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Gene transfer of potent immunostimulatory cytokines such as interleukin-12 (IL-12) is a potential treatment for advanced cancer. Different vectors and IL-12 modifications have been developed to avoid side effects associated with high serum levels of the cytokine, while preserving its antitumor properties. Here we have evaluated two alternative strategies using the Syrian hamster as a model for pancreatic cancer metastatic to the liver. Local administration of an oncolytic adenovirus (OAV) expressing a single-chain version of IL-12 caused transient, very intense elevations of IL-12 in serum, resulting in severe toxicity at sub-therapeutic doses. Anchoring IL-12 to the membrane of infected cells by fusion with the transmembrane domain of CD4 reduced systemic exposure to IL-12 and increased the tolerance to the OAV. However, only a modest increase in the therapeutic range was achieved because antitumor potency was also reduced. In contrast, systemic administration of a helper-dependent adenoviral vector (HDAd) equipped with a Mifepristone-inducible expression system allowed sustained and controlled IL-12 production from the liver. This treatment was well tolerated and inhibited the progression of hepatic metastases. We conclude that HDAds are safer than OAVs for the delivery of IL-12, and are promising vectors for immunogene therapy approaches against pancreatic cancer. [ABSTRACT FROM AUTHOR]

A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects. [ABSTRACT FROM AUTHOR]

Standard methods for producing high-capacity adenoviral vectors (HC-Ads) are based on co-infection with a helper adenovirus (HV). To avoid HV encapsidation, its packaging signal (Ψ) is flanked by recognition sequences for recombinases expressed in the producing cells. However, accumulation of HV and low yield of HC-Ad are frequently observed, due in part to insufficient recombinase expression. We describe here a novel HV (AdTetCre) in which Ψ is flanked by loxP sites that can be excised by a chimeric MerCreMer recombinase encoded in the same viral genome. Efficient modulation of cleavage was obtained by simultaneous control of MerCreMer expression using a tet-on inducible system, and translocation to the nucleus by 4-hydroxytamoxifen (TAM). Encapsidation of AdTetCre was strongly inhibited by TAM plus doxycicline. Using AdTetCre and 293Cre4 cells for the production of HC-Ads, we found that cellular and virus-encoded recombinases cooperate to minimize HV contamination. The method was highly reproducible and allowed the routine production of different HC-Ads in a medium-scale laboratory setting in adherent cells, with titers >1010 infectious units and <0.1% HV contamination. The residual HVs lacked Ψ and were highly attenuated. We conclude that self-inactivating HVs based on virally encoded recombinases are promising tools for the production of HC-Ads. [ABSTRACT FROM AUTHOR]

A partial deletion of the adenovirus E3 region, comprising the overlapping 6.7K/gp19K genes, has been described for the incorporation of therapeutic genes in ‘armed’ oncolytic adenoviruses. This deletion allows the insertion of up to 2.5 kb genetic material into the virus and ensures strong expression of transgenes without reducing the replication and cytolytic potency of viruses in vitro. E3-gp19K and 6.7K proteins are involved in avoiding recognition and elimination of infected cells by the host immune system. Therefore, we have studied the effect of this deletion on the replication and transgene expression of the virus in immunocompetent models based on Syrian hamsters. Tumors were established by intrahepatic injection of pancreatic cancer cells with moderate (HaP-T1, HP-1) or low (H2T) permissivity for adenovirus replication. The wild-type human adenovirus 5 (Ad5) or a modified version containing the luciferase gene in the E3-6.7K/gp19K locus (Ad-WTLuc) were injected intratumorally. We found that elimination of Ad-WTLuc was faster than Ad5 in HaP-T1 and HP-1 tumors. In contrast, no differences were observed when the same tumor was established in severely immunocompromised NOD-scid IL2Rγnull mice. In addition, virus-mediated luciferase expression was more stable in these animals. These results suggest that the lack of E3-6.7K/gp19K genes may accelerate the clearance of oncolytic adenoviruses in some immunocompetent tumor models.Cancer Gene Therapy (2009) 16, 703–712; doi:10.1038/cgt.2009.12; published online 20 February 2009 [ABSTRACT FROM AUTHOR]

Drug-inducible systems allow modulation of the duration and intensity of cytokine expression in liver immuno-based gene therapy protocols. However, the biological activity of the transgene may influence their function. We have analyzed the kinetics of interleukin-12 (IL-12) expression controlled by the doxycycline (Dox)- and the mifepristone (Mif)-dependent systems using two long-term expressing vectors directed to liver: a plasmid administered by hydrodynamic injection and a high-capacity adenoviral vector. Daily administration of Dox or Mif was associated with a progressive loss of inducibility and a decrease of murine IL-12 production. This inhibition occurred at the transcriptional level and was probably caused by an interferon (IFN)-γ-mediated downmodulation of liver-specific promoters that control the expression of transactivators in these systems. Genome-wide expression microarrays studies revealed a parallel downregulation of liver-specific genes in mice overexpressing murine IL-12. However, a promoter naturally induced by IL-12 was also inhibited by this cytokine when placed in a plasmid vector. Interestingly, treatment with sodium butyrate, a class I/II histone deacetylase inhibitor, was able to rescue liver-specific promoter activity solely in the vector. We conclude that biologically active IL-12 can transiently inhibit the function of drug-inducible systems in non-integrative DNA vectors by reducing promoter activity, probably through IFN-γ and protein deacetylation-dependent mechanisms.Gene Therapy (2008) 15, 277–288; doi:10.1038/sj.gt.3303073; published online 22 November 2007 [ABSTRACT FROM AUTHOR]

The promoter for human telomerase reverse transcriptase (hTERTp) is preferentially active in malignant cells. It was recently used to control the expression of the adenoviral E1A gene for the development of oncolytic adenoviruses. To ensure maximal repression in normal cells, the inclusion of additional E-boxes in the proximal region of the core promoter was described. We found that the transcriptional activity of this artificial sequence (T-255-4DEB) is minimal in normal cells, but it is also reduced in all the cancer cell lines tested. The cancer specificity of a new oncolytic adenovirus based in this promoter (AdTE1) was evaluated by direct comparison with wild-type adenovirus type 5 (AdWT) in vitro and in vivo. In all the parameters tested, AdTE1 was attenuated in normal cells, but the efficacy in cancer cells showed a parallel reduction, suggesting a lack of specificity. However, the cytotoxicity of AdTE1 was repressed in senescent cells compared to AdWT. Therefore, we conclude that AdTE1 is preferentially attenuated only in cells that are permanently devoid of telomerase expression such as senescent cells. Further modifications in the telomerase-based promoters should be introduced in order to combine maximal attenuation of oncolytic adenoviruses in normal tissues and enhanced activity in tumors. [ABSTRACT FROM AUTHOR]