Herpes Simplex Virus Thymidine Kinase Gene Research Articles

Suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cell gene delivery vehicles in recurrent glioblastoma multiforme: a first-in-human, dose-escalation, phase I clinical trial

BackgroundGlioblastoma multiforme (GBM) is associated with remarkably poor prognosis, and its treatment is challenging. This investigation aimed to evaluate the safety of suicide gene therapy using allogeneic adipose tissue-derived mesenchymal stem cells (ADSCs) carrying herpes simplex virus-thymidine kinase (HSV-TK) gene for the first time in patients with recurrent GBM.MethodsThis study was a first-in-human, open-label, single-arm, phase I clinical trial with a classic 3 + 3 dose escalation design. Patients who did not undergo surgery for their recurrence were included and received this gene therapy protocol. Patients received the intratumoral stereotactic injection of ADSCs according to the assigned dose followed by prodrug administration for 14 days. The first dosing cohort (n = 3) received 2.5 × 105 ADSCs; the second dosing cohort (n = 3) received 5 × 105 ADSCs; the third dosing cohort (n = 6) received 10 × 105 ADSCs. The primary outcome measure was the safety profile of the intervention.ResultsA total of 12 patients with recurrent GBM were recruited. The median follow-up was 16 (IQR, 14-18.5) months. This gene therapy protocol was safe and well tolerated. During the study period, eleven (91.7%) patients showed tumor progression, and nine (75.0%) died. The median overall survival (OS) was 16.0 months (95% CI 14.3–17.7) and the median progression-free survival (PFS) was 11.0 months (95% CI 8.3–13.7). A total of 8 and 4 patients showed partial response and stable disease, respectively. Moreover, significant changes were observed in volumetric analysis, peripheral blood cell counts, and cytokine profile.ConclusionsThe present clinical trial, for the first time, showed that suicide gene therapy using allogeneic ADSCs carrying the HSV-TK gene is safe in patients with recurrent GBM. Future phase II/III clinical trials with multiple arms are warranted to validate our findings and further investigate the efficacy of this protocol compared with standard therapy alone.Trial registration: Iranian Registry of Clinical Trials (IRCT), IRCT20200502047277N2. Registered 8 October 2020, https://www.irct.ir/.

Potent bystander effect and tumor tropism in suicide gene therapy using stem cells from human exfoliated deciduous teeth.

Herpes simplex virus thymidine kinase (HSVTK)/ganciclovir (GCV) suicide gene therapy has a long history of treating malignant gliomas. Recently, stem cells from human exfoliated deciduous teeth (SHED), which are collected from deciduous teeth and have excellent harvestability, ethical aspects, and self-renewal, have been attracting attention mainly in the field of gene therapy. In the present study, we assessed SHED as a novel cellular vehicle for suicide gene therapy in malignant gliomas, as we have previously demonstrated with various cell types. SHED was transduced with the HSVTK gene (SHEDTK). In vitro experiments showed a significant bystander effect between SHEDTK and glioma cell lines in coculture. Furthermore, apoptotic changes caused by caspase 3/7 activation were simultaneously observed in SHEDTK and glioma cells. Mice implanted with a mixture of U87 and SHEDTK and treated with intraperitoneal GCV survived for longer than 100 days. Additionally, tumors in treatment model mice were significantly reduced in size during the treatment period. SHEDTK implanted at the contralateral hemisphere migrated toward the tumor crossing the corpus callosum. These results suggested that SHEDTK-based suicide gene therapy has potent tumor tropism and a bystander-killing effect, potentially offering a new promising therapeutic modality for malignant gliomas.

Ganciclovir and Its Hemocompatible More Lipophilic Derivative Can Enhance the Apoptotic Effects of Methotrexate by Inhibiting Breast Cancer Resistance Protein (BCRP)

Efflux transporters, namely ATP-binding cassette (ABC), are one of the primary reasons for cancer chemoresistance and the clinical failure of chemotherapy. Ganciclovir (GCV) is an antiviral agent used in herpes simplex virus thymidine kinase (HSV-TK) gene therapy. In this therapy, HSV-TK gene is delivered together with GCV into cancer cells to activate the phosphorylation process of GCV to active GCV-triphosphate, a DNA polymerase inhibitor. However, GCV interacts with efflux transporters that are responsible for the resistance of HSV-TK/GCV therapy. In the present study, it was explored whether GCV and its more lipophilic derivative (1) could inhibit effluxing of another chemotherapeutic, methotrexate (MTX), out of the human breast cancer cells. Firstly, it was found that the combination of GCV and MTX was more hemocompatible than the corresponding combination with compound 1. Secondly, both GCV and compound 1 enhanced the cellular accumulation of MTX in MCF-7 cells, the MTX exposure being 13–21 times greater compared to the MTX uptake alone. Subsequently, this also reduced the number of viable cells (41–56%) and increased the number of late apoptotic cells (46–55%). Moreover, both GCV and compound 1 were found to interact with breast cancer resistant protein (BCRP) more effectively than multidrug-resistant proteins (MRPs) in these cells. Since the expression of BCRP was higher in MCF-7 cells than in MDA-MB-231 cells, and the cellular uptake of GCV and compound 1 was smaller but increased in the presence of BCRP-selective inhibitor (Fumitremorgin C) in MCF-7 cells, we concluded that the improved apoptotic effects of higher MTX exposure were raised mainly from the inhibition of BCRP-mediated efflux of MTX. However, the effects of GCV and its derivatives on MTX metabolism and the quantitative expression of MTX metabolizing enzymes in various cancer cells need to be studied more thoroughly in the future.

Intracerebral Administration of Autologous Mesenchymal Stem Cells as HSV-TK Gene Vehicle for Treatment of Glioblastoma Multiform: Safety and Feasibility Assessment.

Widespread investigation has revealed the promising ability of suicidal genes in the treatment of glioma tumors; nevertheless, promoting their effects relies on the ability to apply suitable vehicles and techniques. In this study, the safety and feasibility of using bone marrow-derived mesenchymal stem cells (MSCs) in combination with prodrug for treatment of patients with primary and secondary glioblastoma multiform (GBM) was assessed. Five GBM patients were recruited. Following gross total resection of the tumor and adjuvant radiotherapy and chemotherapy, intracerebral injection of autologous MSCs transduced with lentivirus containing herpes simplex virus thymidine kinase (HSV-TK) was performed followed by intravenous administration of ganciclovir for 2weeks. The treatment was well tolerated by all patients. Mild-to-moderate fever, headache, and cerebrospinal fluid leukocytosis were evident in three, two, and one patient, respectively. The progression-free survival (PFS) and overall survival (OS) of patients were 95.79 ± 51.40 and 128.85 ± 48.81weeks, respectively. The 1-year PFS and OS were 60% and 100%, respectively, among our patients, and two patients had more than 3years of OS and more than 2years of PFS. It seems that intracerebral administration of bone marrow MSC containing the HSV-TK gene in combination with intravenous ganciclovir would be safe and feasible in the treatment of patients with GBM.

Congress of neurological surgeons systematic review and evidence-based guidelines update on the role of emerging developments in the management of newly diagnosed glioblastoma

These recommendations apply to adult patients with newly diagnosed or suspected glioblastoma. Question What imaging modalities are in development that may be able to provide improvements in diagnosis, and therapeutic guidance for individuals with newly diagnosed glioblastoma? Level III: It is suggested that techniques utilizing magnetic resonance imaging for diffusion weighted imaging, and to measure cerebral blood and magnetic spectroscopic resonance imaging of N-acetyl aspartate, choline and the choline to N-acetyl aspartate index to assist in diagnosis and treatment planning in patients with newly diagnosed or suspected glioblastoma. Question What new surgical techniques can be used to provide improved tumor definition and resectability to yield better tumor control and prognosis for individuals with newly diagnosed glioblastoma? Level II: The use of 5-aminolevulinic acid is recommended to improve extent of tumor resection in patients with newly diagnosed glioblastoma. Level II: The use of 5-aminolevulinic acid is recommended to improve median survival and 2 year survival in newly diagnosed glioblastoma patients with clinical characteristics suggesting poor prognosis. Level III: It is suggested that, when available, patients be enrolled in properly designed clinical trials assessing the value of diffusion tensor imaging in improving the safety of patients with newly diagnosed glioblastoma undergoing surgery. Question What new pathology techniques and measurement of biomarkers in tumor tissue can be used to provide improved diagnostic ability, and determination of therapeutic responsiveness and prognosis for patients with newly diagnosed glioblastomas? Level II: Assessment of tumor MGMT promoter methylation status is recommended as a significant predictor of a longer progression free survival and overall survival in patients with newly diagnosed with glioblastoma. Level II: Measurement of tumor expression of neuron-glia-2, neurofilament protein, glutamine synthetase and phosphorylated STAT3 is recommended as a predictor of overall survival in patients with newly diagnosed with glioblastoma. Level III: Assessment of tumor IDH1 mutation status is suggested as a predictor of longer progression free survival and overall survival in patients with newly diagnosed with glioblastoma. Level III: Evaluation of tumor expression of Phosphorylated Mitogen-Activated Protein Kinase protein, EGFR protein, and Insulin-like Growth Factor-Binding Protein-3 is suggested as a predictor of overall survival in patients with newly diagnosed with glioblastoma. Question What radiation therapy techniques are in development that may be used to provide improved tumor control and prognosis for individuals with newly diagnosed glioblastomas? Level III: It is suggested that patients with newly diagnosed glioblastoma undergo pretreatment radio-labeled amino acid tracer positron emission tomography to assess areas at risk for tumor recurrence to assist in radiation treatment planning. Level III: It is suggested that, when available, patients be with newly diagnosed glioblastomas be enrolled in properly designed clinical trials of radiation dose escalation, altered fractionation, or new radiation delivery techniques. Question What emerging chemotherapeutic agents or techniques are available to provide better tumor control and prognosis for patients with newly diagnosed glioblastomas? Level III: As no emerging chemotherapeutic agents or techniques were identified in this review that improved tumor control and prognosis it is suggested that, when available, patients with newly diagnosed glioblastomas be enrolled in properly designed clinical trials of chemotherapy. Question What new targeted therapy agents are available to provide better tumor control and prognosis for individuals with newly diagnosed glioblastomas? Level III: As no new molecular and targeted therapies have clearly provided better tumor control and prognosis it is suggested that, when available, patients with newly diagnosed glioblastomas be enrolled in properly designed clinical trials of molecular and targeted therapies IMMUNOTHERAPY: Question What emerging immunotherapeutic agents or techniques are available to provide better tumor control and prognosis for patients with newly diagnosed glioblastomas? Level III: As no immunotherapeutic agents have clearly provided better tumor control and prognosis it is suggested that, when available, patients with newly diagnosed glioblastomas be enrolled in properly designed clinical trials of immunologically-based therapies. Question What novel therapies or techniques are in development to provide better tumor control and prognosis for individuals with newly diagnosed glioblastomas? Level II: The use of tumor-treating fields is recommended for patients with newly diagnosed glioblastoma who have undergone surgical debulking and completed concurrent chemoradiation without progression of disease at the time of tumor-treating field therapy initiation. Level II: It is suggested that, when available, enrollment in properly designed studies of vector containing herpes simplex thymidine kinase gene and prodrug therapies be considered in patients with newly diagnosed glioblastoma.

Abstract 953: Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic tumor cells induce anti-tumor immunity

Abstract Immune cells can recognize tumor antigen and elicit an adaptive immune response against tumor cells. It has been reported that apoptotic or necrotic death of tumor cells releases tumor antigen stimulating anti-tumor immunity. However, the immune responses against apoptotic tumor cells in vivo are poorly understood. To investigate whether apoptotic tumor cells affect anti-tumor immunity, local immune responses in tumor were evaluated by induction of apoptotic tumor cells with herpes simplex virus thymidine kinase (HSVtk)/ ganciclovir (GCV) system. HSVtk gene transduced tumor cells were resulted in apoptosis in vitro. Administration of GCV triggered apoptosis in HSVtk transfected tumor cells in vivo and regressed the tumor. Comprehensive gene expression analysis of the bulk tumor showed that IFNγ and IL-12 were upregulated. CD8+ T cells were increased in the treatment group compared to the control group. The tumor regression by induction of GCV-triggered apoptosis was inhibited by anti-CD8 depleting antibody. These findings indicate that apoptotic tumor cells induce CD8+ T cell-dependent anti-tumor immunity. Citation Format: Sho Umegaki, Hidekazu Shirota, Chikashi Ishioka. Herpes simplex virus thymidine kinase/ganciclovir-mediated apoptotic tumor cells induce anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 953.

Engineering and Characterization of Oncolytic Vaccinia Virus Expressing Truncated Herpes Simplex Virus Thymidine Kinase.

Oncolytic viruses are a promising class of anti-tumor agents; however, concerns regarding uncontrolled viral replication have led to the development of a replication-controllable oncolytic vaccinia virus (OVV). The engineering involves replacing the native thymidine kinase (VV-tk) gene, in a Wyeth strain vaccinia backbone, with the herpes simplex virus thymidine kinase (HSV-tk) gene, which allows for viral replication control via ganciclovir (GCV, an antiviral/cytotoxic pro-drug). Adding the wild-type HSV-tk gene might disrupt the tumor selectivity of VV-tk deleted OVVs; therefore, only engineered viruses that lacked tk activity were selected as candidates. Ultimately, OTS-412, which is an OVV containing a mutant HSV-tk, was chosen for characterization regarding tumor selectivity, sensitivity to GCV, and the influence of GCV on OTS-412 anti-tumor effects. OTS-412 demonstrated comparable replication and cytotoxicity to VVtk- (control, a VV-tk deleted OVV) in multiple cancer cell lines. In HCT 116 mouse models, OTS-412 replication in tumors was reduced by >50% by GCV (p = 0.004); additionally, combination use of GCV did not compromise the anti-tumor effects of OTS-412. This is the first report of OTS-412, a VV-tk deleted OVV containing a mutant HSV-tk transgene, which demonstrates tumor selectivity and sensitivity to GCV. The HSV-tk/GCV combination provides a safety mechanism for future clinical applications of OTS-412.

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.

Cell-based interferon gene therapy using proliferation-controllable, interferon-releasing mesenchymal stem cells

An important safety concern on cell-based gene therapy is that few methods have been available to control the proliferation and functioning of therapeutic protein-expressing cells after transplantation. We previously reported that the proliferation and functioning of the cells transfected with herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, can be controlled by administration of ganciclovir. In this study, we tried to control the amount of murine interferon-γ (IFN-γ) secreted from transplanted murine mesenchymal stem cell line C3H10T1/2 cells to achieve safe cell-based IFN-γ gene therapy for cancer. C3H10T1/2 cells were transfected with HSVtk- and murine IFN-γ-expressing plasmid vectors to obtain C3H10T1/2/HSVtk/IFN-γ cells. C3H10T1/2/HSVtk/IFN-γ cells released IFN-γ and were sensitive to ganciclovir. C3H10T1/2/HSVtk/IFN-γ cells significantly suppressed the proliferation of murine adenocarcinoma cell line colon26 cells both in vitro and in vivo. Moreover, subcutaneous administration of ganciclovir to mice transplanted with NanoLuc luciferase-expressing C3H10T1/2/HSVtk cells for three consecutive days reduced the luminescence signals from the transplanted cells. These results indicate that the cell regulation system using HSVtk gene and ganciclovir can be useful for safe and efficient cell-based IFN-γ gene therapy for cancer.

Alpha-Herpesvirus Thymidine Kinase Genes Mediate Viral Virulence and Are Potential Therapeutic Targets.

Alpha-herpesvirus thymidine kinase (TK) genes are virulence-related genes and are nonessential for viral replication; they are often preferred target genes for the construction of gene-deleted attenuated vaccines and genetically engineered vectors for inserting and expressing foreign genes. The enzymes encoded by TK genes are key kinases in the nucleoside salvage pathway and have significant substrate diversity, especially the herpes simplex virus 1 (HSV-1) TK enzyme, which phosphorylates four nucleosides and various nucleoside analogues. Hence, the HSV-1 TK gene is exploited for the treatment of viral infections, as a suicide gene in antitumor therapy, and even for the regulation of stem cell transplantation and treatment of parasitic infection. This review introduces the effects of α-herpesvirus TK genes on viral virulence and infection in the host and classifies and summarizes the current main application domains and potential uses of these genes. In particular, mechanisms of action, clinical limitations, and antiviral and antitumor therapy development strategies are discussed.

Differences in the Likelihood of Acyclovir Resistance-Associated Mutations in the Thymidine Kinase Genes of Herpes Simplex Virus 1 and Varicella-Zoster Virus.

Acyclovir (ACV) resistance-associated mutations in two recombinant herpes simplex virus 1 (HSV-1) clones were compared. Recombinant HSV-1 lacking its thymidine kinase (TK) and expressing varicella-zoster virus (VZV) TK ectopically had no mutations in the VZV TK gene. In contrast, recombinant HSV-1 expressing HSV-1 TK ectopically harbored mutations in the HSV-1 TK gene. These results suggest that the relatively low frequency of ACV-resistant VZV is a consequence of the characteristics of the TK gene.

Noninvasive and Local Delivery of Adenoviral-Mediated Herpes Simplex Virus Thymidine Kinase to Treat Glioma Through Focused Ultrasound-Induced Blood-Brain Barrier Opening in Rats.

Adenoviral-mediated gene therapy has been shown great prospects for tumor treatment. However, it is still a great challenge for its application in the glioma. A main cause is the blood-brain barrier (BBB) limits the delivery of adenoviral vectors, greatly compromising their efficacy. Here, we used focused ultrasound (FUS) induced microbubble cavitation to locally and reversibly open BBB for enhancing gene delivery. In this study, Ad-CMV-TK-IRES-EGFP (Ad-HSV-TK-EGFP) carrying the herpes simplex virus thymidine kinase (HSV-TK) and EGFP transgenes was chose as gendicine and Ad-CMV-IRES-EGFP (Ad-EGFP) as the control. The in vitro experiments showed that Ad-HSV-TK-EGFP had a high infection efficiency for C6 glioma cells, producing good tumor cell killing effects when these cells were exposed to more than 10 μg/ml ganciclovir (GCV). Taking advantage of FUS-induced BBB opening, Ad-HSV-TK-EGFP could be effectively delivered into the brain tumors, getting the overexpression of HSV-TK gene in the tumor cells. After exposure to GCV, the significantly stronger anti-tumor efficacy and longer survival time were observed in tumor-bearing mice treated with Ad-HSV-TK-EGFP + FUS than those treated with Ad-EGFP + FUS or only Ad-HSV-TK-EGFP. Histological examination indicated that the reduced expression level of Ki67 proteins and the increased apoptotic tumor cells in tumor xenografts, causing the inhibition of tumor growth. In conclusion, our study provided a new strategy to efficiently and locally deliver recombinant adenoviral vector-mediated HSV-TK gene into the brain to treat glioma.

New Plasmids for Fusarium Transformation Allowing Positive-Negative Selection and Efficient Cre-loxP Mediated Marker Recycling.

In filamentous fungi such as Fusarium graminearum, disruption of multiple genes of interest in the same strain (e.g., to test for redundant gene function) is a difficult task due to the limited availability of reliable selection markers. We have created a series of transformation vectors that allow antibiotic-based selection of transformants and subsequent negative selection for marker removal using thymidine kinase fusions combined with the Cre-loxP system. The fusion genes contain commonly used C-terminal drug resistance markers, either nptII (G418), nat1 (nourseothricin), or hph (hygromycin B). These resistance genes are fused to the sequence encoding Herpes simplex virus thymidine kinase (HSVtk). Despite the presence of the 1 kb HSVtk gene (about ∼30% increase in total marker size), there is only a slight reduction in transformation efficiency on a molar basis. The fusion genes expressed under the Trichoderma pyruvate kinase (PKI) promoter also confer antibiotic resistance in Escherichia coli, allowing straightforward construction of disruption plasmids. For removal of the loxP flanked resistance cassettes, protoplasts of transformants are directly treated with purified Cre recombinase protein. Loss of the HSVtk containing cassette is selected by restoration of resistance to 5-fluoro-2-deoxyuridine (FdU). As a proof of principle, we demonstrated the efficiency of the HSVtk-based marker removal in Fusarium by reversing the disruption phenotype of the gene responsible for production of the red pigment aurofusarin. We first disrupted the FgPKS12 gene via integration of the loxP-flanked HSVtk-nptII cassette into the promoter or the first intron, thereby generating transformants with a white mycelium phenotype. Using Cre recombinase and FdU, the selection marker was subsequently removed, and the resulting transformants regained red pigmentation despite the remaining loxP site. We also found that it is possible to remove several unselected loxP-flanked cassettes with a single Cre protein treatment, as long as one of them contains a negative selectable HSVtk cassette. The negative selection system can also be used to introduce allele swaps into strains without leaving marker sequences, by first disrupting the gene of interest and then complementing the deletion in situ with genomic DNA containing a different allele.

Reversibly immortalized hepatic progenitor cell line containing double suicide genes.

A large number of functional hepatocytes is required for bioartificial liver therapy. Simian virus 40 T-antigen (SV40T) has been previously reported to improve the immortalized proliferation of primary hepatocytes to generate a sufficient number of cells; however, these long-term immortalized hepatocytes may induce further malignant transformation in vivo. In the present study, the SV40T immortalization gene and two suicide genes, herpes simplex virus thymidine kinase (HSV-tk) and cytosine deaminase (CD), were transducted into primary hepatocytes to construct a novel type of Cre/LoxP-mediated reversible immortalized hepatocyte line. Polymerase chain reaction analysis and western blotting confirmed that the SV40T, HSV-tk and CD genes were successfully inserted into hepatic progenitor cells and their expression was controlled by Cre/LoxP recombination. Total removal of SV40T could be achieved via the ganciclovir (GCV)/HSV-tk suicide system. Cells maintained their biosafety in vivo with CD gene expression and 5-fluoro-cytosine (5-FC) induced cell death. Following transplantation into the carbon tetrachloride (CCl4) model group, the majority of cells had survived after 14 days post-implantation and a number of the cells had transported into the liver parenchyma. When compared with the CCl4 model group, the transplanted cells repaired the liver biochemical index and pathological structure markedly. Thus, the present study reports a novel reversible immortalized hepatocyte with double suicide genes, which exhibited the cellular phenotype and recovery function of normal liver cells. This method maximally guaranteed the biological safety of immortalized hepatocytes for in vivo application, providing a reliable, safe and ideal cell material for the artificial liver technique.

Regulation of proliferation and functioning of transplanted cells by using herpes simplex virus thymidine kinase gene in mice

Though cell transplantation is becoming an attractive therapeutic method, uncontrolled cell proliferation or overexpression of cellular functions could cause adverse effects. These unfavorable outcomes could be avoided by regulating the proliferation or functioning of transplanted cells. In this study, we used a combination of the herpes simplex virus thymidine kinase (HSVtk) gene, a suicide gene, and ganciclovir (GCV) to control the proliferation and functioning of insulin-secreting cells after transplantation in diabetic mice. Mouse pancreatic β cell line MIN6 cells were selected as insulin-secreting cells for transfection with the HSVtk gene to obtain MIN6/HSVtk cells. Proliferation of MIN6/HSVtk cells was suppressed by GCV in a concentration-dependent manner; 0.25 μg/mL GCV maintained a constant number of MIN6/HSVtk cells for at least 16 days. MIN6 or MIN6/HSVtk cells were then transplanted to streptozotocin-induced diabetic mice. Mice transplanted with MIN6 cells exhibited hypoglycemia irrespective of GCV administration. In contrast, normal (around 150 mg/dL) blood glucose levels were maintained in mice transplanted with MIN6/HSVtk cells by a daily administration of 50 mg/kg of GCV. These results indicate that controlling the proliferation and functioning of HSVtk gene-expressing cells by GCV could greatly improve the usefulness and safety of cell-based therapy.

Orthotopic hepatic cancer: radiofrequency hyperthermia-enhanced intratumoral herpes simplex virus-thymidine kinase gene therapy.

PurposeTo validate the feasibility of using interventional radiofrequency hyperthermia(RFH) to enhance herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy of rat orthotopic hepatic cancer.Material and MethodsRat hepatocellular carcinoma cells (MCA-RH-7777) were transduced with lentivirus/luciferase gene for optical imaging. In-vitro experiments with the luciferase cells and in-vivo experiments on rats with orthotopic hepatic tumors were divided into four treatment groups: (i) HSV-TK/GCV-mediated gene therapy combined with RFH; (ii) gene therapy alone; (iii) RFH alone; and (iv) phosphate buffered saline (PBS). Cell viability was evaluated by MTS assay and confocal microscopy, and HSV-TK gene expression in cells and tumors was quantified by western blotting. Bioluminescent optical imaging and ultrasound imaging were used to monitor and compare the photon signal and tumor size changes among different treatment groups overtime, respectively. The imaging findings were correlated with histology.ResultsFor in-vitro experiments, the combination therapy group (gene therapy + RFH) demonstrated the lowest cell proliferation by MTS assay, compared to the gene therapy alone, RFH alone, and PBS (26.1±3.2% vs 50.4±4.6% vs 82.9±6.3% vs 100%, p<0.01). The combination therapy group also showed fewer survived cells by the confocal microscopy and the lowest bioluminescent signal by the optical imaging. For in-vivo experiments, the combination therapy group demonstrated a significantly decreased signal intensity on the bioluminescent optical imaging (0.57±0.09, 1.06±0.10 vs 3.43±0.27 vs 3.85±0.12, p<0.05) and smallest tumor volume by ultrasound imaging (0.28±0.11 vs 1.28±0.23vs 4.64±0.35 vs 6.37±0.36, p<0.05), compared to the other three groups. Additionally, these imaging findings correlated well with the histological confirmation.ConclusionIt is feasible to use RFH to enhance HSV-TK/GCV gene therapy of hepatic tumors in in-vitro and in-vivo settings, as assessed by molecular imaging. This technical development may provide a novel opportunity for effective treatment of liver malignancies by employing simultaneous integration of radiofrequency technology, interventional oncology, and direct intratumoral gene therapy.

Immunotherapy and gene therapy as novel treatments for cancer.

The immune system interacts closely with tumors during the disease development and progression to metastasis. The complex communication between the immune system and the tumor cells can prevent or promote tumor growth. New therapeutic approaches harnessing protective immunological mechanisms have recently shown very promising results. This is performed by blocking inhibitory signals or by activating immunological effector cells directly. Immune checkpoint blockade with monoclonal antibodies directed against the inhibitory immune receptors CTLA-4 and PD-1 has emerged as a successful treatment approach for patients with advanced melanoma. Ipilimumab is an anti-CTLA-4 antibody which demonstrated good results when administered to patients with melanoma. Gene therapy has also shown promising results in clinical trials. Particularly, Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (TK) gene to tumor cells in combination with ganciclovir (GCV) may provide an effective suicide gene therapy for destruction of glioblastomas, prostate tumors and other neoplasias by recruiting tumor-infiltrating lymphocytes into the tumor. The development of new treatment strategies or combination of available innovative therapies to improve cell cytotoxic T lymphocytes trafficking into the tumor mass and the production of inhibitory molecules blocking tumor tissue immune-tolerance are crucial to improve the efficacy of cancer therapy.

Genetically Engineered Multilineage-Differentiating Stress-Enduring Cells as Cellular Vehicles against Malignant Gliomas

Malignant glioma, the most common malignant brain tumor in adults, is difficult to treat due to its aggressive invasive nature. Enzyme/prodrug suicide gene therapy based on the herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system is an efficient strategy for treating malignant gliomas. In the present study, we evaluated treatment with multilineage-differentiating stress-enduring (Muse) cells, which are endogenous non-tumorigenic pluripotent-like stem cells that are easily collectable from the bone marrow as SSEA-3+ cells, as carriers of the HSVtk gene. Human Muse cells showed potent migratory activity toward glioma cells both in vitro and in vivo. HSVtk gene-transduced Muse cells (Muse-tk cells) at a cell number of only 1/32 that of U87 human glioma cells completely eradicated U87 gliomas in nude mouse brains, showing a robust in vivo bystander effect. Pre-existing intracranial U87 gliomas in nude mouse brains injected intratumorally with Muse-tk cells followed by intraperitoneal GCV administration were significantly reduced in size within 2 weeks, and 4 of 10 treated mice survived over 200 days. These findings suggest that intratumoral Muse-tk cell injection followed by systemic GCV administration is safe and effective and that allogeneic Muse-tk cell-medicated suicide gene therapy for malignant glioma is clinically feasible.

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice.

Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHVXP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals (P < 0.01 or P < 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P < 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P < 0.01 or P < 0.05). Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4+ T cells and CD8+ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.

A Modified Adenovirus Vector–Mediated Antibody Screening Method Identifies EphA2 as a Cancer Target

BACKGROUND: We constructed a genetically modified adenovirus vector incorporating an IgG Fc-binding motif from staphylococcal protein A, Z33 (Adv-FZ33). Adv-FZ33 allows an antibody to redirect the vector to a target molecule on the cell surface. We attempted to search for target antigen candidates and antibodies that allowed highly selective gene transduction into malignant tumors. METHODS: Hybridoma libraries producing monoclonal antibodies (mAbs) were screened that increased transduction efficiency in cancer cell lines after cross-linking with Adv-FZ33. Target antigens of the mAbs were identified by immunoprecipitation and mass spectrometry. Of these mAbs, we noted a clone, F2-27, that recognized the receptor tyrosine kinase EphA2. Next, we generated an adenovirus vector, Ax3CMTK-FZ33, that expressed a herpes simplex virus thymidine kinase (HSV-TK). The therapeutic efficacy of F2-27–mediated HSV-TK gene transduction, followed by ganciclovir (GCV) administration, was studied in vitro. The inhibitory effect of F2-27 on cancer cell invasion was investigated by a three-dimensional spheroid formation assay. RESULTS: In vitro reporter gene expression after Adv-FZ33 infection via F2-27 was 146 times higher than with control mAb in EphA2-expressing cancer cell lines. F2-27–mediated Ax3CMTK-FZ33 infection induced the HSV-TK gene in an F2-27–dependent manner and had a highly effective cytotoxic effect in a GCV-dependent manner. Additionally, F2-27 independently inhibited migration of EphA2-positive breast cancer cell lines in three-dimensional culture. CONCLUSION: Our modified adenovirus and hybridoma screening system is useful for the development of targeted cancer therapy, and F2-27 has the potential to be an antibody-based therapy for various EphA2-positive cancers.