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Tumor-specific promoters and cis-regulatory genetic elements are used for transcriptional control of therapeutic transgene expression in cancer gene therapy. HRE (hypoxia response element) and ARE (antioxidant response element) cis-regulatory elements are targets for HIF1 and Nrf2 transcriptional factors, respectively, and mediate activation of gene transcription in a response to hypoxia and oxidative stress, characteristic of most solid tumors. Due to these features HREs and AREs are used in genetic constructs for cancer gene therapy to provide tumor-specific therapeutic transgene expression or replication of oncolytic adenoviruses. In this work on the basis of the tumor-specific promoter hTERT we have constructed hybrid promoters carrying combinations of HRE and ARE. We showed that upon imitation of hypoxia in human lung cancer cell lines the activity of the hybrid promoter HRE-ARE-hTERT is substantially higher compared to promoters carrying only ARE or HRE. Using in vitro suicide cancer gene therapy with the CD: UPRT/5-FC (cytosine deaminase: uracil phosphoribosyl transferase/5-fluorocytosine) enzyme-prodrug system as a model we showed an enhancement of the cytotoxic effect on human lung cancer cells upon imitation of hypoxia when cytosine deaminase: uracil phosphoribosyl transferase was expressed under the control of the HRE-ARE-hTERT promoter compared to HRE-hTERT and ARE-hTERT promoters. The novel hybrid promoter HRE-ARE-hTERT could be used for transcriptional targeting of therapeutic transgene expression or oncolytic adenovirus replication upon development of novel anti-cancer gene therapeutics. [ABSTRACT FROM AUTHOR]

Objectives: To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. Results: Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in kcat/KM with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in kcat/KM versus wild type YfkO in converting CB1954 to a genotoxic drug. Conclusions: The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy. [ABSTRACT FROM AUTHOR]

Abstract: The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.Graphical abstract: [ABSTRACT FROM AUTHOR]

Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s). For this purpose, either sequence of a VEGFR2-specific nanobody or its natural ligand (VEGF) was inserted into the binding site of sindbis virus E2 glycoprotein. In silico modeling data suggested that the inserted targeting motifs were exposed in the context of cSVE2s. Western blot analysis of LVs indicated the incorporation of cSVE2s into viral particles. Capture ELISA demonstrated the specificity/functionality of the incorporated cSVE2s. Transduction of 293/KDR (expressing VEGFR2) or 293T cells (negative control) by constructed LVs followed by fluorescent microscopy and flow cytometric analyses indicated selective transduction of 293/KDR cells (30 %) by both targeting motifs compared to 293T control cells (1-2 %). These results implied similar targeting properties of VEGFR2-specific nanobody compared to the VEGF and indicated the potential for transductional targeting of tumor vasculature by the nanobody displaying LVs. [ABSTRACT FROM AUTHOR]

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity. [ABSTRACT FROM AUTHOR]

Tumor metastasis often confers poor prognosis for cancer patients due to lack of comprehensive strategy in dealing with cells growing in different environment. Current anticancer therapies have incomplete effectiveness because they were designed assuming metastatic tumors behave similarly in different organs. We hypothesize that tumors growing in different sites are biologically heterogeneous in growth potential, as well as in tumor response to anti-cancer therapies. To test this hypothesis, we have developed a multi-organ tumor growth model using the hydrodynamic cell delivery method to establish simultaneous and quantifiable tumor growth in the liver, lungs and kidneys of mice. We demonstrated that growth rate of melanoma tumor in the liver is higher than that of the lungs and kidneys. Tumors in the lungs and kidneys grew minimally at the early stage and aggressively thereafter. Tumors in different organs were also heterogeneous in response to chemotherapy and immune gene therapy using dacarbazine and interferon beta gene, respectively. Lung tumors responded to chemotherapy better than tumors in the liver, but showed minimal response to interferon beta gene therapy, compared to tumors in the liver and kidneys. We also confirmed differential tumor growth of the metastatic colon cancer in mice. Our results point out the importance of a better understanding of the differences in tumor growing in diverse environments. The biological heterogeneity of metastatic tumors demonstrated in this study necessitates establishing new drug screening strategies that take into account the environmental difference at the sites of tumor growth. [ABSTRACT FROM AUTHOR]

The development of antitumor preparations with low toxicity and high selectivity of action is one of the top priorities of cancer gene therapy. Mesenchymal stem cells possess natural tropism towards tumors, a property that makes it possible to use them as vehicles for the targeted delivery of therapeutic genes to tumors of various etiologies. At present, genes that encode enzymes (cytosine deaminase, thymidine kinase, carboxyl esterase), cytokines (IL-2, IL-4, IL-12, and IFN-β), and apoptosis inducing factors (TRAIL) are used as therapeutic genes. Mesenchymal stem cells, as demonstrated using experimental models of tumors of various etiologies, as well as animals with metastases in brain and lungs, are able to successfully deliver therapeutic genes into tumors and produce a significant antitumor effect. However, to effectively use this therapeutic strategy in a clinical setting, a number of technical problems must be solved. [ABSTRACT FROM AUTHOR]

Purpose: To improve gene transducibility mediated by adenovirus (Ad) in cancer cells and further enhance anti-tumor effects by co-delivery. Methods: Calcium-induced phase change method was used to prepare the complex of anionic liposomes and adenovirus (AL/Ad5). Gene expression was qualitatively detected by X-gal staining and quantitatively detected by ELISA. Taking adenovirus-mediated stromal cell-derived factor-1α (Ad5-SDF1α) as therapeutic gene and carmustine (BCNU) as chemotherapeutic agent, a co-delivering system of AL/Ad5-SDF1α/BCNU was prepared and administered to tumor-bearing mice by intratumor injection. Results: Enhanced LacZ gene transduction was obtained in B16 and Lewis lung carcinoma cells in vitro and in vivo. Complexes of AL/Ad5-SDF1α improved SDF1α gene expression and led to accumulation of dendritic cells among the murine B16 melanoma cells in vivo. This co-delivery system of AL/Ad5-SDF1α/BCNU could significantly suppress tumor growth and prolong survival of tumor-bearing mice. Conclusions: Through the co-delivering system, AL/Ad5-SDF1α could synergize with BCNU to improve the antitumor effect. It may be a promising strategy for solid tumor therapy. [ABSTRACT FROM AUTHOR]

Suicide gene therapy is a promising concept in oncology. We have recently introduced a novel suicide gene, TK.007, which was shown to excel established herpes simplex virus thymidine kinase (HSVtk) variants when used for donor-lymphocyte modification in adoptive immunotherapy models. Here, the potential of TK.007 in killing cancer cells was studied. Initially, we transduced tumour cell lines derived from different neoplasias (glioblastoma, melanoma, lung cancer, colon cancer) with lentiviral LeGO vectors encoding TK.007 or the splice-corrected (sc)HSVtk together with an eGFP/Neo-marker. Based on direct in vitro comparison, we found that TK.007 facilitates more efficient tumour cell killing at significantly lower ganciclovir doses in all tumour cell lines tested. Also, using different readout systems, we found a significantly stronger bystander effect of TK.007 as compared to scHSVtk. Importantly, in vitro data were confirmed in vivo using a subcutaneous G62 glioblastoma model in NOD/SCID mice. In mice transplanted with scHSVtk-positive tumours, treatment with low (10 mg/kg) or standard (50 mg/kg) ganciclovir doses resulted only in short-term growth inhibition or transient tumour remission, respectively. In striking contrast, in the TK.007 group, all animals achieved continuous complete remission after both standard and low-dose ganciclovir. Finally, a substantial bystander effect for TK.007 was also confirmed with the G62 model in vivo, where significantly prolonged survival for mice bearing tumours containing only 10% or 50% TK.007-expressing cells was observed. In summary, our data indicate strongly improved anti-tumour activity of TK.007 as compared to conventional HSVtk. We therefore suppose that TK.007 is an excellent candidate for cancer suicide gene therapy. [ABSTRACT FROM AUTHOR]

The inactivation of tumor suppressors and activation of protooncogenes are critical events in malignant cell transformation and tumor progression. Pdcd4 encodes a protein with tumor suppressor functions, which accounts for the increased interest in Pdcd4 as a potential diagnostic and prognostic marker and a target for anti-neoplastic therapy. The review summarizes the well-known properties and functions of Pdcd4 tumor suppressor and the mechanisms of its regulation in tumor tissues, the role of Pdcd4 in cellular transformation and tumor progression, and its potential practical application in oncology. [ABSTRACT FROM AUTHOR]

AbstractAim:Oncolytic adenovirus, also called conditionally replicating adenovirus (CRAD), can selectively propagate in tumor cells and cause cell lysis. The released viral progeny can infect neighboring cancer cells, initiating a cascade that can lead to the ultimate destruction of the tumor. Suicide gene therapy using herpes simplex virus thymidine kinase (HSV-TK) and ganciclovir (GCV) offers a potential treatment strategy for cancer and is undergoing preclinical trials for a variety of tumors. We hypothesized that HSV-TK gene therapy combined with oncolytic adenoviral therapy would have an enhanced effect compared with the individual effects of the therapies and is a potential novel therapeutic strategy to treat liver cancer.Methods:To address our hypothesis, a novel CRAD was created, which consisted of a telomerase-dependent oncolytic adenovirus engineered to express E1A and HSV-TK genes (Ad-ETK). The combined effect of Ad-ETK and GCV was assessed both in vitro and in vivo in nude mice bearing HepG2 cell-derived tumors. Expression of the therapeutic genes by the transduced tumor cells was analyzed by RT-PCR and Western blotting.Results:We confirmed that Ad-ETK had antitumorigenic effects on human hepatocellular carcinoma (HCC) both in vitro and in vivo, and the TK/GCV system enhanced oncolytic adenoviral therapy. We confirmed that both E1A and HSV-TK genes were expressed in vivo.Conclusion:The Ad-ETK construct should provide a relatively safe and selective approach to killing cancer cells and should be investigated as an adjuvant therapy for hepatocellular carcinoma.Acta Pharmacologica Sinica (2009) 30: 617–627; doi: 10.1038/aps.2009.33; published online 13th April 2009 [ABSTRACT FROM AUTHOR]

Background Eukaryotic initiation factor eIF4E, an important regulator of translation, plays a crucial role in the malignant transformation, progression and chemoresistance of many human solid tumors. The overexpression of this gene has been found in a variety of human malignancies including breast carcinoma. In the present study, we attempted to explore the possibility of eIF4E as a therapeutic target for the treatment of human breast carcinoma using breast carcinoma cell line (MCF-7). Materials and methods The survivin promoter-driven eIF4E-shRNA vector was constructed on the basis of pSUPER.retro vector. Then, we established stably transfected MCF-7 and MCF210 (normal human mammary epithelial cell) cells expressing eIF4E-shRNAs or control-shRNAs. Firstly, the changes of eIF4E expression were detected by RT-PCR and Western blot assays. Next, the optimal shRNA vector (eIF4E-shRNA2) was selected to knock down eIF4E expression and investigate the effect of eIF4E-shRNA on eIF4E-regulated gene expression and cell proliferation both in vitro and in vivo. Followingly, the changes of cell cycle and apoptosis in the stably transfectants (MCF-7) were detected by flow cytometry and TUNEL methods, while we also explored possible apoptosis pathways. Finally, we investigated the effect of shRNA targeting eIF4E on the chemosensitivity of breast carcinoma cells to cisplatin in vitro and in vivo. Results Two survivin promoter-driven eIF4E-shRNA vectors were successfully constructed. eIF4E-shRNA2 but not eIF4E-shRNA1 efficiently downregulated the levels of eIF4E expression in the stably transfected MCF-7-s2 cells but not in the stably transfected MCF210-s2 cells, while MCF-7-s2 showed obvious proliferation suppression but MCF210-s2 did not. The downregulation of eIF4E expression significantly reduced the levels of VEGF, FGF-2 and cyclinD1 expression, suppressed cell growth, induced cell cycle arrest in G0/G1 phase and subsequent apoptosis by activating caspase 3 in MCF-7 cells. The results of FCM and TUNEL staining assays indicated that the classic apoptosis characters of the MCF-7 cells stably expressing eIF4E-shRNA2 manifested an apoptosis rate of 18.3%, significantly higher than those in the control groups ( P < 0.05). Morever, we found that downregulation of c-IAP1, c-IAP2 and c-Myc but not Bcl-2 family proteins were involved in the apoptosis induced by eIF4E-shRNA2. In tumorigenicity assay, xenograft tumors developed from MCF-7-s2 cells in mice showed a significant slowdown in the growth speed and formation rate compared with control groups. Furthermore, we also testified that eIF4E-shRNA could synergistically enhance the cytotoxicity effects of cisplatin to MCF-7 cells both in vitro and in vivo. Conclusion Survivin promoter–driven RNA interference system could efficiently and specifically downregulate eIF4E expression in human breast carcinoma cells but not in normal human mammary epithelial cell cells. Thus, eIF4E might play an important role in chemosensitivity to cisplatin, and survivin promoter-driven RNAi targeting eIF4E can be used as adjuvant therapy for human breast carcinomas with tumor specificity and high efficacy. [ABSTRACT FROM AUTHOR]

Despite promising preclinical results, the clinical benefits of cancer gene therapy have been modest heretofore. The main obstacle continues to be the level and persistence of gene delivery to sufficiently large areas of the tumor. One approach for overcoming this might entail extended local virus release. We studied the utility of silica gel monoliths for delivery of adenovirus to advanced orthotopic gastric and pancreatic cancer tumors. Initially, the biochemical properties of the silica-virus matrix were studied and nearly linear release as a function of time was detected. Virus stayed infective for weeks at +37 °C and months at +4 °C, which may facilitate storage and distribution. In vivo, extended release of functional replication deficient and also replication-competent, capsid-modified oncolytic viruses was seen. Treatment of mice with pancreatic cancer doubled their survival (P<0.001). Also, silica gel-based delivery slowed the development of antiadenovirus antibodies.Gene Therapy (2009) 16, 103–110; doi:10.1038/gt.2008.142; published online 28 August 2008 [ABSTRACT FROM AUTHOR]

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-β, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-β was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-β or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-β-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-β therapy correlating the antitumor effect. Systemic level of IFN-β was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-β therapy for prostate cancer lung metastasis.Gene Therapy (2008) 15, 1446–1453; doi:10.1038/gt.2008.101; published online 3 July 2008 [ABSTRACT FROM AUTHOR]

We have generated a thymidine kinase gene-deleted vaccinia virus (VV) (Copenhagen strain) that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. Intratumoral inoculation of this thymidine kinase gene-deleted VV encoding FCU1 (VV-FCU1) in the presence of systemically administered prodrug 5-fluorocytosine (5-FC) produced statistically significant reductions in the growth of subcutaneous human colon cancer in nude mice compared with thymidine kinase gene-deleted VV treatments or with control 5-fluorouracil alone. A limitation of prodrug therapies has often been the requirement for the direct injection of the virus into relatively large, accessible tumors. Here we demonstrate vector targeting of tumors growing subcutaneously following systemic administration of VV-FCU1. More importantly we also demonstrate that the systemic injection of VV-FCU1 in nude mice bearing orthotopic liver metastasis of a human colon cancer, with concomitant administration of 5-FC, leads to substantial tumor growth retardation. In conclusion, the insertion of the fusion FCU1 suicide gene potentiates the oncolytic efficiency of the thymidine kinase gene-deleted VV and represents a potentially efficient means for gene therapy of distant metastasis from colon and other cancers.Gene Therapy (2008) 15, 1361–1371; doi:10.1038/gt.2008.82; published online 15 May 2008 [ABSTRACT FROM AUTHOR]

We have developed a hypoxia-inducible gene therapy approach for the expression of the mature form of human endonuclease G to facilitate cell death in hypoxic regions of the tumor. The chimeric therapeutic gene is placed under the control of a hypoxia response element based promoter and contains a translocation motif linked in frame to an oxygen-dependent degradation domain and the endonuclease G gene. Transient expression of the chimeric therapeutic gene in breast and prostate cancer cell lines resulted in efficient cell death under hypoxia-mimetic conditions. Stable MDA-MB-435 cells expressing the chimeric therapeutic gene under 1% O2 showed an increase in stable HIF-1α protein levels and synthesis of the endonuclease G protein in a time-dependent manner. In normoxic conditions, these stable transgenic cells exhibited no change in growth rate, invasion and motility when compared to parental cells. Moreover, xenografts generated using the transgenic cells exhibited highly significant suppression of tumor growth in a preclinical cancer model compared to the parental cell line. Thus, the hypoxia-modulated endonuclease G expression has the potential to be used as a gene-based-therapy system to kill malignant cells within hypoxic regions of tumors.Cancer Gene Therapy (2008) 15, 645–654; doi:10.1038/cgt.2008.39; published online 13 June 2008 [ABSTRACT FROM AUTHOR]

RNA interference, due to its target specificity, may be highly effective as a novel therapeutic modality, but direct delivery of synthetic small interfering RNA still remains a major obstacle for this approach. To induce long-term expression and specific gene silencing, novel delivery vector system is also required. In this study, we have generated an efficient oncolytic adenovirus (Ad)-based short hairpin (shRNA) expression system (Ad-ΔB7-U6shIL8) against IL-8, a potent proangiogenic factor. To demonstrate IL-8-specificity of this newly engineered Ad-based shRNA, we also manufactured replication-incompetent Ads (Ad-ΔE1-CMVshIL8 and Ad-ΔE1-U6shIL8) under the control of the cytomegalovirus (CMV) and U6 promoters, respectively. Ad-ΔE1-U6shIL8 was highly effective in reducing IL-8 expression, and was much more effective in driving IL-8-specific shRNA than the CMV promoter-driven vector. The reduced IL-8 expression then translated into decreased angiogenesis in vitro as measured by migration, tube formation and rat aortic ring sprouting assays. In addition to its effect on endothelial cells, Ad-ΔE1-U6shIL8 also effectively suppressed the migration and invasion of cancer cells. In vivo, intratumoral injection of Ad-ΔB7-U6shIL8 significantly inhibited the growth of Hep3B and A549 human tumor xenografts. Histopathological analysis of Ad-ΔB7-U6shIL8-treated tumors revealed an increase in apoptotic cells and a reduction in vessel density. Finally, Ad-ΔB7-U6shIL8 was also shown to inhibit the growth of disseminated MDA-MB-231 breast cancer metastases. Taken together, these findings demonstrate the utility and antitumor effectiveness of oncolytic Ad expressing shRNA against IL-8.Gene Therapy (2008) 15, 635–651; doi:10.1038/gt.2008.3; published online 14 February 2008 [ABSTRACT FROM AUTHOR]

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture supernatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture supernatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the G2/M phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02. [ABSTRACT FROM AUTHOR]

Oncolytic adenoviruses have shown some promise in cancer gene therapy. However, their efficacy in clinical trials is often limited, and additional therapeutic interventions have been proposed to increase their efficacies. In this context, molecular imaging of viral spread in tumours could provide unique information to rationalize the timing of these combinations. Here, we use the human sodium iodide symporter (hNIS) as a reporter gene in wild-type and replication-selective adenoviruses. By design, hNIS cDNA is positioned in the E3 region in a wild-type adenovirus type 5 (AdIP1) and in an adenovirus in which a promoter from the human telomerase gene (RNA component) drives E1 expression (AdAM6). Viruses show functional hNIS expression and replication in vitro and kinetics of spread of the different viruses in tumour xenografts are visualized in vivo using a small animal nano-SPECT/CT camera. The time required to reach maximal spread is 48 h for AdIP1 and 72 h for AdAM6 suggesting that genetic engineering of adenoviruses can affect their kinetics of spread in tumours. Considering that this methodology is potentially clinically applicable, we conclude that hNIS-mediated imaging of viral spread in tumours may be an important tool for combined anticancer therapies involving replicating adenoviruses.Gene Therapy (2007) 14, 1731–1738; doi:10.1038/sj.gt.3303043; published online 25 October 2007 [ABSTRACT FROM AUTHOR]

Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435-fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.Gene Therapy (2007) 14, 1278–1286; doi:10.1038/sj.gt.3302982; published online 21 June 2007 [ABSTRACT FROM AUTHOR]

In vivo gene therapy was attempted using poly-(D,L-lactic-co-glycolic acid) microspheres containing the interleukin-12 gene (p2CMVmlL12) in colon adenocarcinoma (CT-26)-bearing Balb/ c mice. Treatment with IL-12-loaded microspheres inhibited tumor growth significantly compared to a control group treated with free IL-12 DNA, though the degree of tumor inhibition was not dependent on the amount of IL-12 gene loaded. Combining gene therapy with 5-fluorou-racil (50 mg/kg) treatment further inhibited tumor growth compared with gene therapy alone. The results indicate that microsphere formulations of pDNA may provide an efficient gene delivery system. [ABSTRACT FROM AUTHOR]

Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved ‘turbo-NTR’ variants from a library of 6.8 × 105 clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.Gene Therapy (2007) 14, 690–698. doi:10.1038/sj.gt.3302919; published online 15 February 2007 [ABSTRACT FROM AUTHOR]

Gene therapy offers an elegant alternative to toxic chemotherapy regimens, mostly without severe side effects. Cancer gene therapy was among the first applications. Following the enthusiasm in the early nineties, a more rationale view is the recent way to look at it. This tutorial review looks upon the tools of gene therapy and the principle elements (vector, promoter, targeting, therapeutic gene). The principles of gene therapy such as gene directed enzyme prodrug therapy (GDEPT) and gene directed tumor vaccination are explained. Further, published protocols and clinical studies for pancreatic carcinoma gene therapy are reviewed. Finally, an outlook is given on the latest developments, some of them beyond conventional gene therapy. [ABSTRACT FROM AUTHOR]

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.Gene Therapy (2007) 14, 503–513. doi:10.1038/sj.gt.3302889; published online 14 December 2006 [ABSTRACT FROM AUTHOR]

For successful cancer gene therapy via intravenous (i.v.) administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and the following processes. We report the development of a PEG-peptide-lipid ternary conjugate (PEG-Peptide-DOPE conjugate (PPD)). In this strategy, the PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (Multifunctional Envelope-type Nano Device: MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the i.v. administration of PPD or PEG/PPD dually-modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus, MEND modified with PPD is a promising device, which has the potential to make in vivo cancer gene therapy achievable.Gene Therapy (2007) 14, 68–77. doi:10.1038/sj.gt.3302843; published online 17 August 2006 [ABSTRACT FROM AUTHOR]

Hyperacute rejection (HAR) is a rapid immunological response to an organ xenotransplant caused by recognition of endothelial galactose(α1,3)galactose (αGal) epitopes and complement-mediated cell lysis by host anti-αGal antibody (‘natural antibody’). The αGal epitope is synthesised by a galactosyl transferase ((α1,3)GT) which humans lack. Because human cells transduced with (α1,3)GT are sensitised to natural antibody/complement-mediated lysis in human serum, delivery of (α1,3)GT to tumour vasculature in patients is a potential therapeutic strategy, by mimicking the pathophysiology of organ rejection. We therefore sought to develop an animal model of HAR for cancer therapy. Nude/(α1,3)GT knock-out mice allowed the growth of human tumour xenografts and the use of ecotropic retrovirus producer cells to generate expression of αGal on tumour vasculature. Lysis of αGal-positive murine endothelial cells with rabbit complement in conjunction with murine anti-αGal antibody in vitro was used to define the conditions necessary for HAR. However, tumour growth retardation and destruction of αGal-positive tumour endothelium were minimal after their administration, despite sera retaining post hoc cytolytic activity with fresh complement. The major limitation of this experimental system, of relevance to other therapeutic approaches, results from the use of a xenograft, in which additional xenoreactivities lead to complement insufficiency. Development of a tractable preclinical model in which to evaluate HAR for cancer therapy requires a syngeneic experimental system. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) induces gene silencing at a level of posttranscription mediated by double stranded RNA. There are numerous methods for delivery of small double-stranded interference RNA (siRNA) to the target cells, including nonviral and viral vectors. Among these methods, viral vectors are the more efficient vehicles. The expression of short hairpin RNA (shRNA) by viral vectors in target cells can be cut by Dicer enzyme to become -21 bp siRNA, which could guide degradation of cognate mRNA. RNAi technology can be directed against cancer using a variety of strategies, including the inhibition of overexpressed oncogenes, promoting apoptosis, regulating cell cycle, antiangiogenesis and enhancing the efficacy of chemotherapy and radiotherapy. Since RNAi technology has become an excellent strategy for cancer gene therapy, this review outlines the latest developments and applications of such a novel technology. [ABSTRACT FROM AUTHOR]

Gene therapy is a new therapeutic approach for the treatment of human cancers. Gene expression systems that can be regulated by drugs have been developed to improve the safety and efficacy of therapeutic transgene delivery. One of the most promising systems is the tetracycline (Tet)-responsive system in the Tet-On configuration. A major problem of the Tet-On system if used in viral vectors is the high basal activity of the Tet response element (TRE) promoter leading to leaky expression of transgenes under uninduced conditions. We therefore evaluated novel TRE promoters for controlling gene expression in an adenovirus vector (AdV) Tet-On system and further investigated them for expression of the pro-apoptotic CD95/Fas ligand (FasL) in human epithelial carcinoma cell line (HeLa) and lung cancer cells. Plasmid-based reporter gene assays showed that modifications within the tetO 7 and minimal immediate early cytomegalovirus promoter (CMV)min sequence of the TRE promoter reduced its leakiness and led to a markedly improved regulatability by doxycycline. Among several TRE promoters tested, a new construct (TRE-Tight1) containing modifications of both the tetO 7 sequence and the CMVmin showed 11-fold reduced leakiness and 1.5-fold increased absolute transgene expression levels after induction, as compared to the original TRE. Under induced conditions, a TRE-Tight1 promoter-dependent AdV expressing the pro-apoptotic CD95L/FasL induced apoptosis and cell lysis in HeLa cells as efficiently as an AdV containing the original TRE promoter. In contrast to the latter, however, the vector with the modified TRE promoter left cells totally unaffected in the absence of the inducer. Stringently regulated induction of apoptosis and cell death by TRE-Tight1-AdV was also demonstrated in three human lung cancer cell lines. These data show that the novel TRE-Tight1 promoter has a high potential for closely controlled and efficient expression of cytotoxic genes in AdV-based anti-cancer approaches. [ABSTRACT FROM AUTHOR]

Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, EΔNLSM, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.EΔNLSM and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.EΔNLSM and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.EΔNLSM and Ad.MEM in vivo. Induction of Ad.EΔNLSM inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. EΔNLSM and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.Gene Therapy (2006) 13, 173–186. doi:10.1038/sj.gt.3302604; published online 1 September 2005 [ABSTRACT FROM AUTHOR]

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a ‘potent bystander apoptosis-inducing effect’ in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this ‘bystander effect’, and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a ‘bystander antitumor effect’ not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-xL and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.Oncogene (2005) 24, 7552–7566. doi:10.1038/sj.onc.1208911; published online 25 July 2005 [ABSTRACT FROM AUTHOR]

Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase (iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine (PE). CMViNOS transfection also reduced response to PE, but by only 52%. A single injection of 25 μg of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO•, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO•. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.Gene Therapy (2005) 12, 1417–1423. doi:10.1038/sj.gt.3302552; published online 19 May 2005 [ABSTRACT FROM AUTHOR]

Gain-of-function mutation of the K-ras gene is one of the most common genetic changes in human tumors. In tumors carrying K-ras mutation, the presence of oncogenic K-Ras is necessary for maintenance of the transformed phenotype. ESXR1 is a human paired-like homeodomain-containing protein expressed primarily in the testis. In cells, the 65-kDa full-length ESXR1 protein is proteolytically processed into an N-terminal 45-kDa fragment containing the homeodomain, which localizes exclusively within the nucleus, and a C-terminal 20-kDa fragment consisting of a proline-rich repeat region, which is located in the cytoplasm. In this work, we demonstrated that the N-terminal ESXR1 fragment specifically recognizes the TAATNNNATTA P3 consensus sequence for the paired-like homeodomain and functions as a sequence-specific transcriptional repressor. We also showed that the N-terminal ESXR1 fragment binds to the TAATGTTATTA sequence present within the first intron of the human K-ras gene and inhibits its expression at both mRNA and protein levels. Ectopic expression of the N-terminal ESXR1 fragment in human carcinoma cells that carry mutated K-ras reduces the level of K-Ras and thereby inhibits the tumor cell proliferation. Identification of ESXR1 as a transcriptional repressor of K-ras has an important implication for the development of cancer therapy that inhibits oncogenic K-Ras expression.Oncogene (2005) 24, 5878–5887. doi:10.1038/sj.onc.1208736; published online 9 May 2005 [ABSTRACT FROM AUTHOR]

The Semliki Forest virus (SFV) vector is an RNA-based suicide expression vector that has been used experimentally for tumour therapy. Recently, a new enhanced vector pSFV10-E has been developed that expresses foreign genes at levels up to 10 times higher than the original vector. Interleukin-12 (IL-12), an immunomodulatory cytokine, plays a key role in the induction of T-helper1 responses. The two IL-12 gene subunits were cloned from mouse splenocytes and inserted into the pSFV10-E and pSFV10 (nonenhanced) vectors. Both constructs expressed and secreted biologically active murine IL-12. Administration of high titre rSFV10-E-IL12 particles intratumourally to treat implanted K-BALB tumours in BALB/c mice demonstrated complete tumour regression in comparison to control or rSFV10-IL12 treated groups. High titre rSFV10-E-IL12 particles were also effective in the CT26 tumour model. Histological and immunohistochemical studies revealed tumour necrosis in addition to aggressive influx of CD4+ and CD8+ T cells and other immune cells. Furthermore, inhibition of primary tumour growth and lung metastases of a metastatic (4T1) tumour model indicated the potential of high titres of rSFV10-E-IL12 particles as an efficient antitumour therapeutic agent.Gene Therapy (2005) 12, 1253–1263. doi:10.1038/sj.gt.3302561; published online 2 June 2005 [ABSTRACT FROM AUTHOR]

Deregulated tumour growth and neovascularization result in an inadequate tumour blood supply, leading to areas of chronic hypoxia and necrosis. Irregular vascular structure and abnormal tumour physiology also cause erratic blood flow in tumour vessels. We reasoned that tumour stroma, including vascular endothelial cells, would consequently experience transient hypoxia that may allow transcriptional targeting as part of an antivascular gene therapy approach to cancer. To exploit hypoxia for transcriptional regulation, retroviral vectors were generated with modified LTRs: a 6-mer of hypoxia response elements in place of the viral enhancer produced near wild-type levels of expression in hypoxia but was functionally inert in normoxia. In a tumour xenograft model, expression was mainly around areas of necrosis, which were shown to be hypoxic; no expression was detected in tumour stroma. Time-course experiments in vitro demonstrated that expression was transient in response to a hypoxic episode, such that a reporter gene would be insensitive to acute hypoxia in vivo. In contrast, a significant therapeutic effect was seen upon ganciclovir administration with a vector expressing thymidine kinase (TK) in the tumour stroma. Expression of TK was more effective when targeted to acute hypoxia in the stroma compared to chronic hypoxia in the poorly vascularized regions of the tumour cell compartment. The data presented here are evidence that hypoxia in the stromal compartment does occur and that transient hypoxia constitutes a valid therapeutic target.Gene Therapy (2005) 12, 1058–1069. doi:10.1038/sj.gt.3302504 Published online 31 March 2005 [ABSTRACT FROM AUTHOR]

Current gene therapy protocols against cancer often have limited target specificity. Here, a novel tumor-specific targeted gene delivery procedure, which is based on Tetrahymena group I intron ribozyme, is presented. This ribozyme can target a cancer-specific transcript and then replace the RNA with new transcripts, resulting in induction of the transgene activity selectively in cancer cells that express the target RNA. The RNA replacement occurs by trans-splicing reaction with high fidelity with the target RNA. In addition, the ribozyme can specifically inhibit function of the targeted gene in the cells expressing the RNA. Moreover, the ribozyme can selectively deliver cytotoxin gene activity into cancer cells expressing the RNA and thereby specifically hampers the survival of these cells. These findings suggest that the trans-splicing ribozyme that targets the cancer-specific RNA could be a potential agent for specific tumor gene therapy. [ABSTRACT FROM AUTHOR]

To assess the potential of immune stimulation in combination with apoptosis induction by Semliki Forest virus (SFV) and its derived vector for tumour treatment, we have utilized the poorly immunogenic and rapidly growing K-BALB and CT26 murine tumour models. Both cell lines underwent apoptosis and expressed viral antigen when infected with the SFV4 strain of SFV, or recombinant SFV (rSFV) virus-like particles (VLPs) encoding the p62-6k viral structural proteins. VLPs were used to immunize groups of BALB/c and BALB/c nu/nu mice prior to subcutaneous tumour induction and treatment. Direct intratumoral injection of VLPs or SFV4 resulted in an immediate and intense inflammatory reaction in immunized groups that was not observed in naïve groups until day 5 of treatment, and was not observed in nu/nu groups. A significantly higher level of tumour growth inhibition was observed in immunocompetent groups than in athymic mice. For K-BALB tumours, SFV4 treated groups showed greater inhibition than that observed in VLP-treated groups, with immunization prior to treatment enhancing the overall antitumour effect and immune response. No significant difference was observed in CT26 tumours between VLP and SFV4-treated groups, but prior immunization considerably enhanced the antitumoural response. It is concluded that use of the inherent apoptosis-inducing capability of SFV or its vector, by perfusion in combination with immune stimulation, may have potential for the treatment of rapidly growing tumours.Gene Therapy (2005) 12, 147-159. doi:10.1038/sj.gt.3302390 Published online 16 September 2004 [ABSTRACT FROM AUTHOR]

We investigated the effectiveness of in vivo electrogene transfer as a means of therapy in rat urinary bladder carcinoma and in mammary carcinoma models in both athymic and syngeneic mice using the herpes simplex virus 1 thymidine kinase (HSVtk) orIL-12genes in combination with ganciclovir (GCV). A significant increase in the levels of tissue apoptosis and necrosis was induced with a single injection of HSVtkvector directly into bladder and mammary tumors followed by in vivo transfection and a regimen of intraperitoneal GCV injection. This procedure induced significant selective tumor cell death, characterized by marked inflammation and peripheral macrophage influx. Active caspase-3 was also strongly expressed in areas of cell death, indicating the initiation of apoptosis. This result was confirmed in corollary in vitro studies on a mouse bladder carcinoma cell line in which elevated caspase-3, -8, and -9 activities and decreased mitochondrial membrane potential were observed as a result of transfection with HSVtkand addition of GCV to the medium. In the syngeneic mouse mammary cancer model, we additionally found both tumor volume and metastasis to lymph nodes and lungs to be significantly reduced throughout the 2-month experiment. However, in contrast to their syngeneic counterparts, HSVtk/GCV therapy did not effectively inhibit mammary tumor growth/metastasis in an athymic mouse model, leading us to believe that T-cell-mediated immune responses may participate via the bystander effect in HSVtk/GCV experimental therapy. We subsequently evaluated the antitumor activity ofIL-12,which can activate T-cell-mediated immune responses involving macrophages, in the syngeneic mammary tumors and found thatIL-12also significantly suppressed mammary tumor growth and metastasis. We thus suggest that in vivo electrogene transfer is a useful transfection tool in cancer gene therapy and, in addition, we show that T-cell-mediated immune responses may be a critical factor in cancer gene therapy using HSVtk/GCV andIL-12. [ABSTRACT FROM AUTHOR]

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN-γ production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication. [ABSTRACT FROM AUTHOR]

Biliary tract cancer, or cholangiocarcinoma, has a poor prognosis. Resection is the only curative treatment, but only a minority of patients are eligible. Chemotherapy and γ-irradiation are merely palliative, as they are unable to remove the malignancy completely. The chicken anemia virus-derived protein apoptin induces apoptosis in a wide range of human tumor cells and is not hindered by mutations inactivating p53 or by overexpression of Bcl-2, changes known to frustrate chemotherapy and radiation therapy. We examined whether apoptin kills human biliary tract cancer cells. Expression of apoptin by means of plasmids caused extensive cell death in three independent cholangiocarcinoma cell lines, CC-LP, CC-SW, and Mz-ChA-1, regardless of their oncogenic mutations, which included inactivated p16 and p53 and the disruption of the transforming growth factor β signaling pathway. In vitro delivery of apoptin by an adenoviral vector completely eradicated cholangiocarcinoma cells. Moreover, coexpression of the broad-spectrum caspase inhibitor p35 with apoptin only delayed the induced cell death. Changes in nuclear morphology still occurred early after transfection, and nuclei eventually disintegrated, suggesting that apoptin-induced cell death in these cells is not blocked by mutations in either the initiation or execution phase of apoptosis. The efficient induction of cell death by apoptin in cholangiocarcinoma cell lines makes apoptin an attractive candidate for molecular therapy of biliary tract cancer. [ABSTRACT FROM AUTHOR]

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G2/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-xL or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.Oncogene (2003) 22, 8758-8773. doi:10.1038/sj.onc.1206891 [ABSTRACT FROM AUTHOR]

Over the past 30 years, a multidisciplinary approach com-bining surgery, chemotherapy, and radiation has led to a dramatic improvement in survival for patients affected by malignant diseases. Cellular therapies, such as stem cell transplantation, have also made a significant contribution. Nonetheless, many patients are still resistant to standard therapies, which also have high and often unacceptable acute and chronic organ toxicity, with an increased risk for secondary malignancies. Therefore new strategies are needed to improve overall survival and decrease treatment-asso-ciated morbidity. Immunotherapy represents one of the most appealing of these approaches, and active immunization in particular has proved to be amongst the most important of these new cellular strategies because of its ability to stimulate the immune system to actively recognize and kill the malignant cells. Identification of antigens expressed on tumor cells (Table 1), together with a better understanding of the molecular and cellular mechanisms involved in the immune response against cancer, have given investigators tools to manipulate the immune system to induce an efficient immune response in the tumor-bearing host [1-5]. The present review describes the approaches used in cancer vaccination that are intended not only to optimize antitu-mor immunity, but also to overcome tumor immune escape. Examples of how these approaches are being applied clinically are also given. [ABSTRACT FROM AUTHOR]

We have developed a new approach to photodynamic therapy based on adenoviral transduction of the rate-limiting enzyme in heme synthesis. Conventional phototherapy uses porphyrin-based chemical photosensitisers, including δ-aminolaevulinic acid (ALA) which is converted to protoporphyrin IX (PpIX) by the enzymes of the heme biosynthetic pathway. The lack of a specific mechanism for targeting chemical photosensitisers and PpIX to tumour cells means that therapeutic irradiation can damage normal tissue and exposure to sunlight following treatment can cause severe burns. The rate limiting enzyme in PpIX synthesis is ALA-synthase (ALA-S). We have developed a new yeast vector system for manipulation of the adeno- virus genome and used it to construct a virus expressing a mutant form of ALA-S lacking the iron response elements which regulate ALA-S translation and the heme regulatory motifs which regulate import of ALA-S into mitochondria. The virus induces a large increase in PpIX expression and confers photosensitivity on cultured cells. Unlike conventional photodynamic therapy, a viral approach makes it possible to restrict photosensitivity by biological rather than purely physical or chemical means. As with HSV thymidine kinase, ALA-S expression is a general mechanism for sensitisation to a therapeutic agent which can easily be adapted to whatever means of gene delivery is most effective. [ABSTRACT FROM AUTHOR]

Background: Interleukin (IL)-12 has potent antitumor effects in animal models. We hypothesized that direct transfer of the IL-12 gene to established tumors would result in tumor regression without significant toxicity. Methods: Liver tumors were established by direct injection of CT26, a murine adenocarcinoma, into the livers of BALB/c mice, followed by three transfections with either murine IL-12, murine granulocyte-macrophage colony-stimulating factor, or luciferase cDNA using particle-mediated gene transfer. To assess the mechanism of this effect, immunohistochemical staining and depletion experiments with anti-CD4 or -CD8 antibodies were performed. Results: Progressive growth of primary tumors and carcinomatosis were present by day 16 after transfection with luciferase or murine granulocyte-macrophage colony-stimulating factor. At 50 days, complete regression of tumor was evident in seven of eight IL-12-treated mice ( P < .001). In IL-12-transfected livers, immunohistochemical staining revealed an increase in CD8+ T cells. Selective depletion of CD4+ or CD8+ T cells was performed before and during transfection with murine IL-12. At 50 days, 75% of control mice were tumor-free. Only 46% of CD4+ cell-depleted mice ( P = .143) and 7% of CD8+ cell-depleted mice ( P < .001) were tumor-free. Conclusions: IL-12 gene transfer using particle-mediated gene transfer results in complete regression of established CT26 liver tumors in 88% of mice; this effect is dependent on CD8+ T cells. [ABSTRACT FROM AUTHOR]

To investigate the potential of the thymidine kinase gene from Varicella zoster virus (VZVtk) to act as a suicide gene, VZVtk was transferred via a dicistronic retroviral construct into MCF7, T-47D and MDA-MB-435 human breast cancer cells. The cytotoxicity of antiviral drugs was then evaluated in vitro on the wild-type and transduced cells. Acyclovir and ganciclovir did not show any selective toxicity for the modified cells. In contrast, (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) was extremely toxic for the VZVtk expressing cells, with IC50 values of 0.6 μ M, 0.1 μ M and 0.06 μ M for MCF7, T-47D and MDA-MB-435 cells, respectively. The selectivity index of BVDU (ie the IC50 value ratio of the wild-type to the VZVtk+ cells) was 400 for MCF7, 750 for T-47D and 2000 for MDA-MB-435 cells. To test the system in vivo, VZVtk carrying MDA-MB-435 cells were inoculated subcutaneously into nude mice. An intraperitoneal treatment with BVDU administered at the emergence of the tumors, led to a prolonged arrest of the tumor growth and a reduced tumor mass. This effect was BVDU dose-dependent. No bystander effect of BVDU killing could be demonstrated in vitro on mixed populations of VZVtk positive and negative MDA-MB-435 cells. However, an important bystander effect was observed in identical experiments performed on 9L rat gliosarcoma cells infected with the VZVtk-carrying vector. These results demonstrate the efficiency of VZVtk as a suicide gene when BVDU is used as prodrug. The bystander effect measured in vitro, depends however on the tumoral cell type used. [ABSTRACT FROM AUTHOR]

Numerous approaches in gene therapy of human cancers are focused on the establishment of cell type specific or inducible expression vectors allowing the targeted and regulated expression of therapeutic genes. Various conditionally active vectors have been created carrying promoters responding to certain factors or therapeutic modalities (eg hormones, irradiation). The promoter of the multidrug resistance gene (mdr1) harbors such responsive elements and two of these elements have been related to drug responsiveness. In earlier studies we and others have characterized the mdr1 drug responsive-element in CAT reporter assays demonstrating its inducibility by MDR-associated drugs. To exploit this property, we linked the mdr1 promoter sequence to the human tumor necrosis factor alpha (TNF) cDNA in a retroviral vector and transduced the vector into human mammary and colon carcinoma cell lines. These cells were treated with various mdr1-associated drugs to induce TNF expression in vitro. We have shown that the mdr1 promoter-driven TNF expression is drug-inducible and that this induction is drug concentration and time dependent. The studies demonstrate the feasibility of the novel vector system for a chemotherapy-inducible expression of a chemosensitizing cytokine that is successful at enhancing cytotoxicity of drugs in cancer therapy. [ABSTRACT FROM AUTHOR]