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// Marianna Di Scala 1 , Irene Gil-Farina 1,3 , Cristina Olague 1 , Africa Vales 1 , Luciano Sobrevals 1 , Puri Fortes 1 , David Corbacho 2 and Gloria Gonzalez-Aseguinolaza 1 1 Gene Therapy and Regulation of Gene Expression Program, Center for Applied Medical Research (CIMA), Pamplona, Spain 2 Imaging Unit and Cancer Imaging Laboratory, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain 3 Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany Correspondence to: Marianna Di Scala, email: // Gloria Gonzalez-Aseguinolaza, email: // Keywords : interleukin-15, side effects, interferon-γ, T cells, genetic transfer, Immunology and Microbiology Section, Immune response, Immunity Received : September 27, 2015 Accepted : June 09, 2016 Published : June 23, 2016 Abstract Interleukin-15 (IL-15) is a cell growth-factor that regulates lymphocyte function and homeostasis. Its strong immunostimulatory activity coupled with an apparent lack of toxicity makes IL-15 an exciting candidate for cancer therapy, somehow limited by its short half-life in circulation. To increase IL-15 bioavailability we constructed a recombinant adeno-associated vector expressing murine IL-15 (AAV-mIL15) in the liver. Mice injected with AAV-mIL15 showed sustained and vector dose-dependent levels of IL-15/IL-15Rα complexes in serum, production of IFN-γ and activation of CD8+ T-cells and macrophages. The antitumoral efficacy of AAV-mIL15 was tested in a mouse model of metastatic colorectal cancer established by injection of MC38 cells. AAV-mIL15 treatment slightly inhibits MC38 tumor-growth and significantly increases the survival of mice . However, mIL-15 sustained expression was associated with development of side effects like hepatosplenomegaly, liver damage and the development of haematological stress, which results in the expansion of hematopoietic precursors in the bone marrow. To elucidate the mechanism, we treated IFN-γ receptor-, RAG1-, CD1d- and µMT-deficient mice and performed adoptive transfer of bone marrow cells from WT mice to RAG1-defcient mice. We demonstrated that the side effects of murine IL-15 administration were mainly mediated by IFN-γ-producing T-cells. Conclusion: IL-15 induces the activation and survival of effector immune cells that are necessary for its antitumoral activity; but, long-term exposure to IL-15 is associated with the development of important side effects mainly mediated by IFN-γ-producing T-cells. Strategies to modulate T-cell activation should be combined with IL-15 administration to reduce secondary adverse events while maintaining its antitumoral effect.

Pancreatic tumors contain cancer stem cells highly resistant to chemotherapy. The identification of therapies that can eliminate this population of cells might provide with more effective treatments. In the current work we evaluated the potential of oncolytic adenoviruses to act against pancreatic cancer stem cells (PCSC). PCSC from two patient-derived xenograft models were isolated from orthotopic pancreatic tumors treated with saline, or with the chemotherapeutic agent gemcitabine. An enrichment in the number of PCSC expressing the cell surface marker CD133 and a marked enhancement on tumorsphere formation was observed in gemcitabine treated tumors. No significant increase in the CD44, CD24, and epithelial-specific antigen (ESA) positive cells was observed. Neoplastic sphere-forming cells were susceptible to adenoviral infection and exposure to oncolytic adenoviruses resulted in elevated cytotoxicity with both Adwt and the tumor specific AduPARE1A adenovirus. In vivo, intravenous administration of a single dose of AduPARE1A in human-derived pancreatic xenografts led to a remarkable anti-tumor effect. In contrast to gemcitabine AduPARE1A treatment did not result in PCSC enrichment. No enrichment on tumorspheres neither on the CD133+ population was detected. Therefore our data provide evidences of the relevance of uPAR-controlled oncolytic adenoviruses for the elimination of pancreatic cancer stem cells.

The continuous identification of molecular changes deregulating critical pathways in pancreatic tumor cells provides us with a large number of novel candidates to engineer gene-targeted approaches for pancreatic cancer treatment. Targets—both protein coding and non-coding—are being exploited in gene therapy to influence the deregulated pathways to facilitate cytotoxicity, enhance the immune response or sensitize to current treatments. Delivery vehicles based on viral or non-viral systems as well as cellular vectors with tumor homing characteristics are a critical part of the design of gene therapy strategies. The different behavior of tumoral versus non-tumoral cells inspires vector engineering with the generation of tumor selective products that can prevent potential toxic-associated effects. In the current review, a detailed analysis of the different targets, the delivery vectors, the preclinical approaches and a descriptive update on the conducted clinical trials are presented. Moreover, future possibilities in pancreatic cancer treatment by gene therapy strategies are discussed.

Glutathione (GSH) plays an important role in the defence of microorganisms and plants against different environmental stresses. To determine the role of GSH under different stresses, such as acid pH, saline shock, and oxidative shock, a GSH-deficient mutant (Bradyrhizobium sp. 6144-S7Z) was obtained by disruption of the gshA gene, which encodes the enzyme γ-glutamylcysteine synthetase. Growth of the mutant strain was significantly reduced in liquid minimal saline medium, and the GSH content was very low, about 4% of the wild-type level. The defect, caused by disruption of the gshA gene in the growth of mutant strain, cannot be reversed by the addition of GSH (up to 100 µmol/L) to the liquid minimal saline medium, and the endogenous GSH level was approximately the same as that observed without the addition of GSH. In contrast, the wild-type strain increased the GSH content under these conditions. However, the growth of the mutant strain in a rich medium (yeast extract – mannitol) increased, suggesting that at least some but not all of the functions of GSH could be provided by peptides and (or) amino acids. The symbiotic properties of the mutant were similar to those found in the wild-type strain, indicating that the mutation does not affect the ability of the mutant to form effective nodules.Key words: glutathione, γ-glutamylcysteine synthetase, Bradyrhizobium sp., peanut.

We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl(4)) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished alpha-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFbeta), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4alpha) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1).IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation.

We investigated whether gene transfer of insulin-like growth factor I (IGF-I) to the hepatic tissue was able to improve liver histology and function in established liver cirrhosis. Rats with liver cirrhosis induced by carbon tetrachloride (CCl4) given orally for 8 weeks were injected through the hepatic artery with saline or with Simian virus 40 vectors encoding IGF-I (SVIGF-I), or luciferase (SVLuc). Animals were sacrificed 8 weeks after vector injection. In cirrhotic rats we observed that, whereas IGF-I was synthesized by hepatocytes, IGF-I receptor was predominantly expressed by nonparenchymal cells, mainly in fibrous septa surrounding hepatic nodules. Rats treated with SVIGF-I showed increased hepatic levels of IGF-I, improved liver function tests, and reduced fibrosis in association with diminished α-smooth muscle actin expression, up-regulation of matrix metalloproteases (MMPs) and decreased expression of the tissue inhibitors of MMPs TIM-1 and TIM-2. SVIGF-I therapy induced down-regulation of the profibrogenic molecules transforming growth factor beta (TGFβ), amphiregulin, platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF), and vascular endothelium growth factor (VEGF) and induction of the antifibrogenic and cytoprotective hepatocyte growth factor (HGF). Furthermore, SVIGF-I-treated animals showed decreased expression of Wilms tumor-1 (WT-1; a nuclear factor involved in hepatocyte dedifferentiation) and up-regulation of hepatocyte nuclear factor 4 alpha (HNF4α) (which stimulates hepatocellular differentiation). The therapeutic potential of SVIGF-I was also tested in rats with thioacetamide-induced liver cirrhosis. Also in this model, SVIGF-I improved liver function and reduced liver fibrosis in association with up-regulation of HGF and MMPs and down-regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1). Conclusion: IGF-I gene transfer to cirrhotic livers induces MMPs and hepatoprotective factors leading to reversion of fibrosis and improvement of liver function. IGF-I gene therapy may be a useful alternative therapy for patients with advanced cirrhosis without timely access to liver transplantation. (HEPATOLOGY 2010;51:912–921.)

// Anabel Jose 1,2 , Luciano Sobrevals 1,2 , Juan Miguel Camacho-Sanchez 3 , Meritxell Huch 2 , Nuria Andreu 2 , Eduard Ayuso 4 , Pilar Navarro 5 , Ramon Alemany 3 , Cristina Fillat 1,2 1 Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona. 2 Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Barcelona. 3 Laboratori de Recerca Traslacional IDIBELL-Institut Catala d’Oncologia, L’Hospitalet de Llobregat. 4 Centre de Biotecnologia Animal i Terapia Genica and Centro de Investigacion Biomedica en Red de Diabetes y Enfermedades Metabolicas Asociadas CIBERDEM, Barcelona. 5 Institut de Recerca Hospital del Mar-IMIM, Barcelona, Spain. Correspondence: Cristina Fillat, email: // Keywords : Pancreatic cancer, adenovirus, orthotopic xenografts, transgenic mice, thymidine kinase. Received : December 21, 2012, Accepted : January 10, 2013, Published : January 12, 2013 Abstract 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 AduPARTK T plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TK T 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

Pancreatic Ductal Adenocarcinoma (PDAC) therapies show limited success./nIrreversible electroporation (IRE) is an innovative loco-regional therapy in which highvoltage pulses are applied to induce plasma membrane defects leading to cellular death. In the present study we evaluated the feasibility of IRE against PDAC. IRE/ntreatment exhibited significant antitumor effects and prolonged survival in mice with orthotopic xenografts. Extensive tumor necrosis, reduced tumor cell proliferation and disruption of microvessels were observed at different days post-IRE. Animals had transient increases in transaminases, amylase and lipase enzymes that normalized at/n24h post-IRE. These results suggest that IRE could be an effective treatment for locally advanced pancreatic tumors. This work was supported by the Spanish/nMinistry of Science and Innovation (MICINN), BIO2008-04692-C03-02/01 and received/npartial support from the Generalitat de Catalunya SGR091527. Anabel José was a/nrecipient of a FPU fellowship from the MICINN. Fillat’s group is also partially funded by/nCIBERER and by IIS10/00014 from Instituto de Salud Carlos III. Ivorra’s research is/ncurrently supported by a Ramón y Cajal fellowship from the Spanish Ministry for/nScience and Innovation and a Marie Curie IRG grant from the European Commission.

Background Combined treatment of oncolytic adenoviruses with chemotherapeutic agents is foreseen as a therapeutic option for cancer. Here we have investigated the potential to use gemcitabine in combination with the oncolytic adenovirus AduPARE1A to treat pancreatic cancer and evaluate the underlying mechanism. Methods We treated pancreatic cancer cell lines BxPC-3 and PANC-1 with AduPARE1A and gemcitabine individually or in combination and analyzed cell viability, combination index, apoptosis and viral production. We also investigated the effects of the combination on tumor growth and mice survival in two xenograft models. Furthermore, we analyzed uPAR promoter activity from different uPAR-controlled adenovirus and studied NF-κB mediated effects. Results Synergistic cell killing from the combination AduPARE1A/Gemcitabine was observed in BxPC-3 and PANC-1 cells. Moreover, the combination treatment produced therapeutic benefits over either individual modality in two mouse models bearing orthotopic tumors, showing reduced tumor progression and significant prolonged mouse survival. Mechanistic studies showed that the synergistic cell death was not due to an increase in viral replication but occurred through an enhancement of apoptotic cell death. Gemcitabine stimulation increased the transcription of uPAR-controlled transgenes through the induction of NF-κB acting on the uPAR promoter. Interestingly, NF-κB gemcitabine-mediated induction of AduPAR adenoviruses interfered with the activation of NF-κB regulated genes, probably as a result of an intracellular competition for NF-κB DNA binding. Consequently, AduPARE1A infection sensitized cells to gemcitabine-induced apoptosis in the combined treatment. Conclusions These data highlights the potential of the combination as a treatment modality for pancreatic cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0413-2) contains supplementary material, which is available to authorized users.