Herpes Simplex Virus Thymidine Research Articles

Editorial Expression of Concern: Herpes simplex virus thymidine kinase/ganciclovir-induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells.

Editorial Expression of Concern: Herpes simplex virus thymidine kinase/ganciclovir-induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells.

Fluoxetine enhances the antitumor effect of olfactory ensheathing cell-thymidine kinase/ganciclovir gene therapy in human glioblastoma multiforme cells through upregulation of Connexin43 levels.

Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma, resulting in poor clinical outcomes. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy is considered a promising strategy for GBM treatment. Since Connexin43 (Cx43) expression is reduced in GBM cells, increasing Cx43 levels could enhance the effectiveness of gene therapy. The present study aims to examine the impact of fluoxetine on HSV-TK/GCV gene therapy in human GBM cells using human olfactory ensheathing cells (OECs) as vectors. The effect of fluoxetine on Cx43 levels was assessed using the western blot technique. GBM-derived astrocytes and OECs-TK were Cocultured, and the effect of fluoxetine on the Antitumor effect of OEC-TK/GCV gene therapy was evaluated using MTT assay and flow cytometry. Our results showed that fluoxetine increased Cx43 levels in OECs and GBM cells and augmented the killing effect of OECs-TK on GBM cells. Western blot data revealed that fluoxetine enhanced the Bax/Bcl2 ratio and the levels of cleaved caspase-3 in the coculture of OECs-TK and GBM cells. Moreover, flow cytometry data indicated that fluoxetine increased the percentage of apoptotic cells in the coculture system. This study suggests that fluoxetine, by upregulating Cx43 levels, could strengthen the Antitumor effect of OEC-TK/GCV gene therapy on GBM cells.

Distinct role of CD8 cells and CD4 cells in antitumor immunity triggered by cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir system.

Immune cells can recognize tumor-associated antigens released from dead tumor cells, which elicit immune responses, potentially resulting in tumor regression. Tumor cell death induced by chemotherapy has also been reported to activate immunity. However, various studies have reported drug-induced immunosuppression or suppression of inflammation by apoptotic cells. Thus, this study aimed to investigate whether apoptotic tumor cells trigger antitumor immunity independent of anticancer treatment. Local immune responses were evaluated after direct induction of tumor cell apoptosis using a Herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system. The inflammatory response was significantly altered at the tumor site after apoptosis induction. The expression of cytokines and molecules that activate and suppress inflammation simultaneously increased. The HSV-tk/GCV-induced tumor cell apoptosis resulted in tumor growth suppression and promoted T lymphocyte infiltration into tumors. Therefore, the role of T cells after inducing tumor cell death was explored. CD8 T cell depletion abrogated the antitumor efficacy of apoptosis induction, indicating that tumor regression was mainly dependent on CD8 T cells. Furthermore, CD4 T cell depletion inhibited tumor growth, suggesting the potential role of CD4 T cells in suppressive tumor immunity. Tumor tissues were evaluated after tumor cell apoptosis and CD4 T cell depletion to elucidate this immunological mechanism. Foxp3 and CTLA4, regulatory T-cell markers, decreased. Furthermore, arginase 1, an immune-suppressive mediator induced by myeloid cells, was significantly downregulated. These findings indicate that tumors accelerate CD8 T cell-dependent antitumor immunity and CD4 T cell-mediated suppressive immunity. These findings could be a therapeutic target for immunotherapy in combination with cytotoxic chemotherapy.

Engineering of mesenchymal stem cells to express cytosine deaminase gene with a selecable marker gene.

e14050 Background: Suicide genes induce apoptosis not only in the cell itself but also in surrounding cells. The former is called the suicide effect and the latter is called the bystander effect. Two major suicide gene strategies currently pursued, cytosine deaminase (CD)/5-fluorocytosine (5-FC) and the herpes simplex virus-thymidine kinase/ganciclovir. Both genes have similar potential to induce suicide effects, but bystander effect is higher in CD/5-FC. Here we show that the level of CD expression/activity influence the anti-cancer activity of CD-expressing cells and further demonstrate the need to use selectable markers during manufacturing of CD-expressing cells. Finally we test whether live microbes harboring the shed selectable marker genes are controllable by commonly used antibiotics. Methods: We introduced a microbial CD gene and puromycin-N-acetyltransferase ( pac) gene to bone-marrow derived mesenchymal stem cells MSCs) using a retroviral vector and selected the transduced cells with 4 ug/mL puromycin for one week. To determine the effect of purity, we varied the ratio of CD-expressing MSCs with naïve MSCs and 5-FC concentration in the culture and on co-cultured glioma cells. To mimic the situation in which the shed pac gene is transmitted to live microbes in vivo, we introduced the pac gene to E. coli and tested the growth of the pac-expressing E. coli using VITEK following the standard protocol M100-S32 in the presence of diverse antibiotics. Results: We found that selection of CD-expressing cells with puromycin during manufacturing process enhanced the bystander (anti-cancer effect). Lowering the purity of CD-expressing MSCs required more 5-FC with higher IC50. Growth of pac-expressing E. coli was completely inhibited by commonly used antibiotics in the hospital. Conclusions: We proved that antibiotic selection is necessary during manufacturing process. We also showed that the risk of transmission of shed pac gene can be manageable with commonly used antibiotics. We propose that using pac selectable marker gene can be beneficial to maintain the purity of engineered cells.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

Putting PLX5622 into perspective: microglia in central nervous system viral infection.

β2-Adrenergic receptor agonist enhances the bystander effect of HSV-TK/GCV gene therapy in glioblastoma multiforme via upregulation of connexin 43 expression

Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma. Gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) is a new strategy for GBM treatment. As the connexin 43 (Cx43) levels are downregulated in GBM cells, it seems that the upregulation of Cx43 could improve the efficacy of the gene therapy. This study aims to evaluate the effect of clenbuterol hydrochloride (Cln) as a β2-adrenergic receptor agonist on HSV-TK/GCV gene therapy efficacy in human GBM cells using olfactory ensheathing cells (OECs) as vectors. The lentivirus containing the thymidine kinase gene was transduced to OECs and the effective dose of GCV on cells was measured by MTT assay. We found that Cln upregulated Cx43 expression in human GBM cells and OECs and promoted the cytotoxic effect of GCV on the co-culture cells. Western blot results showed that Cln increased the cleaved caspase-3 expression and the Bax/Bcl2 ratio in the co-culture of GBM cells and OEC-TK. Also, the flow cytometry results revealed that Cln increased apoptosis in the co-culture of GBM cells and OEC-TK cells. This study showed that Cln via upregulation of Cx43 expression could enhance the bystander effect of HSVTK-GCV gene therapy in human GBM cells.

"PFH/AGM-CBA/HSV-TK/LIPOSOME-Affibody": Novel Targeted Nano Ultrasound Contrast Agents for Ultrasound Imaging and Inhibited the Growth of ErbB2-Overexpressing Gastric Cancer Cells.

ObjectiveGastric cancer is one of the most lethal malignancies in the world. However, the current research on the diagnosis and treatment of nano-ultrasound contrast agents in the field of tumor is mostly focused on breast cancer, ovarian cancer, prostate cancer, liver cancer, etc. Due to the interference of gas in the stomach, there is no report on the treatment of gastric cancer. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) therapy system is the most mature tumor suicide gene in cancer treatment. At the same time, in order to improve its safety and efficiency, we designed a gastric tumor targeted ultrasound-triggered phase-transition nano ultrasound contrast agent PFH/AGM-CBA/HSV-TK/Liposome (PAHL)-Affibody complex.MethodsIn our study, guanidinylated SS-PAAs polymer poly(agmatine/N, N′-cystamine-bis-acrylamide) (AGM-CBA) was used as a nuclear localization vector of suicide gene to form a polyplex, perfluorohexane (PFH) was used as ultrasound contrast agent, liposomes were used to encapsulate perfluorohexane droplets and the polyplexes of AGM-CBA/HSV-TK, and affibody molecules were conjugated to the prepared PAHL in order to obtain a specific targeting affinity to human epidermal growth factor receptor type 2 (ErbB2) at gastric cancer cells. With the aid of ultrasound targeted microbubble destruction technology and the nuclear localization effect of AGM-CBA vector, the transfection efficiency of the suicide gene in gastric cancer cells was significantly increased, leading to significant apoptosis of gastric cancer cells.ResultsIt was shown that PAHL-Affibody complex was nearly spherical with an average diameter of 560 ± 28.9 nm, having higher and specific affinity to ErbB2 (+) gastric cells. In vitro experiments further confirmed that PAHL could target gastric cancer cells expressing ErbB2. In a contrast-enhanced ultrasound scanning study, the prepared ultrasound-triggered phase-change nano-ultrasound contrast agent, PAHL, showed improved ultrasound enhancement effects. With the application of the low-frequency ultrasound, the gene transfection efficiency of PAHL was significantly improved, thereby inducing significant apoptosis in gastric cancer cells.ConclusionThis study constructs PFH/AGM-CBA/HSV-TK/Liposome-Affibody nano ultrasound contrast agent, which provides new ideas for the treatment strategy of ErbB2-positive gastric cancer and provides some preliminary experimental basis for its inhibitory effect.

Engineering silica-polymer hybrid nanosystems for dual drug and gene delivery.

In recent years, it has been shown that a combination of different antitumour strategies involving distinct therapeutic agents, such as chemical compounds and genetic material, could result in an effective therapeutic activity that is much higher than that obtained by conventionally used individual approaches. Therefore, the main goal of this work was to develop a new hybrid nanosystem based on mesoporous silica nanoparticles and polymers to efficiently transport and deliver drug and plasmid DNA into cancer cells. Moreover, its potential to mediate a combinatorial antitumour strategy involving epirubicin and herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy was evaluated. For this purpose, various cationic polymers were assessed, including poly(β-amino ester) homopolymer, gelatine type A, gelatine type B, and poly(ethylene glycol)-b-poly(2-aminoethyl methacrylate hydrochloride) block copolymer. The obtained results show that using different polymers leads to nanosystems with different physicochemical properties and, consequently, different biological activities. The best formulation was obtained for hybrid nanosystems coated with PEG-b-PAMA. They demonstrated the ability to cotransport and codeliver an anticancer drug and plasmid DNA and effectively mediate the combined antitumour strategy in 2D and 3D tumour cell culture models. In summary, we developed a novel silica- and polymer-based nanosystem able to mediate a dual chemotherapeutic and suicide gene therapy strategy with a much higher therapeutic effect than that obtained through the use of individual approaches, showing its potential for cancer treatment.

Therapeutic potential of the cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine suicide system for canine melanoma.

We tested the efficacy of a yeast cytosine deaminase::uracil phosphoribosyl transferase/5-fluorocytosine (CDU/5-FC) non-viral suicide system on eight established canine melanoma cell lines. Albeit with different degree of sensitivity 5 days after lipofection, this system was significantly efficient killing melanoma cells, being four cell lines highly, two fairly and two not very sensitive to CDU/5-FC (their respective IC50 ranging from 0.20 to 800 μM 5-FC). Considering the relatively low lipofection efficiencies, a very strong bystander effect was verified in the eight cell lines: depending on the cell line, this effect accounted for most of the induced cell death (from 70% to 95%). In our assay conditions, we did not find useful interactions either with the herpes simplex thymidine kinase/ganciclovir suicide system (in sequential or simultaneous modality) or with cisplatin and bleomycin chemotherapeutic drugs. Furthermore, only two cell lines displayed limited useful interactions of the CDU/5-FC either with interferon-β gene transfer or the proteasome inhibitor bortezomib respectively. These results would preclude a wide use of these combinations. However, the fact that all the tested cells were significantly sensitive to the CDU/5-FC system encourages further research as a gene therapy tool for local control of canine melanoma.

Neural differentiation of glioblastoma cell lines via a herpes simplex virus thymidine kinase/ganciclovir system driven by a glial fibrillary acidic protein promoter.

Glioblastoma is a malignant brain tumor with poor prognosis that rapidly acquires resistance to available clinical treatments. The herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system produces the selective elimination of HSVtk-positive cells and is a candidate for preclinical testing against glioblastoma via its ability to regulate proliferation and differentiation. Therefore, in this study, we aimed to establish a plasmid encoding the HSVtk/GCV system driven by a glial fibrillary acidic protein (GFAP) promoter and verify its possibility of neural differentiation of glioblastoma cell line under the GCV challenge. Four stable clones—N2A-pCMV-HSVtk, N2A-pGFAP-HSVtk, U251-pCMV-HSVtk, and U251-pGFAP-HSVtk—were established from neuronal N2A and glioblastoma U251 cell lines. In vitro GCV sensitivity was assessed by MTT assay for monitoring time- and dosage-dependent cytotoxicity. The capability for neural differentiation in stable glioblastoma clones during GCV treatment was assessed by performing immunocytochemistry for nestin, GFAP, and βIII-tubulin. Under GFAP promoter control, the U251 stable clone exhibited GCV sensitivity, while the neuronal N2A clones were nonreactive. During GCV treatment, cells underwent apoptosis on day 3 and dying cells were identified after day 5. Nestin was increasingly expressed in surviving cells, indicating that the population of neural stem-like cells was enriched. Lower levels of GFAP expression were detected in surviving cells. Furthermore, βIII-tubulin-positive neuron-like cells were identified after GCV treatment. This study established pGFAP-HSVtk-P2A-EGFP plasmids that successfully ablated GFAP-positive glioblastoma cells, but left neuronal N2A cells intact. These data suggest that the neural differentiation of glioblastoma cells can be promoted by treatment with the HSVtk/GCV system.

Particulate mediators of the bystander effect linked to suicide and interferon-β transgene expression in melanoma cells.

In the context of comparative oncology, melanoma cells derived from companion animal tumors are good models for optimizing and predicting their in vivo response to therapeutic strategies. Here, we report that human, canine, and feline melanoma cells driven to death by bleomycin, interferon-β gene, or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) treatment significantly increased their internal granularity. This fact correlated with the release of a heterogeneous collection of nano- and micro-sized granules as revealed by transmission electron microscopy. While killing lipofected cells, the expressed transgenes and their derived products were incorporated into these granules that were isolated by differential centrifugation. These particulate factors (PFs) were able to transfer, in a dose- and time-dependent manner, appreciable levels of therapeutic genes, related proteins, and drugs. Thus, when recipient cells of SG-carrying PFs were exposed to ganciclovir, this prodrug was efficiently activated, eliminating them. These PFs kept the functionality of their cargo, even after being subjected to adverse conditions, such as the presence of DNase, freezing, or heating. Since our in vitro system did not include any of the immune mechanisms that could provide additional antitumor activity, the chemo-gene treatments amplified by these delivery bags of therapeutic agents offer a great clinical potential.

ApoE-modified liposomes mediate the antitumour effect of survivin promoter-driven HSVtk in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignant tumour with high global morbidity and mortality associated with its multiple aetiologies, poor prognosis, resistance to chemotherapeutic drugs and high rate of recurrence. Here, we evaluated a gene therapy strategy that targets HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. Apolipoprotein E (ApoE)-modified liposomes were used for targeted gene delivery to the tumour tissue, and the survivin promoter was used to drive HSVtk expression in HCC cells. Our results showed that the survivin promoter was specifically activated in tumour cells, and HSVtk was expressed selectively in tumour cells. In combination with GCV treatment, HSVtk expression resulted in the inhibition of HCC cell proliferation via enhanced apoptosis. In addition, tail vein injection of ApoE-HSVtk significantly suppressed the growth of xenograft tumours through an apoptosis-dependent pathway and extended the survival time of tumour-bearing mice. In summary, this study illustrates an effective cancer-specific gene therapy strategy for HCC that can be further developed for future clinical trials.

Golgi membrane protein GP73 modified-liposome mediates the antitumor effect of survivin promoter-driven HSVtk in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide, and there is currently no effective therapeutic strategy in clinical practice. Gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited because of the lack of tumor-specific targets and insufficient gene transfer. The study of targeted transport of therapeutic genes in HCC treatment seems to be very important. In this study, we evaluated a gene therapy approach targeting HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. GP73-modified liposomes targeted gene delivery to the tumor tissue, and the survivin promoter drove HSVtk expression in the HCC cells. Our results showed that the survivin promoter was specifically activated in tumor cells and HSVtk was expressed selectively in tumor cells. Combined with GCV treatment, HSVtk expression resulted in suppression of HCC cell proliferation via enhancing apoptosis. Moreover, tail vein injection of GP73-HSVtk significantly suppressed the growth of xenograft tumors through an apoptosis-dependent pathway and extended the survival of tumor-bearing mice without damaging the mice liver functions. Taken together, this study demonstrates an effective cancer-specific gene therapy strategy using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system for HCC that can be further developed for future clinical trials.

Mechanisms Enhancing the Cytotoxic Effects of Bleomycin plus Suicide or Interferon-β Gene Lipofection in Metastatic Human Melanoma Cells.

Three metastatic human melanoma cell lines generated from patient removed lymph nodes and spleen metastasis were established in our laboratory. To investigate the mechanisms enhancing the cytotoxic effects of Bleomycin (BLM), herpes simplex virus thymidine kinase/ganciclovir Suicide Gene (SG) and human interferon-β gene (hIFNβ) lipofection in early passages of these melanoma cell lines. In these cell lines, we determined: cytotoxicity, bystander effect, lipofection efficiencies, apoptosis, necrosis, senescence, colony forming capacity and mitochondrial membrane depolarization after treatments. The three assayed cell lines displayed sensitivity to single and combined BLM/gene treatments. BLM improved the antitumor and anti-clonogenic effects of SG and hIFNβ genes. Considering the low lipofection efficiencies (<10%), one of the main causes of the SG and hIFNβ gene effectiveness was their bystander effect. In one of these cell lines, this effect eradicated up to 60% of the cells although <1% expressed the transgene. In the three cell lines, BLM alone or combined with SG or hIFNβ gene significantly increased the percentage of cells exhibiting membrane compromise, DNA damage, and senescence. Interestingly, the strong BLM/hIFNβ gene combination was able to generate from 73% to 98% of non-viable cells. The high proportion of senescent cells induced by BLM alone or combined with genes strongly decreased the clonogenic capacity of surviving cells. The presented results indicate that BLM improves the antitumor effects of SG and hIFNβ transgene expression. Altogether, these findings strongly support the clinical potential of these combined approaches.

Suicide Gene Therapy of Oral Squamous Cell Carcinoma and Cervical Carcinoma In Vitro.

Suicide gene therapy induced by the Herpes Simplex Virus thymidine kinase/ganciclovir (HSV-tk/GCV) system has been utilized to successfully treat various cancers. We describe TransfeX-mediated transfection of pCMV.Luc into HeLa cervical carcinoma and HSC-3, FaDu, and H357 oral squamous cell carcinoma (OSCC) cell lines in the presence of 10% serum. This method has proved to be highly efficient, with low nonspecific cytotoxicity. The plasmid pNGVL1-tk encoding HSV-tk under the control of the CMV promoter was delivered to the cells in vitro via TransfeX, a cationic liposomal reagent, followed by treatment with ganciclovir. The Alamar Blue cell viability assay was used to determine levels of thesuicide effect.

P04.67 Assessments for prediction of bystander effect in HSV-tk/GCV gene therapy

Background Enzyme/prodrug gene therapy using stem cells as cellular vehicles is one of the feasible treatment strategies for malignant gliomas, because of the innate tumor tropism of stem cells. Although expectation for its clinical application is high, unrelenting efforts to seek therapeutic effectiveness are imperative. Despite needs for this, useful predictive methods for it have not been reported. Bystander effect which is characterized as a strong tumor killing in herpes simplex virus-thymidine kinase/ganciclovir system (HSV-tk/GCV system) is determined mainly by gap junctional activity. Here, we tried to establish predictive assessment methods for bystander effect measuring functional gap junction using high-content analysis.

RNA Structure Design Improves Activity and Specificity of trans-Splicing-Triggered Cell Death in a Suicide Gene Therapy Approach

Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3′ exon replacement (3’ER), 5′ exon replacement (5’ER) correlated with the thermodynamic stability of the tsRNA 3′ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.

WITHDRAWN: Golgi membrane protein GP73 modified-liposome mediates the antitumor effect of surviving promoter-driven HSVtk in hepatocellular carcinoma

// Fanxiu Meng 1, * , Lina Zhang 1, * , Chunyang Xu 2 , Qi Zhang 1 , Yongfang Li 1 , Yingmin Zhang 1 , Na Zhao 1 , Baofeng Yu 1 , Rui Guo 1 , Hailong Wang 1 , Jun Xie 1 , Gongqin Sun 1, 3 , Gaopeng Li 4 , Jun Xu 4 , Qin Qin 5 and Xiushan Dong 6 1 Department of Biochemistry and Molecular Biology, Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi, China 2 Department of Biological Sciences, The Dietrich School of Arts and Sciences, University Of Pittsburgh, Pittsburgh, PA, USA 3 Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI, USA 4 Department of General Surgery, Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan, Shanxi, China 5 Central Laboratory, Shanxi Provincial People's Hospital, Shanxi Medical University, Taiyuan, Shanxi, China 6 Department of General Surgery, Shanxi Dayi Hospital, Taiyuan, Shanxi, China * These authors contributed equally to this work Correspondence to: Baofeng Yu, email: shanxiyangchengdg@163.com Jun Xu, email: junxuty@163.com Keywords: hepatocellular carcinoma; gene therapy; HSVtk/GCV; GP73; apoptosis Received: July 29, 2017 Accepted: October 28, 2017 Published: January 02, 2018 ABSTRACT Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide, and there is currently no effective therapeutic strategy in clinical practice. Gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited because of the lack of tumor-specific targets and insufficient gene transfer. The study of targeted transport of therapeutic genes in HCC treatment seems very importance. In this study, we evaluated a gene therapy approach targeting HCC using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system in HCC cell lines and in an in vivo human HCC xenograft mouse model. GP73-modified liposomes targeted gene delivery to the tumor tissue, and the survivin promoter drove HSVtk expression in the HCC cells. Our results showed that the survivin promoter was specifically activated in tumor cells and HSVtk was expressed selectively in tumor cells. Combined with GCV treatment, HSVtk expression resulted in suppression of HCC cell proliferation via enhancing apoptosis. Moreover, tail vein injection of GP73-HSVtk significantly suppressed the growth of xenograft tumors through an apoptosis-dependent pathway and extended the survival of tumor-bearing mice without damaging the mice liver functions. Taken together, this study demonstrates an effective cancer-specific gene therapy strategy for HCC that can be further developed for future clinical trials.

Immunogenic Human Papillomavirus Pseudovirus-Mediated Suicide-Gene Therapy for Bladder Cancer.

Bladder cancer is the second most common urological malignancy in the world. In 70% of cases it is initially diagnosed as non-muscle-invasive bladder cancer (NMIBC) and it is amenable to local treatments, with intravesical (IVES) Bacillus-Calmette-Guerin (BCG) immunotherapy being routinely used after transurethral resection of the lesion. However, this treatment is associated with significant side-effects and treatment failures, highlighting the necessity of novel strategies. One potent approach is the suicide-gene mediated therapy/prodrug combination, provided tumor-specificity can be ensured and anti-tumor immune responses induced. Using the mouse syngeneic orthotopic MB49-bladder tumor model, here we show that IVES human papillomavirus non-replicative pseudovirions (PsV) can pseudoinfect tumors with a ten-fold higher efficacy than normal bladders. In addition, PsV carrying the suicide-gene herpes-simplex virus thymidine kinase (PsV-TK) combined to Ganciclovir (GCV) led to immunogenic cell-death of tumor cells in vitro and to MB49-specific CD8 T-cells in vivo. This was associated with reduction in bladder-tumor growth and increased mice survival. Altogether, our data show that IVES PsV-TK/GCV may be a promising alternative or combinatory treatment for NMIBC.

The combination of bleomycin with suicide or interferon-β gene transfer is able to efficiently eliminate human melanoma tumor initiating cells.

We explored the potential of a chemogene therapy combination to eradicate melanoma tumor initiating cells, key producers of recurrence and metastatic spread. Three new human melanoma cell lines, two obtained from lymph nodes and one from spleen metastasis were established and characterized. They were cultured as monolayers and spheroids and, in both spatial configurations they displayed sensitivity to single treatments with bleomycin (BLM) or human interferon-β (hIFNβ) gene or herpes simplex virus thymidine kinase/ganciclovir suicide gene (SG) lipofection. However, the combination of bleomycin with SG or hIFNβ gene transfer displayed greater antitumor efficacy. The three cell lines exhibited a proliferative behavior consistent with melan A and gp100 melanoma antigens expression, and BRAF V600E mutation. BLM and both genetic treatments increased the fraction of more differentiated and treatment-sensitive cells. Simultaneously, they significantly decreased the sub-population of tumor initiating cells. There was a significant correlation between the cytotoxicity of treatments with BLM and gene transfer and the fraction of cells exhibiting (i) high proliferation index, and (ii) high intracellular levels of reactive oxygen species. Conversely, the fraction of cells surviving to our treatments closely paralleled their (i) colony and (ii) melanosphere forming capacity. A very significant finding was that the combination of BLM with SG or hIFNβ gene almost abrogated the clonogenic capacity of the surviving cells. Altogether, the results presented here suggest that the combined chemo-gene treatments are able to eradicate tumor initiating cells, encouraging further studies aimed to apply this strategy in the clinic.