Herpes Simplex Virus Type I Thymidine Kinase Research Articles

Nonviral polymeric nanoparticles for gene therapy in pediatric CNS malignancies

Together, medulloblastoma (MB) and atypical teratoid/rhabdoid tumors (AT/RT) represent two of the most prevalent pediatric brain malignancies. Current treatment involves radiation, which has high risks of developmental sequelae for patients under the age of three. New safer and more effective treatment modalities are needed. Cancer gene therapy is a promising alternative, but there are challenges with using viruses in pediatric patients. We developed a library of poly(beta-amino ester) (PBAE) nanoparticles and evaluated their efficacy for plasmid delivery of a suicide gene therapy to pediatric brain cancer models—specifically herpes simplex virus type I thymidine kinase (HSVtk), which results in controlled apoptosis of transfected cells. In vivo, PBAE-HSVtk treated groups had a greater median overall survival in mice implanted with AT/RT (P = 0.0083 vs. control) and MB (P < 0.0001 vs. control). Our data provide proof of principle for using biodegradable PBAE nanoparticles as a safe and effective nanomedicine for treating pediatric CNS malignancies.

Polymeric nanoparticles for nonviral gene therapy extend brain tumor survival in vivo.

Biodegradable polymeric nanoparticles have the potential to be safer alternatives to viruses for gene delivery; however, their use has been limited by poor efficacy in vivo. In this work, we synthesize and characterize polymeric gene delivery nanoparticles and evaluate their efficacy for DNA delivery of herpes simplex virus type I thymidine kinase (HSVtk) combined with the prodrug ganciclovir (GCV) in a malignant glioma model. We investigated polymer structure for gene delivery in two rat glioma cell lines, 9L and F98, to discover nanoparticle formulations more effective than the leading commercial reagent Lipofectamine 2000. The lead polymer structure, poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-modified with 1-(3-aminopropyl)-4-methylpiperazine, is a poly(β-amino ester) (PBAE) and formed nanoparticles with HSVtk DNA that were 138 ± 4 nm in size and 13 ± 1 mV in zeta potential. These nanoparticles containing HSVtk DNA showed 100% cancer cell killing in vitro in the two glioma cell lines when combined with GCV exposure, while control nanoparticles encoding GFP maintained robust cell viability. For in vivo evaluation, tumor-bearing rats were treated with PBAE/HSVtk infusion via convection-enhanced delivery (CED) in combination with systemic administration of GCV. These treated animals showed a significant benefit in survival (p = 0.0012 vs control). Moreover, following a single CED infusion, labeled PBAE nanoparticles spread completely throughout the tumor. This study highlights a nanomedicine approach that is highly promising for the treatment of malignant glioma.

Valproic acid enhances anti-tumor effect of mesenchymal stem cell mediated HSV-TK gene therapy in intracranial glioma

Suicide gene therapy of glioma based on herpes simplex virus type I thymidine kinase (HSV-TK) and prodrug ganciclovir (GCV) suffers from the lack of efficacy in clinical trials, which is mostly due to low transduction efficacy and absence of bystander effect in tumor cells. Recently, stem cells as cellular delivery vehicles of prodrug converting gene has emerged as a new treatment strategy for malignant glioma. In this study, we evaluated the anti-glioma effect of suicide gene therapy using human bone marrow mesenchymal stem cells expressing HSV-TK (MSCs-TK) combined with valproic acid (VPA), which can upregulate the gap junction proteins and may enhance the bystander effect of suicide gene therapy. Expression of HSV-TK in MSCs was confirmed by RT-PCR analysis and the sensitivity of MSCs-TK to GCV was assessed. A bystander effect was observed in co-cultures of MSCs-TK and U87 glioma cells by GCV in a dose-dependent manner. VPA induced the expression of the gap junction proteins connexin (Cx) 43 and 26 in glioma cell and thereby enhanced the bystander effect in co-culture experiment. The enhanced bystander effect was inhibited by the gap junction inhibitor 18-β-glycyrrhetinic acid (18-GA). Moreover, the combined treatment with VPA and MSCs-TK synergistically enhanced apoptosis in glioma cells by caspase activation. In vivo efficacy experiments showed that combination treatment of MSCs-TK and VPA significantly inhibited tumor growth and prolonged the survival of glioma-bearing mice compared with single-treatment groups. In addition, TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in the combination treated group compared with single-treatment groups. Taken together, these results provide the rational for designing novel experimental protocols to increase bystander killing effect against intracranial gliomas using MSCs-TK and VPA.

Characterization of HIV-1 TAT peptide as an enhancer of HSV-TK/GCV cancer gene therapy

Cancer suicide gene therapy based on herpes simplex virus type I thymidine kinase (HSV-TK) and ganciclovir (GCV) suffers from the lack of efficacy in clinical use, which is mostly due to low gene-transfer efficiency and absence of bystander effect in tumors. We have previously demonstrated the enhancement of GCV cytotoxicity by fusing the HSV-TK with the cell penetrating peptide from HIV-1 transactivator protein transduction domain (TAT PTD). Despite the earlier promising results, we found that the triple fusion protein HIV-1 transactivator protein transduction domain-thymidine kinase suicide gene-green fluorescent protein marker gene (TAT-TK-GFP) increased GCV cytotoxicity only in 3/12 of different human tumor cell lines. Extended GCV exposure enhanced the cytotoxic effect of HSV-TK/GCV gene therapy, but the difference between TK-GFP and TAT-TK-GFP was not statistically significant. The modest improvement on cell killing mediated by TAT PTD in Chinese hamster ovary cells appeared to be associated with cell-surface heparan sulfate proteoglycan (HSPG) composition. However, TAT-mediated increased cell death did not correlate with the density of cell-surface HSPG expression in different tumor cell lines. In conclusion, although some degree of enhancement by TAT was shown in certain tumor cells in vitro, it is unlikely that TAT peptide linked to a suicide protein could be a useful booster of in vivo gene therapy trials.

807. Addition of HU to GCV Induces Bystander Cytotoxicity in Co-Cultures of HSVTK-Expressing and Non-Expressing HeLa Cells

Expression of the herpes simplex virus type I thymidine kinase (HSVTK) gene in tumor cells sensitizes them to the antiviral agent ganciclovir (GCV). Incubation with GCV results not only in cytotoxicity from cells that express the transgene, but neighboring bystander cells as well. Bystander cytotoxicity has been attributed to the transfer of phosphorylated GCV metabolites from HSVTK-expressing to non-expressing cells through protein channels known as gap junctions. We have previously reported good bystander cytotoxicity in the U251 glioblastoma and SW620 colon carcinoma cell lines, which have 84% and 39% gap junction intercellular communication (GJIC), respectively. However, in the HeLa cell line (23% GJIC), no bystander cytotoxicity resulted from incubating a co-culture of HSVTK-expressing and bystander HeLa cells with GCV. Further analysis revealed that lack of bystander cytotoxicity in these cells was not due to an inability to transfer phosphorylated GCV, but resulted from a low level of transfer along with a rapid half-life of GCVTP in bystander cells (3.1 hrs) when compared to the HSVTK-expressing cells (12.3 hrs). We hypothesized that reduction of the endogenous competitor for GCVTP incorporation into DNA, dGTP, would increase levels of GCV incorporation into DNA resulting in the induction of bystander cytotoxicity. To that end, the effect of hydroxyurea (HU), a ribonucleotide reductase inhibitor, on GCV cytotoxicity in cultures of 100% HSVTK-expressing HeLa cells and co-cultures of HSVTK-expressing and bystander HeLa cells was examined. Isobologram analysis of the interaction between GCV and HU demonstrated an additive-antagonistic effect on 100% cultures of HSVTK-expressing HeLa cells, while exhibiting a synergistic effect on the HSVTK-expressing and bystander cells in a co-culture. To determine the mechanism for the synergistic cytotoxicity demonstrated in each of the populations from the co-culture when incubated with GCV and HU, GCVTP and dNTP pools were quantified and the results were compared to 100% cultures of HSVTK-expressing HeLa cells. The results demonstrated a significant increase in the GCVTP:dGTP values in cultures of 100% HSVTK-expressing cells (>2.5 fold) and in co-cultures of HSVTK-expressing cells (>1.4 fold) and bystander cells (>1.8 fold) at 4 hours. However, at 12 hours the GCVTP:dGTP value for 100% cultures of HSVTK-expressing HeLa cells was at control levels (1.3 0.5 fold) while the values for HSVTK-expressing cells (>2.7 fold) and bystander cells (>4.9 fold) in co-culture were still significantly higher. These data suggest that the prolonged increase in the GCVTP:dGTP value in co-cultures incubated with GCV and HU leads to an increase in the levels of GCVTP incorporated into DNA resulting in synergistic cytotoxicity.

Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas

Gene therapy of glioma based on viral delivery of herpes simplex virus type I thymidine kinase (HSV-TK) has failed in the clinic because of low transduction efficacy. To circumvent this problem, this study evaluated highly migratory HSV-TK-transduced neural stem cells (NSC) for their ability to kill untransduced glioma cells by a gap junction-mediated bystander effect. The admixture of HSV-TK-transduced NSC to U87MG and LN-18 human malignant glioma cell lines at ratios of 1:10 or 1:1 eliminated more than 50% or 90% of glioma cells in the presence of ganciclovir (25 μM). Glioma cell cytotoxicity required cell–cell contact. Similarly, tumor cell cytotoxicity was observed in two of three primary glioblastoma cell cultures, and the presence of this bystander effect correlated with the expression of connexin 43 in the untransduced glioma target cells. In conclusion, we delineate a role for migratory HSV-transfected NSC to eliminate glioma cells purely by means of the bystander effect.

Expression of mutant non-cleavable Fas ligand on retrovirus packaging cells causes apoptosis of immunocompetent cells and improves prodrug activation gene therapy in a malignant glioma model

Recombinant retroviruses (RV) have been widely used as vectors for clinical gene therapy of malignant brain tumors. Because of the very limited stability of these vectors in vivo, RV producing cells (VPC) are routinely used for intratumoral RV release. The host immune system, however, recognizes the intratumorally grafted allogeneic or xenogeneic VPC, and mounts an immune response against them. Humoral and cellular immune responses eventually result in reduction of VPC numbers and in limited success of RV mediated gene therapy approaches. This study presents a non-pharmacological and spatially limited approach for protection of VPC grafted in the CNS against destruction by host immune responses. Murine fibroblast-derived VPC expressing herpes-simplex-virus type I thymidine kinase (HSV-tk) were genetically modified to co-express a human Fas ligand (CD95L) deletion mutant (ΔFasL) resistant to enzymatic cleavage and shedding. Direct interactions between Fas (CD95) on lymphocytes and ΔFasL on VPC upon cell-cell contact rapidly caused apoptosis in lymphocytes. In addition, cultured malignant brain tumor cells (U87, LN18, LN229) transduced with ΔFasL-RV were rendered apoptotic by Fas/ΔFasL interaction. ΔFasL-expressing VPC grafted in a 9L rat brain tumor model survived in significantly higher numbers compared with control VPC, and did not cause an increase in neutrophil infiltration of tumors. Gene therapy of tumor bearing animals grafted with the modified ΔFasL-VPC and given the prodrug Ganciclovir resulted in significantly increased survival rates compared to treatment with control VPC and Ganciclovir. In conclusion, prolonged intratumoral presence of ΔFasL-VPC seems to be a direct consequence of the expression of the membrane-bound mutant FasL, and may result in increased total RV output and improved tumor transduction with RV.

Ectopic expression of herpes simplex virus-thymidine kinase gene in human non-small cell lung cancer cells conferred caspase-activated apoptosis sensitized by ganciclovir.

Human non-small cell lung cancer (NSCLC) cells were transfected with recombinant prodrug herpes simplex virus type I thymidine kinase (HSV-tk) cDNA, and the selected clones underwent apoptosis in response to induction by antiviral ganciclovir (GCV). The efficiency of GCV-induced growth inhibition and the extent of the bystander effect were associated with the expression level of HSV-TK in stable transfectants. Development in the HSV-tk/GCV system toward cell death was initiated with cell-cycle accumulation at S and G(2)/M phases, immediately followed by the appearance of sub-G(0)/G(1) cells after drug exposure. To investigate the regulation of cell-cycle modulators during drug treatment, we analyzed release of the apoptosis initiator cytochrome c and activation of the downstream effectors caspase-9, caspase-3 and poly(ADP-ribose)polymerase 16 hr after GCV sensitization, followed by transient escalation of tumor-suppressor p53 and cell-cycle modulators cyclin A and B(1) before committing to programmed cell death. Furthermore, tumor regression was proportional to the degree of ectopic expression of the transferred HSV-tk gene. Our results demonstrate that the HSV-tk/GCV system effectively inhibits the proliferation of NSCLC cells in vitro and in vivo through potent induction of apoptosis, thus providing a rationale for further development.

Experimental study on pancreatic cancer gene therapy using the HSV-TK gene mediated by a retroviral vector

OBJECTIVE: To study the value of the herpes simplex virus type I thymidine kinase (HSV-TK) gene mediated by a retroviral vector in the treatment of human pancreatic cancer cell line 8988. METHODS: The HSV-TK gene was directionally cloned into a site following the SV40 promoter that was adjacent to the 5′ LTR (long terminal repeats) and the neomycin phosphotransferase gene in the retroviral vector pMNSM. This enabled the TK gene to integrate into the chromatin of the host cell. The recombinant plasmid pMNS-TK-M was then transfected into the retrovirus-packaging cell Pa317. Finally, the HSV-TK gene was transfected into pancreatic cancer cell line 8988 by the recombinant retrovirus after selection with G418. RESULTS: The HSV-TK gene was stably integrated into the host cell and its expression confirmed by Southern blotting and drug-sensitivity tests. In vitro studies showed that the acyclovir (ACV) sensitivity level in the 8988/TK+ cells was higher than that of the parent cells. The cells that were transfected with the TK gene were significantly susceptible to ACV. In vivo studies in nude mice showed that intraperitoneal injection of ACV might postpone the formation of implanted tumors and produce a treatment effect on the tumors. CONCLUSIONS: This study demonstrated that the HSV-TK/ACV retroviral system could be used in vivo to treat pancreatic cancer.

Combined suicide and cytokine gene therapy for peritoneal carcinomatosis

BACKGROUNDGene therapy is a novel approach for the treatment of cancers, and tumours disseminated in the peritoneal cavity are suitable for in situ delivery of a therapeutic gene.AIMSThe efficacy of...

Cell-specific targeting of a thymidine kinase/ganciclovir gene therapy system using a recombinant Sindbis virus vector.

Transfer of the herpes simplex virus type I thymidine kinase (HSV-TK) gene into tumor cells using virus-based vectors in conjunction with ganciclovir (GCV) exposure provides a potential gene therapy strategy for the treatment of cancer. The possibility of using a novel targetable Sindbis virus expression vector containing the HSV-TK gene was examined. Baby hamster kidney (BHK) cells and several human tumor cells infected with a Sindbis virus containing the HSV-TK gene showed strong expression of HSV-TK protein. Cells transduced with the HSV-TK gene exhibited increased TK activity, ranging from 3- to 20-fold over an average baseline level. The human HeLa-CD4+ cells infected with recombinant Sindbis virus containing the HSV-TK gene were sensitive to low concentrations of GCV (0.1-1 microg/ml) and the 50% growth inhibitory concentration (IC50) was 0.6 microg/ml. We also demonstrated applications of cell type-specific Sindbis virus-mediated antigen-antibody targeting of the HSV-TK/GCV system in vitro. Sindbis virus containing the HSV-TK gene packaged in a helper virus displaying the IgG-binding domain of protein A on its envelope could infect various tumor cell lines in the presence of specific antibodies that recognize antigens on their surfaces. HSV-TK-transduced tumor cell lines exhibited sensitivity to GCV. Our data suggest the potential for targeted gene therapy of the HSV-TK/GCV system using a cell type-specific recombinant Sindbis virus vector-antibody system.

Gene transfer of herpes simplex virus type I thymidine kinase gene as a drug sensitivity gene into human lung cancer cell lines using retroviral vectors.

One of the recent strategies for gene therapy as a cancer control is the targeted introduction of a drug-sensitivity gene into tumor cells. We investigated the gene transfer of herpes simplex virus type I thymidine kinase (HSV-TK) gene as a drug-sensitivity gene into human lung cancer cell lines. We used a recombinant retroviral vector derived from Moloney murine leukemia virus (MuLV) as one of potential vectors for gene therapy. The amphotropic retroviral vector consisted of the HSV-TK gene and the neomycin-resistant gene under Rous sarcoma virus (RSV) promoter control. The antiherpes drugs, acyclovir (ACV) and ganciclovir (GCV), were chosen for testing the activity of HSV-TK that was transferred into human lung cancer cell lines. ACV and GCV are nucleoside analogs specifically converted by HSV-TK to a toxic form capable of inhibiting DNA synthesis. The cytotoxicity was determined by using a tetrazolium-based colorimetric assay (MTT assay). The results obtained from our experiments demonstrated that the retroviral vector-mediated HSV-TK gene transfer leads to ACV- and GCV-dependent cytotoxicity in human lung cancer cell lines, which were both small cell carcinoma and non-small cell carcinoma established from human specimens. These findings suggest that the gene transfer of HSV-TK gene into tumor cells would be one of the models for the use of gene therapy to control lung cancer.