activity and was found to inhibit the growth of PSA producing prostate cancer cells in vivo.