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Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Gene-directed enzyme-prodrug therapy (GDEPT) is a promising approach for cancer therapy, but it suffers from poor targeted delivery in vivo. Polyethylenimine (PEI) is a cationic polymer efficient in delivering negatively charged nucleic acids across cell membranes; however, it is highly toxic in vivo. Hence, we efficiently reduced PEI toxicity without compromising its transfection efficiency by conjugating it with poly(d,l-lactic- co -glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) as triblock copolymers through a multistep synthetic process. The synthesized nanoparticles showed efficient delivery of loaded nucleic acids to tumor cells in vitro and in vivo in mice. We used this nanoparticle to deliver a rationally engineered thymidine kinase (TK)-p53-nitroreductase (NTR) triple therapeutic gene against hepatocellular carcinoma (HCC), where p53 tumor suppressor gene is mutated in more than 85% of cancers. TK-p53-NTR triple gene therapy restores p53 function and potentiates cancer cell response to delivered prodrugs (ganciclovir (GCV) and CB1954). We used SP94 peptide-functionalized PLGA-PEG-PEI nanoparticles for the optimal delivery of TK-p53-NTR therapeutic gene in vivo. The nanoparticles prepared from the conjugated polymer showed high loading efficiency for the DNA and markedly enhanced TK-NTR-mediated gene therapy upon the simultaneous coexpression of p53 by the concurrent rescue of the endogenous apoptotic pathway in HCC cells of both p53-mutant and wild-type phenotypes in vitro. In vivo delivery of TK-p53-NTR genes by SP94-targeted PLGA-PEG-PEI NP in mice resulted in a strong expression of suicide genes selectively in tumors, and subsequent administration of GCV and CB1954 led to a decline in tumor growth, and established a superior therapeutic outcome against HCC. We demonstrate a highly efficient approach that exogenously supplements p53 to enable synergy with the outcome of TK-NTR suicide gene therapy against HCC.

After 6 years of follow-up treating 364 canine melanoma patients, we present here results about the proof-of-concept, safety, and efficacy of a new surgery adjuvant combined gene therapy. The adjuvant treatment (AT) group was divided in three arms as follows: (i) complete surgery plus vaccine (CS-V), (ii) complete surgery plus combined treatment (CS-CT), and (iii) partial surgery plus combined treatment (PS-CT). Besides the genetic vaccines composed by tumor extracts and lipoplexes carrying human interleukin-2 and granulocyte-macrophage colony-stimulating factor genes, the patients were subjected to combined treatment received in the post-surgical bed injections of lipid-complexed thymidine kinase suicide gene plus ganciclovir and canine interferon-β gene plus bleomycin. As compared with surgery-only treated controls (So), CS-CT and CS-V treatments significantly increased the fraction of local disease-free (from 20 to 89 and 74%) and distant metastases-free patients (M0: from 45 to 87 and 84%). Although less effective than CS arms, PS-CT arm demonstrated a significantly improved control of metastatic disease (M0: 80%) compared with So (M0: 44%). In addition, AT produced a significant 9.3- (CS-CT), 6.5- (CS-V), and 5.4-fold (PS-CT) increase of overall survival as compared with their respective So controls. In general terms, the AT changed a lethal disease into a chronic disease where 70% of CS-CT, 51% of CS-V, and 14% of PS-CT patients died of melanoma unrelated causes. These surgery adjuvant treatments delayed or prevented post-surgical recurrence and distant metastasis, and improved disease-free and overall survival while maintaining quality of life. These successful outcomes encourage assaying a similar scheme for human melanoma.

We conducted a phase 1 trial for single-dose intravenous Ad5CRT, a replication-defective adenovirus vector expressing HSVtk (herpes simplex virus thymidine kinase) modulated by a specific trans-splicing ribozyme that targets human telomerase reverse transcriptase (hTERT)-encoding RNAs. Dose-limiting toxicities (DLTs) were evaluated in 15 patients at dose levels of 0.1-2 × 10 12 virus particles. Patients well tolerated study treatment. During the DLT evaluation period, none of the 15 patients developed any grade 4 toxicities or treatment discontinuation that was related to agents investigated by this trial. The most frequent treatment-related adverse event was fever/chill (26.7%). Of the 18 patients, no patients achieved a partial or complete response, and the median progression-free survival for 18 patients was 1.1 months (95% CI, 1.0-1.3) and the results suggest no clinical benefit from this treatment. Ad5CRT's circulating virus half-life was approximately 10 min. Maximum tolerated dose was 2 × 10 12 virus particles. Single-dose intravenous Ad5CRT was feasible and well tolerated in patients with gastrointestinal cancer liver metastasis. Ad5CRT did not provide meaningful clinical benefit, and the reason for the lack of efficacy was not entirely clear because no pharmocodynamic assessment was made.

Cancer is a devastating disease characterized by uncontrolled and aggressive cell growth. Suicide gene therapy (SGT) facilitating induction of malignancy-specific cell death represents a novel therapeutic approach to treat cancer, which has been investigated in several cancer types with very promising results. In addition, SGT has been suggested as a safeguard in adoptive immunotherapy and regenerative-medicine settings. Generally, SGT consists of two steps-vector-mediated delivery of suicide genes into tumors and subsequent activation of the suicide mechanism, e.g., by administration of a specific prodrug. This chapter provides a framework of protocols for basic and translational research using the Herpes-simplex-virus thymidine kinase (HSV-TK)/ganciclovir (GCV) system, the most widely used suicide gene approach. The protocols provide standard guidelines for the preparation of high-titer third-generation lentiviral vectors encoding a genetically improved HSV-TK version known as TK.007 and its application in in vitro and in vivo treatment setups.

Suicide gene therapy has been tested for the treatment of a variety of cancers, including oral cancer. Among the various suicide gene therapy approaches that have been reported, the Herpes Simplex Virus thymidine kinase (HSV-tk)/ganciclovir (GCV) system is one of the most extensively studied systems, holding great promise in cancer therapy. In this chapter, we describe methods to use the HSV-tk/GCV system to achieve antitumor activity, both in cultured oral cancer cells and in orthotopic and subcutaneous murine models of oral squamous cell carcinoma, using ligand-associated lipoplexes for enhancing therapeutic delivery.

Suicide gene therapy using the herpes simplex virus thymidine kinase (HSV-tk) gene, combined with the prodrug ganciclovir (GCV) medication, is a promising approach for the treatment of malignant tumors, including prostate cancer. The success of this therapeutic strategy requires tissue- or tumor-specific gene expression and efficient gene delivery. In this chapter, we describe the experimental protocols of key methodologies, including promoter construction, reporter assay, adenoviral vector construction and preparation, HSV-tk enzymatic assay and cytotoxicity assay to evaluate the specificity and efficacy of osteonectin promoter-mediated HSV-tk/GCV suicide gene therapy of prostate cancer.

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 the suicide effect.

Despite its low transfer efficiency, suicide gene therapy with HSV-TK is known for its bystander killing effect. The connexin-based gap junction is believed to mediate the bystander effect. Recently, we found that resveratrol, a polyphenol compound, increased the expression of Cx26 and Cx43, which are connexins and important constituents of gap junctions, in murine hepatoma cells. Hypothetically, the resveratrol-induced upregulation of gap junctions may improve the bystander effect that HSV-TK/GCV has on hepatoma cells. Our present investigation revealed that resveratrol could enhance intercellular communication at the gap junctions in CBRH7919 hepatoma cells and thereby enhance the bystander killing effect of GCV on CBRH7919 TK cells. However, inhibition of gap junction using its long-term inhibitor alpha-glycyrrhetinic acid had a negative influence on the bystander effect of gene therapy with HSV-TK/GCV. In addition, combined resveratrol and GCV treatment in tumor-bearing mice with CBRH7919 TK and CBRH7919 WT cells at a ratio of 2:3 resulted in a significant decrease in the volume and weight of the tumor in comparison to GCV or only resveratrol. The present results demonstrate that resveratrol can enhance the bystander effect exerted by the HSV-TK/GCV system by enhancing connexin-mediated gap junctional communication.

"Tumor chemosensitivity" can be achieved by the expression of the herpes simplex virus thymidine kinase gene in cells, followed by the conversion of the "prodrug" ganciclovir into the therapeutic drug inside the cells. This system presaged other combinations of suicide genes and prodrugs, including cytosine deaminase/5-fluorocytosine, purine nucleoside phosphorylase/6-methylpurine deoxyriboside, and horseradish peroxidase/indole-3-acetic acid.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Triple-negative breast cancer (TNBC) represents approximately 20% of all breast cancers and appears resistance to conventional cytotoxic chemotherapy, demonstrating a particularly poor prognosis and a significantly worse clinical outcome than other types of cancer. Suicide gene therapy has been used for the in vivo treatment of various solid tumors in recent clinical trials. In tumor microenvironment, STAT3/NF-κB pathways are constitutively activated in stromal cells as well as in cancer stem cells (CSCs). In this study, we have cloned a novel STAT3/NF-κB-based reporter system to drive the expression of herpes simplex virus thymidine kinase (HSV-TK) against breast cancer. Lentiviral vector expressing HSV-TK under the regulation of STAT3/NF-κB fused response element was developed. In this setting, we exploited the constitutive STAT3/NF-κB activation in tumors to achieve higher transgene expression than that driven by a constitutively active CMV promotor in vivo . An orthotropic MDA-MB-231 triple negative breast cancer mouse model was used for evaluating the feasibility of STAT3-NF-κB-TK/GCV suicide gene therapy system. The basal promoter activity of Lenti-CMV-TK and Lenti-STAT3-NF-κB-TK in MDA-MB-231 cells was compared by 3 H-FEAU uptake assay. The Lenti-CMV-TK showed ~5 fold higher 3 H-FEAU uptake then Lenti -STAT3-NF-κB-TK. In clonogenic assay, cells expressing Lenti-CMV-TK were 2-fold more sensitive to GCV than Lenti-STAT3-NF-κB-TK transduced cells. In vitro effect of STAT3-NF-κB-induced transgene expression was determined by 10ng/mL TNF-α induction and confirmed by western blot analysis and DsRedm fluorescent microscopy. In vivo evaluation of therapeutic effect by bioluminescence and [ 18 F]FHBG microPET imaging indicated that Lenti-STAT3-NF-κB-TK showed more tumor growth retardation than Lenti-CMV-TK when GCV (20 mg/kg) was administered. The invasiveness and expression of cancer stem cell markers were both decreased after STAT3/NF-κB-regulated HSV-TK/GCV therapy. Moreover, STAT3/NF-κB signaling targeting could further sensitize tumor cells to cisplatin. This study successfully established a theranositic approach to treat triple-negative breast cancer via STAT3-NF-κB responsive element-driven suicide gene therapy. This platform may also be an alternative strategy to handle with drug-resistant cancer cells.

Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific T cell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 T cell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Olfactory ensheathing cells (OECs) of human olfactory mucosa are a type of glial-like cells that possess good migratory and tropism properties. We believe that neuronal-derived vehicle may have better capability to receive to the site of injury. In addition to, obtaining of such vehicle from the patient reduces risk of unwanted complications. So, in this study, we investigate whether human olfactory ensheathing cells can be used as a cell source for the first time in gene delivery to assay the tumoricidal effect of herpes simplex virus thymidine kinase gene (HSV-tk) on glioblastoma multiforme (GBM). We obtained OECs from superior turbinate of human nasal cavity mucosa, and cell phenotype was confirmed by the expression of cell-specific antigens including low-affinity nerve growth factor receptor (p75 neurotrophin receptor), microtubule-associated protein-2 (MAP2), and S100 calcium binding protein B (S100-beta) using immunocytochemistry. Then, these cells were transduced by lentiviral vector for transient and stable expression of the herpes simplex virus thymidine kinase gene (OEC-tk). The migratory capacity of OEC-tk, their potency to convert prodrug ganciclovir to toxic form, and cytotoxic effect on astrocyte cells were assayed in vitro. The OECs showed fibroblast-like morphology and expressed specific antigens such as p75 neurotrophin receptor, S100-beta, and MAP2. Our results indicated that OECs-tk were able to migrate toward primary cultured human glioblastoma multiforme and affected survival rate of tumor cells according to exposure time and concentration of ganciclovir. Also, OECs-HSV-tk was capable of inducing apoptosis in tumor cells. Our findings suggest that human OECs could employ as a possible tool to transfer anticancer agent in gene therapy of brain tumor.

Unlabelled: The design of polymeric nanoparticles for gene therapy requires engineering of polymer structure to overcome multiple barriers, including prolonged colloidal stability during formulation and application. Poly(β-amino ester)s (PBAEs) have been shown effective as polymeric vectors for intracellular DNA delivery, but limited studies have focused on polymer modifications to enhance the stability of PBAE/DNA polyplexes. We developed block copolymers consisting of PBAE oligomer center units and poly(ethylene glycol) (PEG) end units. We fabricated a library of PEG-PBAE polyplexes by blending PEGylated PBAEs of different PEG molecular weights and non-PEGylated PBAEs of different structures at various mass ratios of cationic polymer to anionic DNA. Non-PEGylated PBAE polyplexes aggregated following a 24h incubation in acidic and physiological buffers, presenting a challenge for therapeutic use. In contrast, among 36 PEG-PBAE polyplex formulations evaluated, certain polyplexes maintained a small size under these conditions. These selected polyplexes were further evaluated for transfection in human small cell lung cancer cells (H446) in the presence of serum, and the best formulation transfected ∼40% of these hard-to-transfect cells while preventing polymer-mediated cytotoxicity. When PEG-PBAE polyplex delivered Herpes simplex virus thymidine kinase plasmid in combination with the prodrug ganciclovir, the polyplexes killed significantly more H446 cancer cells (35%) compared to healthy human lung fibroblasts (IMR-90) (15%). These findings indicate that PEG-PBAE polyplexes can maintain particle stability without compromising their therapeutic function for intracellular delivery to human small cell lung cancer cells, demonstrate potential cancer specificity, and have potential as safe materials for small cell lung cancer gene therapy., Statement of Significance: Many natural and synthetic biomaterials have been investigated as non-viral vectors to deliver nucleic acids for cancer therapy. However, there are multiple hurdles to successful transfection including achieving particle stability, efficient delivery to cancer cells, and low cytotoxicity. In particular, engineering the physicochemical surface properties of a nanoparticle to improve stability is often offset by a decrease in the cellular entry and transfection efficiency. We developed stable polymeric nanoparticles that demonstrate high transfection efficiency by modifying synthetic biodegradable cationic polymers and engineering nanoparticle formulations using a combinatorial approach. The results of this study show the potential of biodegradable surface-modified polymeric nanoparticles as clinically translatable, biomaterial-based vehicles for cancer gene therapy., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Gene therapeutic strategies with suicide genes are currently investigated in clinical trials for brain tumors. Previously, we have shown that lentiviral vectors delivering the suicide gene HSV-Tk to experimental brain tumors promote a highly significant treatment effect and thus are promising vectors for clinical translation., Methods: In the present study, we tested lentiviral vectors delivering the suicide gene HSV-Tk.007, a highly active mutant of HSV-Tk, to rat brains as a preclinical toxicity study. We injected 10(6) vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped functional lentiviral particles harboring the suicide gene HSV-Tk.007 into the brain of healthy, immunocompetent rats. During prodrug treatment with ganciclovir (GCV), we measured weight and assessed the behavior of the rats in an open field test. After 14 days of GCV treatment, we analyzed HSV-Tk.007 expression in different brain cell populations, as well as inflammatory responses and apoptosis., Results: During prodrug treatment with GCV, behavior experiments did not reveal differences between the treated rats and the control groups. Analysis of HSV-Tk expression in different brain cell populations showed that transduced normal brain cells survived GCV treatment. There were no statistically significant differences in the number of transduced cells between treatment and control groups. Furthermore, inflammatory responses and apoptosis of brain cells were not observed., Conclusions: We show that HSV-Tk.007-mediated suicide gene therapy is not toxic to normal brain cells. This observation is of high relevance for the translation of lentivirus-mediated suicide gene therapies into the clinic for the treatment of brain tumor patients. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

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.

Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.

Suicide gene therapy using herpes simplex virus-1 thymidine kinase (HSV-TK) in combination with ganciclovir (GCV) has emerged as a potential new method for treating cancer. We hypothesize that the efficacy of HSV-TK/GCV therapy is at least partially dependent on p53 status in hepatocellular carcinoma (HCC) patients. Using recombinant adenoviral vectors (rAdV), TK, p53, and ASPP2 were overexpressed individually and in combination in Hep3B (p53 null) and HepG2 (p53 wild-type) cell lines and in primary HCC tumor cells. p53 overexpression induced death in Hep3B cells, but not HepG2 cells. ASPP2 overexpression increased rAdV-TK/GCV-induced HepG2 cell death by interacting with endogenous p53. Similarly, ASPP2 reduced survival in rAdV-TK/GCV-treated primary HCC cells expressing p53 wild-type but not a p53 R249S mutant. Mutated p53 was unable to bind to ASPP2, suggesting that the increase in rAdV-TK/GCV-induced cell death resulting from ASPP2 overexpression was dependent on its interaction with p53. Additionally, γ-H2AX foci, ATM phosphorylation, Bax, and p21 expression increased in rAdV-TK/GCV-treated HepG2 cells as compared to Hep3B cells. This suggests that the combined use of HSV-TK, GCV, rAdV-p53 and rAdV-ASPP2 may improve therapeutic efficacy in HCC patients lacking functional p53.

Introduction: Apoptosis has been suggested as a cytocidal mechanism of the HSV1-tk-expressing cells when exposed to ganciclovir (GCV). This study evaluated the efficacy of (111)In-labeled Annexin V for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and GCV., Materials and Methods: Annexin V was conjugated to DOTA using N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), labeled with (111)In-InCl3 and purified using size exclusion chromatography to give (111)In-DOTA-Annexin V conjugate. The radiochemical yield and the radiochemical purity of (111)In-DOTA-Annexin V were 74±12% and 98±3%, respectively (n=10). (111)In-DOTA-BSA was prepared similarly. An in vitro study to demonstrate the apoptosis of NG4TL4-STK cells after GCV treatment has been performed. Mice bearing NG4TL4-STK and NG4TL4-WT tumors were treated with GCV (10 mg/kg daily) by i.p. injection for 7 consecutive days. Before and during the GCV treatment, biodistribution studies and scintigraphic imaging were performed at 2h post injection of the radiotracers., Results: The uptake of (111)In-DOTA-Annexin V in treated cells (13.41±1.30%) was 4.1 times higher than that in untreated cells (3.21±0.37%). The GCV-induced cell apoptosis in NG4TL4-STK tumor resulted in a significantly increasing accumulation of (111)In-DOTA-Annexin V (1.92±0.32%ID/g at day 0, 4.79±0.86%ID/g at day 2, 4.56±0.58%ID/g at day 4) was observed, but not for that of (111)In-DOTA-BSA. During consecutive GCV treatment, scintigraphic imaging with (111)In-DOTA-Annexin V revealed high uptake in NG4TL4-STK tumor compared with that in NG4TL4-WT tumor. However, no specific (111)In-DOTA-BSA accumulation in NG4TL4-STK and NG4TL4-WT tumors was observed throughout the course of GCV treatment., Conclusions: This study demonstrated that (111)In-DOTA-Annexin V can be used for monitoring tumor cell apoptosis during prodrug activation gene therapy with HSV1-tk and GCV for cancer treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Gene-directed enzyme prodrug therapy (GDEPT) is a promising therapeutic approach for treating cancers of various phenotypes. This strategy is independent of various other chemotherapeutic drugs used for treating cancers where the drugs are mainly designed to target endogenous cellular mechanisms, which are different in various cancer subtypes. In GDEPT an external enzyme, which is different from the cellular proteins, is expressed to convert the injected prodrug in to a toxic metabolite, that normally kill cancer cells express this protein. Theranostic imaging is an approach used to directly monitor the expression of these gene therapy enzymes while evaluating therapeutic effect. We recently developed a dual-GDEPT system where we combined mutant human herpes simplex thymidine kinase (HSV1sr39TK) and E. coli nitroreductase (NTR) enzyme, to improve therapeutic efficiency of cancer gene therapy by simultaneously injecting two prodrugs at a lower dose. In this approach we use two different prodrugs such as ganciclovir (GCV) and CB1954 to target two different cellular mechanisms to kill cancer cells. The developed dual GDEPT system was highly efficacious than that of either of the system used independently. In this chapter, we describe the complete protocol involved for in vitro and in vivo imaging of therapeutic cancer gene therapy evaluation.

A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.

Introduction: Free flap gene therapy exploits a novel therapeutic window when viral vectors can be delivered into a flap ex vivo. The authors investigated the therapeutic potential of an adenovirally-delivered thymidine kinase/ganciclovir prodrug system expressed following vector delivery into a free flap., Methods: The authors demonstrated direct in vitro cytotoxicity by treating a panel of malignant cell lines with the thymidine kinase/ganciclovir system and demonstrated significant cell kill proportional to the multiplicity of infection of adenoviral vector expressing thymidine kinase. Bystander cytotoxicity was demonstrated using conditioned media from producer cells (expressing adenovirally-delivered thymidine kinase and treated with ganciclovir) to demonstrate cytotoxicity in naive tumor cells. The authors investigated the effect of adenoviral vector expressing thymidine kinase/ganciclovir therapy in vivo, using models of microscopic and macroscopic residual disease in a rodent superficial inferior epigastric artery flap model., Results: The authors observed retardation of tumor volume growth in both microscopic (p = 0.0004) and macroscopic (p = 0.0005) residual disease models and prolongation of animal survival. Gene expression studies demonstrated that viral genomic material was found predominantly in flap tissues but declined over time., Conclusions: The authors describe the utility of virally delivered enzyme/prodrug therapy, using a free flap as a vehicle for delivery. They discuss the merits and limitations of this approach and the unique role of therapeutic free flaps among reconstructive techniques available to the plastic surgeon.

Suicide gene therapy of oral squamous cell carcinoma (OSCC) may be a viable approach to the treatment of this cancer. However, human OSCC cells are relatively resistant to efficient transfection by non-viral vectors. To identify an optimal vector for gene delivery, we compared the transfection activities and efficiencies of Glycofect, Metafectene, Metafectene Pro, Metafectene Easy and FuGENE HD, using the OSCC cell line, HSC-3, and the cervical carcinoma cell line, HeLa. The size distribution and ζ-potential of the complexes of these vectors with plasmid DNA were assessed by nanoparticle tracking analysis and electrophoretic mobility measurements, respectively. Metafectene Easy and FuGENE HD mediated the highest transfection activity (measured as luciferase expression) and efficiency (measured as the percentage of cells transfected with ß-galactosidase). These vectors were used to deliver a plasmid encoding herpes simplex virus thymidine kinase, followed by ganciclovir treatment. By day 9, HeLa cell viability was 22±3% of controls with FuGENE HD and 26±3% with Metafectene Easy. The viability of HSC-3 cells was 42±25% with FuGENE HD, and 58±28% with Metafectene Easy. The reduction in viability was statistically significant in both cases (p⩽0.005; average of 3 independent experiments), although there was considerable variability between experiments with the HSC-3 cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following two consecutive doses of a combination of GCV (40 mg/kg) and CB1954 (40 mg/kg) administered at 5 day intervals. In contrast, the respective treatment condition in animals expressing HSV1-sr39TK or NTR separately, showed minimal or no effect on tumor reduction as measured by bioluminescence (tumor mass) and [(18)F]-FHBG microPET (TK expression) imaging. These highlight the strong therapeutic effect of the dual fusion prodrug therapy and its use in theranostic imaging of tumor monitoring in living animals by multimodality molecular imaging.

'Ex vivo' gene therapy using viral vectors to overexpress BMP-2 is shown to heal critical-sized bone defects in experimental animals. To increase its safety, we constructed a dual-expression lentiviral vector to overexpress BMP-2 or luciferase and an HSV1-tk analog, Δtk (LV-Δtk-T2A-BMP-2/Luc). We hypothesized that administering ganciclovir (GCV) will eliminate the transduced cells at the site of implantation. The vector-induced expression of BMP-2 and luciferase in a mouse stromal cell line (W-20-17 cells) and mouse bone marrow cells (MBMCs) was reduced by 50% compared with the single-gene vector. W-20-17 cells were more sensitive to GCV compared with MBMCs (90-95% cell death at 12 days with GCV at 1 μg ml(-1) in MBMCs vs 90-95% cell death at 5 days by 0.1 μg ml(-1) of GCV in W-20-17 cells). Implantation of LV-Δtk-T2A-BMP-2 transduced MBMCs healed a 2 mm femoral defect at 4 weeks. Early GCV treatment (days 0-14) postoperatively blocked bone formation confirming a biologic response. Delayed GCV treatment starting at day 14 for 2 or 4 weeks reduced the luciferase signal from LV-Δtk-T2A-Luc-transduced MBMCs, but the signal was not completely eliminated. These data suggest that this suicide gene strategy has potential for clinical use in the future, but will need to be optimized for increased efficiency.

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT).

Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.

Unlabelled: Suicide gene modified donor T cells can improve immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), but can be eliminated in the event of graft versus host disease (GVHD) through the administration of prodrug. Here we report the production and first-in-man use of mismatched donor T cells modified with a gamma-retroviral vector expressing a herpes simplex thymidine kinase (HSVTK):truncated CD34 (tCD34) suicide gene/magnetic selection marker protein. A stable packaging cell line was established to produce clinical grade vector pseudotyped with the Gibbon Ape Leukaemia Virus (GALV). T cells were transduced in a closed bag system following activation with anti-CD3/CD28 beads, and enriched on the basis of CD34 expression. Engineered cells were administered in two escalating doses to three children receiving T-depleted, CD34 stem cell selected, mismatched allogeneic grafts. All patients had pre-existing viral infections and received chemotherapy conditioning without serotherapy. In all three subjects cell therapy was tolerated without acute toxicity or the development of acute GVHD. Circulating gene modified T cells were detectable by flow cytometry and by molecular tracking in all three subjects. There was resolution of virus infections, concordant with detectable antigen-specific T cell responses and gene modified cells persisted for over 12 months. These findings highlight the suitability of tCD34 as a GMP compliant selection marker and demonstrate the feasibility, safety and immunological potential of HSVTK-tCD34 suicide gene modified donor T cells., Trial Registration: ClinicalTrials.gov NCT01204502

Our aim was to specifically transfer the cytosine deaminase (CD) and thymidine kinase (TK) genes into mucin 1 (MUC1)-positive leukemia cells by anti-MUC1 antibody directed infection of replication-defective lentivirus and to evaluate the targeted cytotoxicity of double suicide genes to leukemia. The target gene vector (containing CD and TK) and envelope (containing GFP and anti-MUC1) and packaging plasmids were cotransfected into 293T cells to produce the recombinant lentivirus. Suicide genes in virus-infected leukemia cells (U937, Jurkat, and K562) were detected by western blot. The cytotoxicity and bystander effect in vitro and the therapeutic effect in vivo were detected after treatment with the prodrugs. The results revealed that combined treatment with prodrug 5-fluorocytosine (5-FC) and ganciclovir (GCV) inhibited leukemia cell growth and caused significant bystander effect than treatment with either prodrug alone. TK/GCV treatment alone induced degeneration and cell death while the effect of CD/5-FC alone mainly caused vacuolar degeneration and necrosis. The addictive effects of combinatorial use of GCV and 5-FC mainly induced swelling of the mitochondria followed by necrosis of the leukemia cells. In vivo experiments revealed that both single and combinatorial prodrug treatments could prolong the survival time of leukemic mice. In summary, anti-MUC1 antibody directed lentiviral vector successfully transduced dual suicide genes and exerted targeted cytotoxicity against MUC1 positive leukemia cells. This targeted lentiviral dual suicide gene delivering system provides a promising approach for clinical treatment of leukemia in future.

Cancer vaccines have recently been shown to induce some clinical benefits. The relationship between clinical activity and anti-vaccine T cell responses is somewhat controversial. Indeed, in many trials it has been documented that the induction of vaccine-specific T cells exceeds the clinical responses observed. Here, we evaluate immunological and clinical responses in 23 MAGE-A3(+) melanoma patients treated with autologous lymphocytes genetically engineered to express the tumor antigen MAGE-A3 and the viral gene product thymidine kinase of the herpes simplex virus (HSV-TK). HSV-TK was used as safety system in case of adverse events and as tracer antigen to monitor the immune competence of treated patients. The increase of anti-TK and anti-MAGE-A3 T-cells after vaccination was observed in 90 and 27% of patients, respectively. Among 19 patients with measurable disease, we observed a disease control rate of 26.3%, with one objective clinical response, and four durable, stable diseases. Three patients out of five with no evidence of disease (NED) at the time of vaccination remained NED after 73+, 70+ and 50+ months. Notably, we report that only patients experiencing MAGE-A3-specific immune responses showed a clinical benefit. Additionally, we report that responder and non-responder patients activate and expand T cells against the tracer antigen TK in a similar way, suggesting that local rather than systemic immune suppression might be involved in limiting clinically relevant antitumor immune responses., (Copyright © 2012 UICC.)

Cancer gene therapy has great potential for decreasing tumor-induced mortality but has been clinically limited by non-targeted and insufficient gene transfer. We evaluated gene therapy targeting hepatocellular carcinoma (HCC) using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system and the tissue inhibitor of metalloproteinase 3 (Timp3) gene. Ultrasound-targeted microbubble destruction (UTMD) targeted gene delivery to the tumor tissue, and the α-fetoprotein promoter targeted HSVtk expression to the HCC cells. Human HepG2 cells transfected with the HSVtk or Timp3 gene demonstrated a reduction in cell viability by >40% compared with the vector control. Cell viability was further inhibited by over 50% with co-transfection of the genes. HepG2 cells were inoculated subcutaneously into athymic mice to induce tumors. UTMD-mediated delivery of HSVtk or Timp3 suppressed tumor growth by >45% and increased survival of tumor-bearing animals (P<0.01 vs vector control). Co-delivery of the genes resulted in a further 30% improvement in tumor suppression and significant extension of animal survival (P<0.01 vs vector control). Targeted gene delivery increased the number of apoptotic cells and decreased the vascular density of the tumors. Targeted co-delivery of the genes synergistically improved the antitumor effects and may provide an effective therapy for HCC.

Failure of clinical trials of nonviral vector-mediated gene therapy arises primarily from either an insufficient transgene expression level or immunostimulation concerns caused by the genetic information carrier (e.g., bacteria-generated, double-stranded DNA (dsDNA)). Neither of these issues could be addressed through engineering-sophisticated gene delivery vehicles. Therefore, we propose a systemic delivery of chemically modified messenger RNA (mRNA) as an alternative to plasmid DNA (pDNA) in cancer gene therapy. Modified mRNA evaded recognition by the innate immune system and was less immunostimulating than dsDNA or regular mRNA. Moreover, the cytoplasmic delivery of mRNA circumvented the nuclear envelope, which resulted in a higher gene expression level. When formulated in the nanoparticle formulation liposome-protamine-RNA (LPR), modified mRNA showed increased nuclease tolerance and was more effectively taken up by tumor cells after systemic administration. The use of LPR resulted in a substantial increase of the gene expression level compared with the equivalent pDNA in the human lung cancer NCI-H460 carcinoma. In a therapeutic model, when modified mRNA encoding herpes simplex virus 1-thymidine kinase (HSV1-tk) was systemically delivered to H460 xenograft-bearing nude mice, it was significantly more effective in suppressing tumor growth than pDNA.

Although the combination of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with ganciclovir (GCV) has been shown as a promising suicide gene treatment strategy for glioma, the almost immunodepressive dose of GCV required for its adequate in vivo efficacy has hampered its further clinical application. Therefore, In order to reduce the GCV dose required, we aim to compare the therapeutic efficacy of HSV1-sr39TK, an HSV1-TK mutant with increased GCV prodrug catalytic activity, with wildtype TK in C6 glioma cells. Accordingly, rat C6 glioma cells were first transfected with pCDNA-TK and pCDNA-sr39TK, respectively, and the gene transfection efficacy was verified by immunocytochemistry and western blot analysis. Then the in vivo sensitivity of these transfected C6-TK and C6-sr39TK cells to GCV was determined by 3-(4,5)-dimethylthiahiazo-(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) colorimetric assay and Hoechst-propidium iodide (PI) staining. Finally, a subcutaneously C6 xenograft tumor model was established in the nude mice to test the in vitro efficacy of TK/GCV gene therapy. Our results showed that, as compared with wildtype TK, HSV1-sr39TK/GCV demonstrated a stronger therapeutic efficacy against C6 glioma both in vitro and in vivo, which, by reducing the required GCV dose, might warrant its future use in the treatment of glioma under clinical setting.

Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p less than 0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor that regulates protection against a wide variety of toxic insults to cells, including cytotoxic cancer chemotherapeutic drugs. Many lung cancer cells harbor a mutation in either Nrf2 or its inhibitor Keap1 resulting in permanent activation of Nrf2 and chemoresistance. In this study, we sought to examine whether this attribute could be exploited in cancer suicide gene therapy by using a lentiviral (LV) vector expressing herpes simplex virus thymidine kinase (HSV-TK/GCV) under the regulation of antioxidant response element (ARE), a cis-acting enhancer sequence that binds Nrf2. In human lung adenocarcinoma cells in which Nrf2 is constitutively overexpressed, ARE activity was found to be high under basal conditions. In this setting, ARE-HSV-TK was more effective than a vector in which HSV-TK expression was driven by a constitutively active promoter. In a mouse xenograft model of lung cancer, suicide gene therapy with LV-ARE-TK/GCV was effective compared with LV-PGK-TK/GCV in reducing tumor size. We conclude that ARE-regulated HSV-TK/GCV therapy offers a promising approach for suicide cancer gene therapy in cells with high constitutive ARE activity, permitting a greater degree of therapeutic targeting to those cells.

The tumor-specific Grp78 promoter is overexpressed in aggressive tumors. Cancer patients would benefit greatly from application of this promoter in gene therapy and molecular imaging; however, clinical benefit is limited by lack of strategies to target the systemic delivery of Grp78-driven transgenes to tumors. This study aims to assess the systemic efficacy of Grp78-guided expression of therapeutic and imaging transgenes relative to the standard cytomegalovirus (CMV) promoter. Combination of ligand and Grp78 transcriptional targeting into a single vector would facilitate systemic applications of the Grp78 promoter. We generated a dual tumor-targeted phage containing the arginine-glycine-aspartic acid tumor homing ligand and Grp78 promoter. Next, we combined flow cytometry, Western blot analysis, bioluminescence imaging of luciferase, and HSVtk/ganciclovir gene therapy and compared efficacy to conventional phage carrying the CMV promoter in vitro and in vivo in subcutaneous models of rat and human glioblastoma. We show that double-targeted phage provides persistent transgene expression in vitro and in tumors in vivo after systemic administration compared with conventional phage. Next, we showed significant tumor killing in vivo using the HSVtk/ganciclovir gene therapy and found a systemic antitumor effect of Grp78-driven HSVtk against therapy-resistant tumors. Finally, we uncovered a novel mechanism of Grp78 promoter activation whereby HSVtk/ganciclovir therapy upregulates Grp78 and transgene expression via the conserved unfolded protein response signaling cascade. These data validate the potential of Grp78 promoter in systemic cancer gene therapy and report the efficacy of a dual tumor targeting phage that may prove useful for translation into gene therapy and molecular imaging applications.

The INSM1 gene encodes a transcriptional repressor that is exclusively expressed in neuronal and neuroendocrine tissue during embryonic development that is re-activated in neuroendocrine tumors. Using the 1.7 kbp INSM1 promoter, an adenoviral HSV thymidine kinase gene therapy was tested for the treatment of neuroendocrine tumors. An unforeseen interference on the INSM1 promoter specificity from the adenoviral genome was observed. Attempts were made to protect the INSM1 promoter from the influence of essential adenoviral sequences and to further enhance the tissue specificity of the INSM1 promoter region. Using the chicken β-globin HS4 insulator sequence, we eliminated off-target tissue expression from the Ad-INSM1 promoter-luciferase2 constructs in vivo. In addition, inclusion of two copies of the mouse nicotinic acetylcholine receptor (n(AchR)) neuronal-restrictive silencer element (NRSE) reduced nonspecific activation of the INSM1 promoter both in vitro and in vivo. Further, inclusion of both the HS4 insulator with the n(AchR) 2 × NRSE modification showed a two log increase in luciferase activity measured from the NCI-H1155 xenograft tumors compared with the original adenovirus construct. The alterations increase the therapeutic potential of adenoviral INSM1 promoter-driven suicide gene therapy for the treatment of a variety of neuroendocrine tumors.

Recent clinical trials for malignant glioma have drawn attention to the potential therapeutic efficacy of herpes simplex virus-thymidine kinase (HSV-tk) suicide gene therapy. Nevertheless, because of the nature of these tumors, it is believed that no single treatment alone is able to combat this fatal disease. Combination therapies may provide a solution to further improve therapies against malignant gliomas. We have recently demonstrated that 15-lipoxygenase-1 (15-LO-1) is able to inhibit tumor angiogenesis as well as enhance apoptosis in tumors. As a result, we studied the potential additive/synergistic effects of 15-LO-1 gene therapy when combined with HSV-tk gene therapy for the treatment of malignant gliomas. For that, BT4C malignant glioma cells were implanted into BDIX male rats. Fourteen days after tumor cell implantation, animals were transduced using adenoviral vectors either with HSV-tk alone or in combination with 15-LO-1. The results show that the combination gene therapy neither improved inhibition of tumor growth nor did it show any benefit on survival. Instead, a profound effect on the migratory properties of the tumor cells was found, resulting in decreased survival. Similar to conventional therapies, the combination of two therapeutic genes may result in unexpected side effects, not seen when given alone.

An established rat intracranial glioma was successfully treated through the tumoricidal bystander effect generated by intratumoral injection of rat bone marrow stromal cells (BMSCs) transduced with the herpes simplex virus-thymidine kinase gene (BMSCtk cells) followed by systemic ganciclovir administration. In the present study, we tested the bystander effect of this treatment strategy when using human BMSCs as the vector cells. Human BMSCtk cells were mixed with various kinds of brain tumor cell lines (human and rat glioma cells) and examined in vitro and in vivo tumoricidal bystander effects, by co-culture study and co-implantation study in the nude mouse, respectively. A significant in vitro bystander effect was observed between human BMSCtk cells and any of the tumor cells examined in the ganciclovir-containing medium. A potent in vivo bystander effect against human and rat glioma cells was also demonstrated when ganciclovir was administered. Migratory activity of the human BMSCs toward the tumor cells was enhanced by the conditioned media obtained from both human and rat glioma cells compared to the fresh media. The results of this study have demonstrated that the bystander effect generated by BMSCtk cells and ganciclovir is not cell type-specific, suggesting that the strategy would be quite feasible for clinical use., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aim: We constructed a recombinant adenovirus construct Ad5-sr39tk-IRES-VEGF(165) (Ad5-SIV) that contained a mutant herpes viral thymidine kinase reporter gene (HSV1-sr39tk) and the human vascular endothelial growth factor 165 (VEGF(165)) gene for noninvasive imaging of gene expression. The recombinant adenovirus Ad5-SIV was transfected into rat bone marrow-derived mesenchymal stem cells (MSCs), and we measured the expression of HSV1-sr39tk and VEGF(165) to evaluate the feasibility of monitoring VEGF(165) expression using reporter gene expression., Methods: The MSCs were infected with Ad5-SIV at various levels of infection (MOI), ranging from 0 to 100 infectious units per cell (IU/cell). The mRNA and protein expression levels of the reporter and therapeutic genes were determined using real-time RT-PCR, Western blot, ELISA and immunofluorescence. The HSV1-sr39tk expression in the MSCs was also detected in vitro using a cellular uptake study of the reporter probe (131)I-FIAU. Gene expression was also evaluated in vivo by micro-Positron Emission Tomography/Computed Tomography (micro-PET/CT) imaging 1day after injecting Ad5-SIV-tranfected MSCs into the left foreleg of the rat. The right foreleg was injected with non-transfected MSCs and served as an internal control., Results: The real-time RT-PCR results demonstrated a good correlation between the expression levels of HSV1-sr39tk mRNA and VEGF(165) mRNA (R(2)=0.93, P<0.05). The cellular uptake of (131)I-FIAU increased with increasing viral titers (R(2)=0.89; P<0.05), and in the group that received an MOI of 100, a peak value of 30.15%±1.11% was found at 3 hours of incubation. The uptake rates increased rapidly between 30 and 150 minutes and reached a plateau after 150 minutes. The uptake rates of (131)I-FIAU by the Ad5-SIV-infected cells were significantly higher than by the Ad5-EGFP-infected cells for all time points (t=18.43-54.83, P<0.05). Moreover, the rate of VEGF(165) protein secretion was highly correlated with the uptake rate of (131)I-FIAU (R(2)=0.84, P<0.05). The radioactivity on the micro-PET/CT images was significantly higher in the left foreleg (which received the transfected MSCs) compared with the control foreleg., Conclusions: These results suggest that radionuclide reporter gene imaging may be used to monitor gene expression in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Pancreatic ductal adenocarcinoma is a deadly malignancy resistant to current therapies. It is critical to test new strategies, including tumor-targeted delivery of therapeutic agents. This study tested the possibility to target the transfer of a suicide gene in tumor cells using an oncotropic lentiviral vector., Results: Three cell surface markers were evaluated to target the transduction of cells by lentiviruses pseudotyped with a modified glycoprotein from Sindbis virus. Only Mucin-4 and the Claudin-18 proteins were found efficient for targeted lentivirus transductions in vitro. In subcutaneous xenografts of human pancreatic cancer cells models, Claudin-18 failed to achieve efficient gene transfer but Mucin-4 was found very potent. Human pancreatic tumor cells were modified to express a fluorescent protein detectable in live animals by bioimaging, to perform a direct non invasive and costless follow up of the tumor growth. Targeted gene transfer of a bicistronic transgene bearing a luciferase gene and the herpes simplex virus thymidine kinase gene into orthotopic grafts was carried out with Mucin-4 oncotropic lentiviruses. By contrast to the broad tropism VSV-G carrying lentivirus, this oncotropic lentivirus was found to transduce specifically tumor cells, sparing normal pancreatic cells in vivo. Transduced cells disappeared after ganciclovir treatment while the orthotopic tumor growth was slowed down., Conclusion: This work considered for the first time three aspect of pancreatic adenocarcinoma targeted therapy. First, lentiviral transduction of human pancreatic tumor cells was possible when cells were grafted orthotopically. Second, we used a system targeting the tumor cells with cell surface antigens and sparing the normal cells. Finally, the TK/GCV anticancer system showed promising results in vivo. Importantly, the approach presented here appeared to be a safer, much more specific and an as efficient way to perform gene delivery in pancreatic tumors, in comparison with a broad tropism lentivirus. This study will be useful in future designing of targeted therapies for pancreatic cancer.

Background: To explore the anti-tumor effect of high-intensity focused ultrasound (HIFU) combined with herpes simplex virus thymidine kinase (HSV-TK) gene-loaded ultrasound-targeted microbubbles on VX2 rabbit liver tumors., Methods: Seventy-five New Zealand white rabbits were randomly divided into five groups after the models of VX2 rabbit liver tumors were established: (a) HIFU group; (b) HIFU and HSV-TK group (HIFU + HSV-TK); (c) HIFU, HSV-TK and ultrasound group (HIFU + HSV-TK + US); (d) HIFU, HSV-TK gene-loaded microbubbles and ultrasound group (HIFU + HSV-TK-MBs + US); and (e) HSV-TK gene-loaded microbubbles and ultrasound group (HSV-TK-MBs + US). After 2 weeks of VX2 liver tumor implantation, rabbits in groups (a), (b), (c) and (d) received HIFU to establish rabbit models of residual tumor by ablating 80% of the tumor volume. After HIFU ablation, rabbits in different groups received MBs wrapped around HSV-TK or HSV-TK solution via marginal ear veins and/or local ultrasonic irradiation to the tumor. Six rabbits in each group were sacrificed 48 h after the corresponding treatment, and tumors were extracted for in vitro experiments. Thymidine kinase mRNA was detected by the real-time polymerase chain reaction. The green fluorescent protein expression in liver tumor was detected by western blotting and immunohistochemistry. Tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. The growth curves of VX2 liver tumors and survival curves of rabbits were compared., Results: Forty-eight hours after treatment, TK mRNA and protein were the highest in the HIFU + HSV-TK + US + MBs group and the HSV-TK + US + MBs group (p < 0.05). At 48 h after treatment, the apoptotic index of tumor cells in HIFU + HSV-TK-MBs + US group was the highest (p < 0.05). Compared to other groups, HIFU combined with MBs wrapped HSV-TK suicide gene significantly inhibited tumor growth in vivo (p < 0.05) and prolonged the survival time of animals (p < 0.05)., Conclusions: HIFU combined with HSV-TK gene-loaded ultrasound-targeted MBs significantly inhibited the growth of VX2 rabbit liver tumors in vivo and prolonged the survival time of the animals, providing a novel gene delivery method and a novel strategy for liver tumor treatment., (Copyright © 2012 John Wiley & Sons, Ltd.)

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. The emergence of cancer gene therapy potentially offers a number of exciting treatments. The majority of approaches involve strategies to suppress the function of activated oncogenes to restore the expression of functional tumour suppressor genes or to initiate tumour self-destruction. One gene therapy approach against tumours that holds great promise is suicide gene therapy. Herpes simplex virus thymidine kinase (HSV-TK) phosphorylates ganciclovir (GCV), which in turn interacts with cellular DNA polymerase and interferes with DNA synthesis to cause death of rapidly dividing cells. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, we investigated the suppression effect of AAV-mediated HSV-TK/GCV system on the bladder cancer cells and in mice xenograft models of bladder cancer. Our data demonstrate that rAAV-HSV-TK system controlled tumour cell growth and achieves strong antitumour efficacy in vivo. These findings provide a foundation for the development of potential targeted clinical therapies for bladder cancer in humans.

The genetic modification of T cells with a suicide gene grants a mechanism of control of adverse reactions, allowing safe infusion after partially incompatible hematopoietic stem cell transplantation (HSCT). In the TK007 clinical trial, 22 adults with hematologic malignancies experienced a rapid and sustained immune recovery after T cell-depleted HSCT and serial infusions of purified donor T cells expressing the HSV thymidine kinase suicide gene (TK+ cells). After a first wave of circulating TK+ cells, the majority of T cells supporting long-term immune reconstitution did not carry the suicide gene and displayed high numbers of naive lymphocytes, suggesting the thymus-dependent development of T cells, occurring only upon TK+ -cell engraftment. Accordingly, after the infusions, we documented an increase in circulating TCR excision circles and CD31+ recent thymic emigrants and a substantial expansion of the active thymic tissue as shown by chest tomography scans. Interestingly, a peak in the serum level of IL-7 was observed after each infusion of TK+ cells, anticipating the appearance of newly generated T cells. The results of the present study show that the infusion of genetically modified donor T cells after HSCT can drive the recovery of thymic activity in adults, leading to immune reconstitution.

Suicide gene therapy using the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system combined with monocyte chemoattractant protein-1 (MCP-1) provides significant antitumor efficacy. The current study was designed to evaluate the antitumor immunity of a newly developed membrane-bound form of MCP-1 (mMCP-1) in an immunocompetent mouse model of hepatocellular carcinoma (HCC). A recombinant adenovirus vector (rAd) harboring the human MCP-1 gene and the membrane-spanning domain of the CX3CL1 gene was used. Large amounts of MCP-1 protein were expressed and accumulated on the tumor cell surface. The growth of subcutaneous tumors was markedly suppressed when tumors were treated with mMCP-1, as compared with soluble MCP-1, in combination with the HSV-tk/GCV system (P<0.01). The numbers of Mac-1-, CD4- and CD8a-positive cells were significantly higher in tumor tissues (P<0.05), and tumor necrosis factor (TNF) mRNA expression levels with mMCP-1 were almost five-fold higher than those with soluble MCP-1. These results indicate that the delivery of the mMCP-1 gene greatly enhanced antitumor effects following the apoptotic stimuli by promoting the recruitment and activation of macrophages and T lymphocytes, suggesting a novel strategy of immune-based gene therapy in the treatment of patients with HCC.

Activity and specificity of chemotherapeutic agents against solid tumors can be augmented via the targeted or localized delivery of 'suicide' genes. Selective activation of specific prodrugs in cells expressing the 'suicide' gene drives their elimination by apoptosis, while also enabling the killing of adjacent bystander cells. Strong bystander effects can compensate for poor 'suicide' gene delivery, and depend on the prodrugs used and mechanisms for the acquisition of activated drug by the bystander population, such as the presence of gap junctional intercellular communications. Although a number of 'suicide' gene therapies for cancer have been developed and characterized, such as herpes simplex virus-derived thymidine kinase (HSV-tk)-based activation of ganciclovir, their limited success highlights the need for the development of more robust approaches. Limiting activation kinetics and evolution of chemoresistance are major obstacles. Here we describe 'suicide' gene therapy of cancer based on the lentivirus-mediated delivery of a thymidine-active human deoxycytidine kinase variant. This enzyme possesses substrate plasticity that enables it to activate a multitude of prodrugs, some with distinct mechanisms of action. We evaluated the magnitude and mechanisms of bystander effects induced by different prodrugs, and show that when used in combination, they can synergistically enhance the bystander effect while avoiding off-target toxicity.

Objective: To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system., Methods: A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis., Results: The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]., Conclusion: HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.