Increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector

An improved viral vector for cancer gene therapy should be capable of infecting tumors with high efficiency, inducing specific and high-level expression of transgene in the tumor and selectively destroying tumor cells. In the design of such a vector to treat hepatocellular carcinoma, we took advantage of (a) the high infectivity of adenoviruses for hepatic cells, (b) the high level of protein expression and proapoptotic properties that characterize Semliki Forest virus (SFV) replicon, and (c) tumor selectivity provided by alpha-fetoprotein (AFP) promoter. We constructed a hybrid viral vector composed of a helper-dependent adenovirus containing an SFV replicon under the transcriptional control of AFP promoter and a transgene driven by SFV subgenomic promoter. Hybrid vectors containing murine interleukin-12 (mIL-12) genes or reporter gene LacZ showed very specific and high-level expression of transgenes in AFP-expressing hepatocellular carcinoma cells, both in vitro and in an in vivo hepatocellular carcinoma animal model. Infected hepatocellular carcinoma cells were selectively eliminated due to the induction of apoptosis by SFV replication. In a rat orthotopic liver tumor model, treatment of established tumors with a hybrid vector carrying mIL-12 gene resulted in strong antitumoral activity without accompanying toxicity. This new type of hybrid vectors may provide a potent and safe tool for cancer gene therapy

Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice

BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 μg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine

The involvement of ADAM10 in acantholysis in mucocutaneous pemphigus vulgaris depends on the autoantibody profile of each patient

Background Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non‐Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. Objectives Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). Methods We used three PV‐IgG fractions from different patients containing high or low levels of anti‐Dsg1 and anti‐Dsg3 antibodies, and the presence or not of anti‐desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. Results Although all of the PV‐IgG fractions produced suprabasal acantholysis, only those containing anti‐Dsg1/3, but not anti‐Dsc2/3 antibodies, induced ADAM10 activation in a Src‐dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti‐Dsc2/3 antibodies, in addition to anti‐Dsg1/3, triggered earlier and ADAM10‐independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. Conclusions All PV‐IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti‐Dsg antibodies or the presence of non‐Dsg antibodies, such as anti‐Dsc, more severe cell–cell epidermal detachment will occur at different times, and in an ADAM10‐dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non‐Dsg proteins, and therefore more specific therapeutic approaches in PV should be used

Protection against liver damage by cardiotrophin-1: a hepatocyte survival factor up-regulated in the regenerating liver in rats

BACKGROUND & AIMS: Cardiotrophin-1 (CT-1) is a member of the interleukin 6 (IL-6) family of cytokines, which protect cardiac myocytes against thermal and ischemic insults. In this study, we investigated the expression of CT-1 by liver cells and its possible hepatoprotective properties. METHODS: We analyzed the production, signaling, and antiapoptotic properties of CT-1 in hepatocytes and the expression of this cytokine during liver regeneration. We also investigated whether CT-1 might exert protective effects in animal models of liver damage. RESULTS: We found that CT-1 is up-regulated during liver regeneration and exerts potent antiapoptotic effects on hepatocytic cells. Hepatocytes cultured under serum starvation or stimulated with the pro-apoptotic cytokine transforming growth factor beta (TGF-beta) produce CT-1, which behaves as an autocrine/paracrine survival factor. Treatment with an adenovirus encoding CT-1 efficiently protects rats against fulminant liver failure after subtotal hepatectomy, an intervention that causes 91% mortality in control animals whereas 54% of those receiving CT-1 gene therapy were long-term survivors. This protective effect was associated with reduced caspase-3 activity and activation of the antiapoptotic signaling cascades signal transducer and activator of transcription (Stat-3), extracellular regulated kinases (Erk) 1/2, and Akt in the remnant liver. Gene transfer of CT-1 to the liver also abrogated Concanavalin A (Con-A) liver injury and activated antiapoptotic pathways in the hepatic tissue. Similar protection was obtained by treating the animals with 5 microg of recombinant CT-1 given intravenously before Con-A administration. CONCLUSIONS: We show that CT-1 is a hepatocyte survival factor that efficiently reduces hepatocellular damage in animal models of acute liver injury. Our data point to CT-1 as a new promising hepatoprotective therapy

Liver Damage using Suicide Genes A Model for Oval Cell Activation

Liver regeneration from the facultative hepatic stem cells, the oval cells, takes place in situations in which liver regeneration from pre-existing hepatocytes is prevented. Different models have been used to stimulate oval cell response. Many of them involve the use of carcinogenic agents with or without partial hepatectomy. In this study we show that adenovirus-mediated gene transfer of the suicide gene thymidine kinase followed by ganciclovir administration caused hepatotoxicity of variable intensity. Rats with moderate elevation in serum transaminases recovered normal liver architecture few weeks after adenovirus injection. In contrast, rats with severe liver damage exhibited a marked and persisting activation of oval cells accompanied by ductular hyperplasia. In some rats, such lesion eventually evolved to cholangiofibrosis and in one rat to cholangiocarcinoma. Deposition of fibronectin and increased number of hepatic stellate cells were found in association with oval cells and cholangiofibrotic lesions. Hepatocyte growth factor was hyperexpressed in the livers with intense oval cell response or ductular proliferation, suggesting a participation of this factor in those lesions. In summary, our data demonstrate activation of oval cell response after gene transfer of thymidine kinase followed by ganciclovir administration. These findings indicate that high doses of this therapy causes liver damage together with an impairment in hepatocellular regeneration

Self-inactivating helper virus for the production of high-capacity adenoviral vectors

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