Heterogeneity and viral replication fitness of HSV-1 clinical isolates with mutations in the thymidine kinase and DNA polymerase.

BACKGROUND: Prolonged antiviral therapy in immunocompromised individuals can result in the emergence of (multi)drug-resistant herpes simplex virus 1 (HSV-1) infections, forming a therapeutic challenge. OBJECTIVES: To evaluate spatial and temporal differences in drug resistance of HSV-1 samples from a HSCT recipient and to determine the effect of resistance mutations on viral replication fitness. PATIENTS AND METHODS: Five HSV-1 isolates were recovered from a HSCT recipient who suffered from persistent HSV-1 lesions, consecutively treated with aciclovir, foscarnet, cidofovir and a combination of ganciclovir and cidofovir. Spatial and temporal differences in HSV-1 drug resistance were evaluated genotypically [Sanger sequencing and next-generation sequencing (NGS) of the viral thymidine kinase (TK) and DNA polymerase (DP)] and phenotypically (plaque reduction assay). Viral replication fitness was determined by dual infection competition assays. RESULTS: Rapid evolution to aciclovir and foscarnet resistance was observed due to acquisition of TK (A189V and R222H) and DP (L778M and L802F) mutations. Virus isolates showed heterogeneous populations, spatial virus compartmentalization and minor viral variants in three out of five isolates (detectable by NGS but not by Sanger sequencing). Mutations in the TK and DP genes did not alter replication fitness without drug pressure. TK and/or DP mutants influenced replication fitness under antiviral pressure and showed increased fitness under pressure of the drug they showed resistance to. CONCLUSIONS: The use of NGS and dual infection competition assays revealed rapid evolution of HSV-1 drug resistance in a HSCT recipient with spatial and temporal compartmentalization of viral variants that had altered replication fitness under antiviral pressure.

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers.

Suicide gene therapies provide a unique ability to target cancer cells selectively, often based on modification of viral tropism or transcriptional regulation of therapeutic gene expression. We designed a novel suicide gene therapy approach wherein the gene product (herpes simplex virus thymidine kinase or yeast cytosine deaminase) is phosphorylated and stabilized in expression by the extracellular signal-regulated kinase (ERK), which is overactive in numerous cancers with elevated expression or mutation of receptor tyrosine kinases or the GTPase RAS. In contrast to transcriptional strategies for selectivity, regulation of protein stability by ERK allows for high copy expression via constitutive viral promoters, while maintaining tumor selectivity in contexts of elevated ERK activity. Thus, our approach turns a signaling pathway often coopted by cancer cells for survival into a lethal disadvantage in the presence of a chimeric protein and prodrug, as highlighted by a series of in vitro and in vivo examples explored here.

Step-by-Step Immune Activation for Suicide Gene Therapy Reinforcement.

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM-CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT-GM-CSF-danger signal system by means of artificial cancer specific promoters or a modified delivery system.

Molecular imaging-monitored radiofrequency hyperthermia-enhanced intratumoral herpes simplex virus-thymidine kinase gene therapy for rat orthotopic ovarian cancer.

Objective: To establish the technique of intratumoral combination therapy of radiofrequency hyperthermia (RFH) with herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy for rat ovarian cancers.Material and methods: This study consisted of three parts: (1) in vitro experiments to establish the 'proof of principal' that combination of RFH and HSV-TK gene therapy has the synergistic effect on human ovarian cancer cells; (2) creation of bioluminescence imaging-detectable rat ovarian cancer model; and (3) in vivo experiments using this rat model to validate the technical feasibility of the combination therapy. Cells and nude rats were divided into four groups: (i) combination therapy (HSV-TK/GCV + RFH); (ii) RFH; (iii) HSV-TK/GCV; and (iv) phosphate-buffered saline (PBS). Data were analyzed using Dunnett t-test or Kruskal-Wallis test.Results: Cell proliferation assay demonstrated significantly greater reduction in viable cells with the combination therapy [0.52 (0.43, 0.61)] compared to other treatments [RFH 0.90 (0.84, 0.96), HSV-TK/GCV 0.71 (0.53, 0.88), PBS 1 (1, 1); p < .05]. For 24 rat models with bioluminescence imaging-detectable orthotopic ovarian cancer (n = 6 per group), optical imaging demonstrated significantly decreased relative bioluminescence signal with the combination therapy [0.81 (0.52, 1.08)] compared to other treatments [RFH 3.60 (2.34, 4.86), HSV-TK/GCV 2.21 (1.71, 2.71), PBS 3.74 (3.19, 4.29); p < .001]. Ultrasound imaging demonstrated the smallest relative tumor volume with the combination therapy [0.78 (0.45, 1.11) versus 3.50 (2.67, 4.33), 2.10 (0.83, 3.37), 3.70 (1.79, 5.61); p < .05].Conclusion: The feasibility of intratumoral RFH-enhanced HSV-TK/GCV gene therapy was established on a unique rat model with molecular imaging-detectable orthotopic ovarian cancer.

Molecular docking and dynamics supported investigation of antiviral activity of Lichen metabolites of Roccella montagnei: an in silico and in vitro study.

Lichens are symbiotic organisms that have been traditionally used for treating different kinds of ailments. As there are only a few reports on the antiviral activity of lichens, we thought of evaluating the anti-Herpes simplex virus-1 (HSV-1) activity of methanolic extract of Roccella montagnei and their isolated compounds. Fractionation of crude methanolic extract of Roccella montagnei by column chromatography isolated two pure compounds. Antiviral activity was assessed using a CPE inhibition assay at non-cytotoxic concentrations on Vero cells. Molecular docking and dynamics studies were carried out against Herpes simplex type-1 thymidine kinase to understand the binding interactions of the isolated compounds with reference to acyclovir. Isolated compounds were characterized as methyl orsellinate and montagnetol by spectral methods. Methanolic extract of Roccella montagnei exhibited an EC50 value of 56.51 µg/ml, while the compounds methyl orsellinate and montagnetol offered EC50 values of 13.50 µg/ml and 37.52 µg/ml, respectively, against HSV-1 viral infection on Vero cell lines. The selectively index (SI) of montagnetol (10.93) was found to be higher when compared to that of methyl orsellinate (5.55), indicating its better anti-HSV-1 activity. The docking and dynamics studies showed montagnetol was stable throughout the 100 ns, having better interactions and docking scores with HSV-1 thymidine kinase than methyl orsellinate, as well as the standard. To understand the mechanism of montagnetol's anti-HSV-1 activity, more research is required, and this could lead to the discovery of new and effective antiviral agents.Communicated by Ramaswamy H. Sarma.

[Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1].

OBJECTIVE: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). METHODS: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. RESULTS: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) µg/ml, which was significantly lower than (0.301±0.04) µg/ml of CA46-C23-KNC cells and (0.338±0.05) µg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle. CONCLUSION: The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

Dioscin potentiates the antitumor effect of suicide gene therapy in melanoma by gap junction intercellular communication-mediated antigen cross-presentation.

Dioscin (Dio), steroid saponin, exists in several medicinal herbs with potent anticancer efficacy. This study aimed to explore the effect of Dio on the immune-related modulation and synergistic therapeutic effects of the herpes simplex virus thymidine kinase/ganciclovir (HSV-Tk/GCV) suicide gene therapy system in murine melanoma, thereby providing a research basis to improve the potential immunomodulatory mechanism underlying combination therapy. Using both in vitro and in vivo experiments, we confirmed the immunocidal effect of Dio-potentiated suicide gene therapy on melanoma. The results showed that Dio upregulated connexin 43 (Cx43) expression and improved gap junction intercellular communication (GJIC) in B16 cells while increasing the cross-presentation of antigens by dendritic cells (DCs), eventually promoting the activation and antitumor immune killing effects of CD8+ T lymphocytes. In contrast, inhibition or blockade of the GJIC function (overexpression of mutant Cx43 tumor cells/Gap26) partially reversed the potentiating effect. The significant synergistic effect of Dio on HSV-Tk/GCV suicide gene therapy was further investigated in a B16 xenograft mouse model. The increased number and activation ratio of CD8+ T lymphocytes and the levels of Gzms-B, IFN-γ, and TNF-α in mice reconfirmed the potential modulatory effects of Dio on the immune system. Taken together, Dio targets Cx43 to enhance GJIC function, improve the antigens cross-presentation of DCs, and activate the antitumor immune effect of CD8+ T lymphocytes, thereby providing insights into the potential immunomodulatory mechanism underlying combination therapy.

Iron Oxide Nanoparticles Promote Cx43-Overexpression of Mesenchymal Stem Cells for Efficient Suicide Gene Therapy during Glioma Treatment.

Background: Mesenchymal stem cells (MSCs) have been applied as a promising vehicle for tumour-targeted delivery of suicide genes in the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy against malignant gliomas. The efficiency of this strategy is largely dependent on the bystander effect, which relies on high suicide gene expression levels and efficient transportation of activated GCV towards glioma cells. However, up to now, the methods to enhance the bystander effect of this strategy in an efficient and safe way are still lacking and new approaches to improve this therapeutic strategy are required. Methods: In this study, MSCs were gene transfected using magnetosome-like ferrimagnetic iron oxide nanochains (MFIONs) to highly express HSV-tk. Both the suicide and bystander effects of HSV-tk expressed MSCs (MSCs-tk) were quantitatively evaluated. Connexin 43 (Cx43) expression by MSCs and glioma cells was measured under different treatments. Intercellular communication between MSCs and C6 glioma cells was examined using a dye transfer assay. Glioma tropism and the bio-distribution of MSCs-tk were observed. Anti-tumour activity was investigated in the orthotopic glioma of rats after intravenous administration of MSCs-tk followed by intraperitoneal injection of GCV. Results: Gene transfection using MFIONs achieved sufficient expression of HSV-tk and triggered Cx43 overexpression in MSCs. These Cx43 overexpressing MSCs promoted gap junction intercellular communication (GJIC) between MSCs and glioma cells, resulting in significantly inhibited growth of glioma through an improved bystander effect. Outstanding tumour targeting and significantly prolonged survival with decreased tumour size were observed after the treatment using MFION-transfected MSCs in glioma model rats. Conclusion: Our results show that iron oxide nanoparticles have the potential to improve the suicide gene expression levels of transfected MSCs, while promoting the GJIC formation between MSCs and tumour cells, which enhances the sensitivity of glioma cells to HSV-tk/GCV suicide gene therapy.

Prognostic role of serum thymidine kinase 1 activity in patients with hormone receptor-positive metastatic breast cancer: Analysis of the randomised phase III Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT).

BACKGROUND: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome. PATIENTS AND METHODS: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression. RESULTS: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker. CONCLUSION: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.

HSV­TK/GCV can induce cytotoxicity of retinoblastoma cells through autophagy inhibition by activating MAPK/ERK.

Retinoblastoma is an severe ophthalmic disease and the most common type intraocular malignant tumor, particularly in infants. Currently, few drugs and therapies are available. Gene therapy has been considered to be a potential treatment to cure cancer effectively and Herpes simplex virus type 1 thymidine kinase/ganciclovir (HSV­TK/GCV) is one type of suicide gene therapy that has been extensively studied. Numerous in vitro and in vivo studied have shown that this system can kill tumor cells, including liver and lung cancer cells. GCV is used as an antiviral drug, and the thymidine kinase, HSV­TK can phosphorylate GCV to GCV­TP, a competitive inhibitor of DNA synthesis, instead of guanine­5'­triphosphate in the process of DNA synthesis. This process prevents DNA chain elongation causing cell death via apoptosis. However, the toxic effects of HSV­TK/GCV on retinoblastoma cells remain unknown, and the molecular mechanisms of its therapeutic effects have not been fully elucidated. Our results suggest that HSV­TK/GCV can significantly cause the death of retinoblastoma cell lines, HXO­RB44 and Y79. Further studies have reported that this cell death is induced by the inhibition of autophagy by activating the MAPK/ERK (mitogen­activated protein kinase/ERK) signaling pathway. The mTOR inhibitor Torin1 can partially block the toxic effects of HSV­TK/GCV on HXO­RB44 cells. The above results demonstrate that the mechanism undertaken by HSV­TK/GCV to exhibit therapeutic effects mechanism may inhibit autophagy by activating MAPK/ERK. The findings of the present study may provide novel insight for the exploration of HSV­TK/GCV in the treatment of retinoblastoma.

[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells].

OBJECTIVE: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. METHODS: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated. RESULT: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect). CONCLUSION: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

Synergistic anticancer effects of ganciclovir/thymidine kinase and 5-fluorocytosine/cytosine deaminase gene therapies.

BACKGROUND: A bacterial enzyme, Escherichia coli cytosine deaminase, which converts the prodrug 5-fluorocytosine into the toxic drug 5-fluorouracil, and a viral enzyme, herpes simplex virus thymidine kinase, which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining these prodrug-activating gene therapies might result in enhanced anticancer effects. METHODS: Rat 9L gliosarcoma cells were transfected with plasmids containing the E. coli cytosine deaminase gene (9L/CD cells), with plasmids containing the herpes simplex virus thymidine kinase gene (9L/TK cells), or with both expression plasmids (9L/CD-TK cells). The drug sensitivities of the cell lines were evaluated; in addition, the sensitivities of 9L and 9L/CD-TK cells mixed in varied proportions were measured. The effects of prodrug treatment on 9L/CD-TK tumor growth (i.e., size and volume) in nude mice were monitored. The isobologram method of Loewe and the multiple drug-effect analysis method of Chou-Talalay were used to measure the interaction between the two prodrug-activating gene therapies. To elucidate the mechanism of interaction, the phosphorylation of ganciclovir in 9L/CD-TK cells after varying prodrug treatments was studied. RESULTS AND CONCLUSIONS: The presence of transfected cytosine deaminase and thymidine kinase genes in 9L gliosarcoma cells reduced cell survival, both in vitro and in vivo, following treatment with the relevant prodrugs; the effects of the two components appeared to be synergistic and related mechanistically to the enhancement of ganciclovir phosphorylation by thymidine kinase following 5-fluorouracil treatment.

Assessment of estrogenic endocrine-disrupting chemical actions in the brain using in vivo somatic gene transfer.

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element-thymidine kinase-luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17ss-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 microg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Adenoviral-mediated delivery of herpes simplex virus thymidine kinase results in tumor reduction and prolonged survival in a SCID mouse model of human ovarian carcinoma.

The herpes simplex virus thymidine kinase gene is the most widely utilized toxin for selective killing of carcinoma cells. Expression of the viral thymidine kinase gene renders cells sensitive to the toxic effects of nucleoside analogs such as ganciclovir. An advantage of this system is the "bystander effect" whereby thymidine kinase transduced tumor cells elicit a toxic effect on surrounding nontransduced tumor cells. Ovarian carcinoma appears to be an ideal candidate for gene therapy as the majority of women present with advanced stage disease, have poor prognosis for long-term survival and have the disease confined within the peritoneal cavity. Therefore the utility of an adenoviral vector to elicit an in vitro bystander effect in ovarian carcinoma cells and the therapeutic efficacy of such a system in vivo was undertaken. Immunocompetent animals were utilized to determine the maximum dose of adenovirus that could be administered without any undesirable side effects and that preimmunization had no effects on subsequent challenge. SCID mice were orthotopically transplanted with human ovarian carcinoma cells and, after establishment of tumor, given a recombinant adenovirus expressing either the herpes simplex virus thymidine kinase or the Escherichia coli beta-galactosidase gene. Half the animals from each viral group were treated with either a ganciclovir regiment (50 mg/kg daily for 14 days) or an equal volume of serum-free media. A subset of mice were killed following drug treatment and analyzed for tumor reduction. The remaining animals were followed daily for survival. The animals treated with the recombinant adenovirus expressing the herpes simplex virus thymidine kinase gene and ganciclovir had significant reduction in overall tumor burden and demonstrated statistically significant prolongation in overall survival.

Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir-mediated cytotoxicity and the "bystander effect".

Transfer of the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene into tumor cells followed by ganciclovir (GCV) administration, will provide selective tumor cell killing. We studied the effect of herpes simplex virus thymidine kinase (HSV-tk) expression level on the HSV-tk/GCV-mediated "bystander effect." Clones of HSV-tk-transduced rat glioma cells (9L) were isolated that stably expressed with different levels of HSV-tk. All clones studied had similar sensitivity to ganciclovir with IC50 values ranging from 0.45 to 1.3 microM. Within certain enzyme level thresholds, in vitro evaluation of the bystander effect has shown that clones with higher level of HSV-tk expression exhibited a better bystander effect. Interestingly, the bystander effect was observed between different cell types. Both the transduction efficiency and bystander effect are essential factors for the success of the antitumor effect by the HSV-tk/prodrug GCV suicide gene system.

Gene therapy for peritoneal dissemination of pancreatic cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene.

Peritoneal dissemination is one of the most common complications of the malignancies of the digestive system, such as gastric or pancreatic cancers. Yet, no effective therapy has been established so far to alleviate this devastating and often fatal end-stage condition. Here we describe a novel approach of intraperitoneal (i.p.) lipofection of a suicidal gene to the pancreatic cancer cells in a mouse peritoneal dissemination model. A human pancreatic cancer cell line, PSN-1, was inoculated into the peritoneal cavity of nude mice. Eight days later, a herpes simplex virus thymidine kinase (HSV-TK) gene expression plasmid under a potent hybrid promoter CAG was injected as a DNA-lipopolyamine complex. Ganciclovir (GCV) was then administered for 8 days, and the mice were examined for tumor development at the 24th day after the tumor inoculation. Although all 24 control mice showed macroscopic peritoneal dissemination and solid tumors on the pancreas, 8 of the 14 mice treated with HSV-TK and GCV were free of tumors, and only a few small tumors were observed in the remaining 6 mice. Treatment-related toxicity was not observed. The semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis suggested that the HSV-TK transgene was expressed in about 10% of tumor cells but not in the normal pancreas or in the small intestine. When the lacZ gene was transduced in place of the HSV-TK gene, the blue-stained cells were identified only in tumor nodules and not in normal organs. This preclinical study suggests the therapeutic feasibility of the i.p. lipofection-based suicidal gene/prodrug strategy for peritoneal dissemination of pancreatic cancer.

Retrovirus-mediated suicide gene transduction in the vitreous cavity of the eye: feasibility in prevention of proliferative vitreoretinopathy.

In proliferative vitreoretinopathy (PVR), retinal pigment epithelial cells, fibroblasts, or other proliferating cells form contractile membranes in the vitreous cavity of the eye, resulting in traction retinal detachment. Retroviral vector-mediated transfer is a suitable method to transduce the herpes simplex virus thymidine kinase (HSV-tk) gene into proliferating cells in PVR, allowing for the selective killing of these cells. To determine the potential of gene transduction in the environment of the vitreous cavity, we evaluated the effect of vitreous humor on retroviral vector-mediated gene transduction of rabbit dermal fibroblasts in vitro and studied in vivo transduction in rabbit experimental PVR with retroviral vector G1BgSvNa. In addition, we studied the bystander effect in vitro and in vivo in a rabbit model of PVR, with low percentages of HSV-tk-positive cells. Finally, we evaluated the efficacy of intravitreal administration of HSV-tk retroviral vector G1TkSvNa followed by ganciclovir (GCV) in the prevention of experimental PVR. Vitreous humor reduced gene transfer efficiency in vitro in a dose-dependent manner. LacZ expression was found in cells of preretinal or intravitreal membranes of animals of both in vivo and in vitro transduction groups; however, in vivo transduction resulted in a decreased number of transduced cells, with a relative transduction efficiency of approximately 2%. Transduction of HSV-tk was associated with a powerful bystander effect both in vitro and in vivo with significant effects even when HSV-tk-positive cells represented only 1% of the population. In vivo transduction with G1TkSvNa followed by GCV significantly inhibited the development of PVR (p < 0.05). These results suggest that retroviral vector-mediated transfer of HSV-tk into the proliferating cells in PVR may be feasible and may provide a novel therapeutic strategy for this disease.

Enhancement of the herpes simplex virus thymidine kinase/ganciclovir bystander effect and its antitumor efficacy in vivo by pharmacologic manipulation of gap junctions.

Apigenin, a flavinoid, and lovastatin, an HMG-CoA reductase inhibitor, upregulated gap junction (GJ) function and dye transfer in tumors expressing GJ and were inactive in the GJ-negative tumor line N2a. N2a cells transfected with the connexin 43 gene showed restored cell-to-cell dye transfer, which could then be improved nearly fourfold by addition of apigenin. To test the drugs in HSV thymidine kinase/ganciclovir (HSV-tk/GCV) tumor killing, mixtures of 90% wild-type (WT) with 10% HSV-tk gene-modified MCA38 adenocarcinoma cells were exposed in vitro to GCV +/- apigenin or lovastatin. A significant bystander effect (BSE) was seen following GCV treatment alone, while neither apigenin or lovastatin alone had any effect on the recovery of viable tumor colonies. However, GCV-treated cultures also exposed to apigenin or lovastatin showed an increased BSE and reduced tumor cell recovery. Thirty percent of mice bearing tumors from the same mixture of 90% WT and 10% HSV-tk MCA38 cells treated with GCV alone became tumor free. Tumor-bearing mice given only two or three injections of lovastatin or apigenin during GCV treatment had a doubling of the antitumor response rate, with 60-70% of the mice achieving complete remission. These results support the hypothesis that the transfer of phosphorylated GCV from HSV-tk gene-expressing cells to neighboring WT tumor cells is a major component of the BSE and that pharmacological manipulation of GJ function with lovastatin or apigenin can result in striking improvement in the antitumor response in mice with tumors modified to contain as few as 10% HSV-tk cells.

Synergy between the herpes simplex virus tk/ganciclovir prodrug suicide system and the topoisomerase I inhibitor topotecan.

An established principle of antineoplastic chemotherapy is that multidrug regimens are generally superior to single-agent therapy. This prompted us to elucidate whether the topoisomerase inhibitor topotecan (TPT) could enhance the efficacy of the herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) system for the treatment of cancer. We assessed the interaction between these two treatments in murine MC38 and human HT-29 colon carcinoma cell lines that were genetically modified to constitutively express HSV-tk, sensitizing them to GCV. Synergistic cell killing was observed in a clonogenic assay over most of the cytotoxic dose range by the median-effect principle of Chou and Talalay (Adv. Enzyme Regul. 1984; 22:27-55). Subcutaneous tumor models, using the same cell lines in C57BL/6 and athymic nude mice, respectively, demonstrated that the combination of GCV and TPT resulted in statistically significant enhanced survival relative to single-agent treatment. In addition, nude mice bearing HT-29 tumor xenografts were treated with an Ad5 E1b Mr 55,000 attenuated replication-competent adenovirus expressing HSV-tk (Ad.TK(RC)) either alone or in combination with GCV and/or TPT. These experiments demonstrated that Ad.TK(RC) followed by GCV and TPT was more efficacious than any other treatment tested. Our results suggest that for antineoplastic therapy, molecular chemotherapy based on the HSV-tk/GCV system combined with traditional chemotherapy is a logical and practical future direction to pursue. Suicide gene therapy is the approach whereby genetically altering a cell makes it susceptible to an otherwise relatively nontoxic prodrug. By this approach it is possible to achieve relatively high concentrations of the toxic metabolites in the transduced cells while maintaining low systemic levels of the active drug. The most often used metabolic suicide gene transfer system is the HSV-tk/GCV paradigm, which is currently being used in cancer therapy or as a safety modality. The low response rate observed in the early clinical HSV-tk cancer trials may be due to failure in achieving adequate transduction efficiency and/or prodrug concentration within the tumor. The combination of such suicide gene prodrug systems with adjunctive drugs resulting in synergistic cytotoxicity might improve the clinical utility of this approach.

Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain.

We investigated the therapeutic efficacy of adenovirus-mediated gene therapy to treat malignant mammary tumors in vitro and in vivo in the brain. A mammary adenocarcinoma cell line derived from Fischer rats (13762 MAT B III; MAT-B) was used. In vitro studies demonstrated that the MAT-B cells could be efficiently transduced with a replication-defective adenovirus (ADV) vector that carried the herpes simplex virus gene for thymidine kinase (ADV-tk), and that ADV-tk transduction rendered the MAT-B cells sensitive to killing, in a dose-dependent manner, with ganciclovir (GCV). An animal model of a mammary tumor metastatic to the brain was produced by injecting MAT-B cells into the caudate nucleus of Fischer rats. Seven days after MAT-B cell injection, when the tumors were approximately 5 mm2 in cross-sectional size, the tumors were injected with ADV-tk or a control adenovirus vector containing the beta-galactosidase (beta-Gal) gene (ADV-beta gal). After vector injection the animals were treated with GCV or with saline for 6 days. Sixteen days after tumor cell injection, the brains were examined histologically. The rats that were injected with ADV-beta gal and treated with GCV or saline, and those that were injected with ADV-tk and treated with saline had large tumors, whereas the rats that were injected with ADV-tk and treated with GCV had no visible tumor tissue at the site of tumor cell injection. In survival studies animals treated with ADV-tk+GCV survived a significantly longer time than animals treated with ADV-beta gal+GCV. Our results demonstrate that the recombinant adenoviral vector containing the tk gene confers GCV cytotoxic sensitivity to mammary tumor cells in vitro and in the brain, and suggest that this treatment strategy may be useful in treating somatic tumors that metastasize to the brain.