Herpes Simplex Virus Thymidine Kinase Expression Research Articles

Improved safety of induced pluripotent stem cell-derived antigen-presenting cell-based cancer immunotherapy

The tumorigenicity and toxicity of induced pluripotent stem cells (iPSCs) and their derivatives are major safety concerns in their clinical application. Recently, we developed granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing proliferating myeloid cells (GM-pMCs) from mouse iPSCs as a source of unlimited antigen-presenting cells for use in cancer immunotherapy. As GM-pMCs are generated by introducing c-Myc and Csf2 into iPSC-derived MCs and are dependent on self-produced GM-CSF for proliferation, methods to control their proliferation after administration should be introduced to improve safety. In this study, we compared the efficacy of two promising suicide gene systems, herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) and inducible caspase-9 (iCasp9)/AP1903, for safeguarding GM-pMCs in cancer immunotherapy. The expression of HSV-TK or iCasp9 did not impair the fundamental properties of GM-pMCs. Both of these suicide gene-expressing cells selectively underwent apoptosis after treatment with the corresponding apoptosis-inducing drug, and they were promptly eliminated in vivo. iCasp9/AP1903 induced apoptosis more efficiently than HSV-TK/GCV. Furthermore, high concentrations of GCV were toxic to cells not expressing HSV-TK, whereas AP1903 was bioinert. These results suggest that iCasp9/AP1903 is superior to HSV-TK/GCV in terms of both safety and efficacy when controlling the fate of GM-pMCs after priming antitumor immunity.

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.

Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect.

Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.

Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene.

In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2.

Regulation of HSVtk gene by endogenous microRNA-122a in liver cell lines as suicide gene therapy.

Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs. Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation. We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines. Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively. Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression.

Combined delivery of temozolomide and the thymidine kinase gene for treatment of glioblastoma

Glioblastoma is the most malignant form of brain tumor. In this study, combination therapy with temozolomide (TMZ) and the herpes simplex virus thymidine kinase (HSVtk) gene was evaluated in glioblastoma models. The R7L10 peptide was used as a carrier of TMZ and the HSVtk gene. TMZ was loaded into R7L10 micelles using the oil-in-water emulsion/solvent evaporation method. The TMZ-loaded R7L10 (R7L10-TMZ) micelles formed a complex with the HSVtk gene, pHSVtk. The formation of the R7L10-TMZ/pHSVtk complex was confirmed by gel retardation and heparin competition assays. An in vitro transfection assay demonstrated that the transfection efficiency of R7L10-TMZ was similar to that of R7L10 in C6 glioblastoma cells. R7L10-TMZ had greater anti-tumor effects than TMZ alone in C6 cells in vitro, suggesting that R7L10 is an efficient carrier of TMZ. The in vivo efficacy of the R7L10-TMZ/pHSVtk complex was evaluated in the intracranial glioblastoma model. HSVtk expression in tumors was confirmed by immunohistochemistry. Furthermore, a greater anti-tumor effect was observed in the R7L10-TMZ/pHSVtk group compared with the TMZ or R7L10/pHSVtk single injection group. In conclusion, combined delivery of TMZ and the HSVtk gene using R7L10 peptides may be useful for the treatment of glioblastoma.

Tumor-specific promoter-driven adenoviral therapy for insulinoma.

Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors. We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, βTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays. Using in vitro insulinoma-derived cell lines and an invivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone. INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.

Combined antitumor gene therapy with herpes simplex virus-thymidine kinase and short hairpin RNA specific for mammalian target of rapamycin.

A proof-of-concept study is presented using dual gene therapy that employed a small hairpin RNA (shRNA) specific for mammalian target of rapamycin (mTOR) and a herpes simplex virus-thymidine kinase (HSV-TK) gene to inhibit the growth of tumors. Recombinant adeno-associated virus (rAAV) vectors containing a mutant TK gene (sc39TK) were transduced into HeLa cells, and the prodrug ganciclovir (GCV) was administered to establish a suicide gene-therapy strategy. Additionally, rAAV vectors expressing an mTOR-targeted shRNA were employed to suppress mTOR-dependent tumor growth. GCV selectively induced death in tumor cells expressing TK, and the mTOR-targeted shRNA altered the cell cycle to impair tumor growth. Combining the TK-GCV system with mTOR inhibition suppressed tumor growth to a greater extent than that achieved with either treatment alone. Furthermore, HSV-TK expression and mTOR inhibition did not mutually interfere with each other. In conclusion, gene therapy that combines the TK-GCV system and mTOR inhibition shows promise as a novel strategy for cancer therapy.

Editorial: Improving the safety of cell therapy products by suicide gene transfer.

Editorial: Improving the safety of cell therapy products by suicide gene transfer.

A GSDMB enhancer-driven HSV thymidine kinase-expressing vector for controlling occult peritoneal dissemination of gastric cancer cells.

BackgroundGastric cancer (GC) is one of the major malignant diseases worldwide, especially in Asia, and Japan and Korea have the highest incidence in the world. Because most of the cases that are refractory to therapies die due to peritoneal dissemination (PD) of the cancer cells, controlling PD is important for patient survival. GSDMB is a member of the gasdermin gene family. Because GSDMB is expressed in many types of cancer, including GC, it is likely that the gene contains a regulatory region that is utilized for therapy of occult PD through cancer cell-specific expression of cytotoxic genes.MethodsWe performed reporter assays to identify the regulatory region for the cancer cell-specific expression. We also constructed a lentiviral therapeutic vector that expresses herpes simplex virus thymidine kinase (HSVtk) in a GC cell-specific manner, and tested it in a mouse model of PD.ResultsWe identified the regulatory region at +496 to +989 from the GSDMB transcription start site and designated it as a GSDMB enhancer. The lentiviral therapeutic vector suppressed proliferation of a GC cell line, 60As6, in vitro in the presence of ganciclovir, and intraperitoneal administration of the vector prolonged the survival term of mice that were intraperitoneally inoculated with 60As6 one week prior to the administration.ConclusionsThe GSDMB-driven HSVtk expression vector had a therapeutic effect on the occult PD model mice. This strategy can potentially be used to treat GC patients with PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1436-1) contains supplementary material, which is available to authorized users.

Improving the safety of cell therapy with the TK-suicide gene.

While opening new frontiers for the cure of malignant and non-malignant diseases, the increasing use of cell therapy poses also several new challenges related to the safety of a living drug. The most effective and consolidated cell therapy approach is allogeneic hematopoietic stem cell transplantation (HSCT), the only cure for several patients with high-risk hematological malignancies. The potential of allogeneic HSCT is strictly dependent on the donor immune system, particularly on alloreactive T lymphocytes, that promote the beneficial graft-versus-tumor effect (GvT), but may also trigger the detrimental graft-versus-host-disease (GvHD). Gene transfer technologies allow to manipulate donor T-cells to enforce GvT and foster immune reconstitution, while avoiding or controlling GvHD. The suicide gene approach is based on the transfer of a suicide gene into donor lymphocytes, for a safe infusion of a wide T-cell repertoire, that might be selectively controlled in vivo in case of GvHD. The herpes simplex virus thymidine kinase (HSV-TK) is the suicide gene most extensively tested in humans. Expression of HSV-TK in donor lymphocytes confers lethal sensitivity to the anti-herpes drug, ganciclovir. Progressive improvements in suicide genes, vector technology and transduction protocols have allowed to overcome the toxicity of GvHD while preserving the antitumor efficacy of allogeneic HSCT. Several phase I-II clinical trials in the last 20 years document the safety and the efficacy of HSV-TK approach, able to maintain its clear value over the last decades, in the rapidly progressing horizon of cancer cellular therapy.

Suicide HSVtk gene delivery by neurotensin-polyplex nanoparticles via the bloodstream and GCV Treatment specifically inhibit the growth of human MDA-MB-231 triple negative breast cancer tumors xenografted in athymic mice.

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55–60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus‐infected cells reduces virus titers upon treatment with ganciclovir in vitro

Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

Co-expression of herpes simplex virus thymidine kinase and Escherichia coli nitroreductase by an hTERT-driven adenovirus vector in breast cancer cells results in additive anti-tumor effects

Breast cancers especially in the late and metastatic stages remain refractory to treatment despite advances in surgical techniques and chemotherapy. Tumor-specific promoter-directed suicide gene therapy and adenoviral technology can be promising strategies for such advanced disease. Previous studies suggested that combining herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) with Escherichia coli nitroreductase (Coli.NTR) and 5-(azaridin-1-yl)-2, 4-dinitrobenzamide (CB1954) by a recombinant retrovirus delivery system resulted in a co-operative killing effect in vitro. We constructed a bicistronic adenovirus type 5 (Ad5)-based vector which co-expresses herpes HSV-TK and Coli.NTR under the control of the human telomerase reverse transcriptase (hTERT) promoter and SV40 enhancer. NTR gene expression mediated by an internal ribosome entry site (IRES) was inserted after the hTERT and HSV-TK sequences. Anti-tumor activities of the novel vector, Ad-hT-TK/NTR-enh, combined with prodrugs were evaluated in human breast cancer cells (ZR-75-30, MCF-7) in vitro and invivo. We showed that expression of HSV-TK and NTR genes by Ad-hT-TK/NTR-enh in combination with GCV and CB1954 resulted in specific and significant cytotoxic effects in breast cancer cells in vitro. The anti-tumor activity of this system was more efficient than that from a single suicide gene, and only slightly lower than by HSV-TK and NTR driven from separate hTERT promoters in vitro and in vivo while the total amount of adenovirus of Ad-hT-TK/NTR-enh was half that of Ad-hT-TK-enh+Ad-hT-NTR-enh. These results suggest that suicide genes HSV-TK and NTR mediated by a single adenovirus vector under the control of an enhanced hTERT promoter results in additive anti-tumor effects and may provide a relatively safe strategy for the treatment of breast cancer by tumor-specific targeting.

Adeno-Associated Virus Liver Transduction Efficiency Measured by in Vivo [18F]FHBG Positron Emission Tomography Imaging in Rodents and Nonhuman Primates

Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.

Illuminating progression: tracking melanoma metastasis in a gene therapy setting

Illuminating progression: tracking melanoma metastasis in a gene therapy setting

Ultrasound-targeted microbubble destruction mediated herpes simplex virus-thymidine kinase gene treats hepatoma in mice

ObjectiveThe purpose of the study was to explore the anti-tumor effect of ultrasound -targeted microbubble destruction mediated herpes simplex virus thymidine kinase (HSV-TK) suicide gene system on mice hepatoma.MethodsForty mice were randomly divided into four groups after the models of subcutaneous transplantation tumors were estabilished: (1) PBS; (2) HSV-TK (3) HSV-TK+ ultrasound (HSV-TK+US); (4) HSV-TK+ultrasound+microbubbles (HSV-TK+US+MB). The TK protein expression in liver cancer was detected by western-blot. Applying TUNEL staining detected tumor cell apoptosis. At last, the inhibition rates and survival time of the animals were compared among all groups.ResultsThe TK protein expression of HSV-TK+MB+US group in tumor-bearing mice tissues were significantly higher than those in other groups. The tumor inhibitory effect of ultrasound-targeted microbubble destruction mediated HSV-TK on mice transplantable tumor was significantly higher than those in other groups (p < 0.05), and can significantly improve the survival time of tumor-bearing mice.ConclusionUltrasound-targeted microbubble destruction can effectively transfect HSV-TK gene into target tissues and play a significant inhibition effect on tumors, which provides a new strategy for gene therapy in liver cancer.

Comparative analysis of herpes simplex virus thymidine kinase gene expression potentiation via HIV-1 Tat-TAR system and cancer-specific promoters in P53(+) and P53(-) cells

Tumor-specific promoters are predominantly active and ensure expression of the gene under control exclusively in cancer cells. However, a low activity of the promoters is an essential disadvantage for their therapy usage. To achieve a higher expression level of the therapeutic gene, herpes simplex virus thymidine kinase (HSV-tk), the Tat-TAR-system being utilized by HIV-1 for increasing own gene expression was developed. A potentiating activity of tat gene under control of two different cancer-specific gene (human survivin gene and human telomerase reverse transcriptase) promoters for increasing of the HSV-tk gene expression being regulated by TAR-element was evaluated, and activity of the cancer-specific promoters in the Tat-TAR-system was compared. Co-transfection of the cells with the both constructions led to the tat protein synthesis and its affect the HIV-1 TAR-element. An expression level of HSV-tk gene ensured by the both promoters in the binary system was close to that for strong non-specific cytomegalovirus (CMV) promoter. Enzymatic activity of HSV-tk protein in cells having both elements of Tat-TAR-system was two orders of magnitude higher than that in the cells transfected with HSV-tk gene under control of the cancer-specific promoter. Notably, the effect was independent of p53-status of transfected cells: HSV-tk expression level was almost the same in p53(+) and p53(-) cells. The obtained results show that system may be used for therapy of different cancer types both p53-defective and p53-positive ones inhibiting cancer-specific promoters activity.

Long-term and stable expression of transgene mediated by piggyBac transposon in gynecological malignant tumor cells

To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer. Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK). By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B. The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency. The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR). The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay. The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line. The insertion sites of foreign gene transferred by PB transposon in genome were analyzed by inverse PCR. (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully. (2) Using three different transfective reagents, PB transposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7+/-9.2)%] was higher than that of lipofectamine 2000 [(54.1+/-11.4)%] and jetPEI [(46.5+/-7.4)%, all P<0.05]; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfection efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7+/-9.2)%, (74.4+/-8.9)% and (83.2+/-9.7)% respectively, which all were higher than that on HEC-1B [(39.5+/-8.7)%, P<0.05]. (3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines. (4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 microg/ml respectively. Inhibitory effect of GCV (10 microg/ml) on SKOV3 transfected with pPB/TK was (86+/-9)%, which was superior to that transfected with pORF-HSVtk alone [(52+/-12)%, P<0.05]. (5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites. mRFP1 expression still could be detected in three months after transfected. PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression. It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

INSM1Promoter-Driven Adenoviral Herpes Simplex Virus Thymidine Kinase Cancer Gene Therapy for the Treatment of Primitive Neuroectodermal Tumors

The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs.