Publications by authors named "Juan R Rodriguez-Madoz"

21 Publications

  • Page 1 of 1

Chromatin activation as a unifying principle underlying pathogenic mechanisms in multiple myeloma.

Genome Res 2020 Sep 20;30(9):1217-1227. Epub 2020 Aug 20.

Centro de Investigación Biomédica en Red de Cáncer, CIBERONC, 28029 Madrid, Spain.

Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (), a major regulator of cellular redox status and, in addition, identified as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.265520.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545147PMC
September 2020

Generation of an induced pluripotent stem cell line (CIMAi001-A) from a compound heterozygous Primary Hyperoxaluria Type I (PH1) patient carrying p.G170R and p.R122* mutations in the AGXT gene.

Stem Cell Res 2019 12 18;41:101626. Epub 2019 Oct 18.

Regenerative Medicine Program, CIMA Universidad de Navarra, Pamplona, Spain. Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Navarra, Spain. Electronic address:

Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive metabolic disorder characterized by defects in enzymes involved in glyoxylate metabolism. PH1 is a life-threatening disease caused by the absence, deficiency or mistargeting of the hepatic alanine-glyoxylate aminotransferase (AGT) enzyme. A human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a PH1 patient being compound heterozygous for the most common mutation c.508G>A (G170R), a mistargeting mutation, and c.364C>T (R122*), a previously reported nonsense mutation in AGTX. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2019.101626DOI Listing
December 2019

CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I.

Nat Commun 2018 12 21;9(1):5454. Epub 2018 Dec 21.

Regenerative Medicine Program, Center for Applied Medical Research (CIMA), University of Navarra, IdiSNA, Pamplona, 31008, Spain.

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1 mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07827-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303323PMC
December 2018

Generation of Macaca fascicularis iPS cell line ATCi-MF1 from adult skin fibroblasts using non-integrative Sendai viruses.

Stem Cell Res 2017 05 16;21:1-4. Epub 2017 Mar 16.

Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Health Research Institute of Navarra, Pamplona, Spain. Electronic address:

We generated ATCi-MF1 induced pluripotent stem (iPS) cell line from Macaca fascicularis adult skin fibroblasts using non-integrative Sendai viruses carrying OCT3/4, KLF4, SOX2 and c-MYC. Once established, ATCi-MF1 cells present a normal karyotype, are Sendai virus-free and express pluripotency associated markers. Microsatellite markers analysis confirmed the origin of the iPS cells from the parental fibroblasts. Pluripotency was tested with the in vivo teratoma formation assay. ATCi-MF1 cell line may be a useful primate iPS cell model to test different experimental conditions where the use of human cells can imply ethical issues, as microinjection of pluripotent stem cells in pre-implantational embryos.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2017.03.008DOI Listing
May 2017

Generation and characterization of human iPSC lines derived from a Primary Hyperoxaluria Type I patient with p.I244T mutation.

Stem Cell Res 2016 Jan 28;16(1):116-9. Epub 2015 Dec 28.

Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra, IdiSNA, Pamplona, Spain. Electronic address:

In this work we describe for the first time the generation and characterization of human induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMCs) and dermal fibroblasts of a Primary Hyperoxaluria Type I (PH1)-diagnosed patient with p.I244T mutation, which is highly prevalent in Canary Islands due to founder effect. Cell reprogramming was performed using non-integrative Sendai viruses containing the Yamanaka factors and the generated PH1-hiPSC lines (PH1-PBMCs-hiPSC4F1 and PH1-Fib-hiPSC4F1) showed normal karyotypes, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers and in vivo differentiation ability to the three germ layers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2015.12.014DOI Listing
January 2016

Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

Stem Cell Res 2016 Jan 2;16(1):20-3. Epub 2015 Dec 2.

Cell Therapy Program, Center for Applied Medical Research (CIMA), University of Navarra, Instituto de Investigación Sanitaria de Navarra, Pamplona, Spain. Electronic address:

In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2015.12.002DOI Listing
January 2016

Whole-epigenome analysis in multiple myeloma reveals DNA hypermethylation of B cell-specific enhancers.

Genome Res 2015 Apr 2;25(4):478-87. Epub 2015 Feb 2.

Unidad de Hematopatología, Servicio de Anatomía Patológica, Hospital Clínic, Universitat de Barcelona, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain;

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.180240.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381520PMC
April 2015

Strict requirement for vector-induced type I interferon in efficacious antitumor responses to virally encoded IL12.

Cancer Res 2015 Feb 19;75(3):497-507. Epub 2014 Dec 19.

Center for Applied Medical Research (CIMA) and Clínica Universidad de Navarra, University of Navarra, Navarra, Spain.

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-13-3356DOI Listing
February 2015

A quick and efficient method to generate mammalian stable cell lines based on a novel inducible alphavirus DNA/RNA layered system.

Cell Mol Life Sci 2014 Dec 3;71(23):4637-51. Epub 2014 May 3.

3P Biopharmaceuticals S.L., Polígono Mocholí, C/Mocholí 2, 31110, Noain, Spain.

We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00018-014-1631-2DOI Listing
December 2014

Short-term intratumoral interleukin-12 expressed from an alphaviral vector is sufficient to induce an efficient antitumoral response against spontaneous hepatocellular carcinomas.

Hum Gene Ther 2014 Feb 7;25(2):132-43. Epub 2014 Jan 7.

1 Division of Gene Therapy, School of Medicine, Center for Applied Medical Research, University of Navarra , Pamplona 31008, Spain .

Interleukin-12 (IL-12) is an immunostimulatory cytokine that has shown strong antitumor effects in animal models of liver cancer. In order to overcome the severe toxicity associated with its systemic administration, we had previously tested different strategies based on IL-12 gene transfer to tumor cells or to the surrounding liver tissue. We obtained promising results both with a recombinant Semliki Forest virus (SFV) vector expressing high levels of IL-12 (SFV-IL-12) after intratumoral injection and with a plasmid vector [pTonL2(T)-mIL12] that allows liver-specific and inducible IL-12 expression. The aim of the present study was to compare the antitumor responses induced by both systems in a clinically relevant animal model of hepatocellular carcinoma (HCC) developed in L-PK/c-myc transgenic mice. These animals overexpress the c-myc oncogene in their livers, giving rise to spontaneous hepatic tumors with latency, histopathology, and genetic characteristics similar to human HCCs. We observed that intratumoral inoculation of SFV-IL-12 induced growth arrest in most tumors, providing 100% survival rate, in contrast to no survival in control animals. Similar results were obtained with hydrodynamic injection of pTonL2(T)-mIL12 after long-term induction of IL-12 expression in the liver. However, tumor arrest was less evident in plasmid-treated mice and the survival rate was slightly lower, despite higher and more sustained levels of IL-12 and IFN-γ in serum. The fact that SFV-IL-12 was able to induce both apoptosis and a type-I IFN response specifically in the tumor could explain why short-term IL-12 expression from this vector was sufficient to mediate an antitumoral response comparable with long-term IL-12 expression driven by pTonL2(T)-mIL12. Since SFV-IL-12 could reduce the possible toxicity associated with long-term IL-12 expression, we believe that this vector could have a potential application for HCC gene therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2013.080DOI Listing
February 2014

Eradication of liver-implanted tumors by Semliki Forest virus expressing IL-12 requires efficient long-term immune responses.

J Immunol 2013 Mar 11;190(6):2994-3004. Epub 2013 Feb 11.

Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona 31008, Navarra, Spain.

Semliki Forest virus vectors expressing IL-12 (SFV-IL-12) were shown to induce potent antitumor responses against s.c. MC38 colon adenocarcinomas in immunocompetent mice. However, when MC38 tumors were implanted in liver, where colon tumors usually metastasize, SFV-IL-12 efficacy was significantly reduced. We reasoned that characterization of immune responses against intrahepatic tumors in responder and nonresponder animals could provide useful information for designing more potent antitumor strategies. Remarkably, SFV-IL-12 induced a high percentage of circulating tumor-specific CD8 T cells in all treated animals. Depletion studies showed that these cells were essential for SFV-IL-12 antitumor activity. However, in comparison with nonresponders, tumor-specific cells from responder mice acquired an effector-like phenotype significantly earlier, were recruited more efficiently to the liver, and, importantly, persisted for a longer period of time. All treated mice had high levels of functional specific CD8 T cells at 8 d posttreatment reflected by both in vivo killing and IFN-γ-production assays, but responder animals showed a more avid and persistent IFN-γ response. Interestingly, differences in immune responses between responders and nonresponders seemed to correlate with the immune status of the animals before treatment and were not due to the treatment itself. Mice that rejected tumors were protected against tumor rechallenge, indicating that sustained memory responses are required for an efficacious therapy. Interestingly, tumor-specific CD8 T cells of responder animals showed upregulation of IL-15Rα expression compared with nonresponders. These results suggest that SFV-IL-12 therapy could benefit from the use of strategies that could either upregulate IL-15Rα expression or activate this receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1201791DOI Listing
March 2013

DENV inhibits type I IFN production in infected cells by cleaving human STING.

PLoS Pathog 2012 4;8(10):e1002934. Epub 2012 Oct 4.

Department of Microbiology and the Global Health and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York City, New York, United States of America.

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1002934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464218PMC
April 2013

Recombinant influenza A viruses with enhanced levels of PB1 and PA viral protein expression.

J Virol 2012 May 7;86(10):5926-30. Epub 2012 Mar 7.

Department of Microbiology, Mount Sinai School of Medicine, New York, New York, USA.

Influenza A viruses containing the promoter mutations G3A/C8U in a given segment express increased levels of the corresponding viral protein during infection due to increased levels of mRNA or cRNA species. The replication of these recombinant viruses is attenuated, and they have an enhanced shedding of noninfectious particles and are incapable of antagonizing interferon (IFN) effectively. Our findings highlight the possibility of increasing influenza virus protein expression and the need for a delicate balance between influenza viral replication, protein expression, and assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.06384-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347261PMC
May 2012

Inhibition of the type I interferon response in human dendritic cells by dengue virus infection requires a catalytically active NS2B3 complex.

J Virol 2010 Oct 21;84(19):9760-74. Epub 2010 Jul 21.

Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA.

Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-alpha/beta production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01051-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937777PMC
October 2010

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

N Biotechnol 2010 May 24;27(2):138-48. Epub 2010 Feb 24.

Center for Applied Medical Research, University of Navarra, Pamplona, Spain.

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nbt.2010.02.005DOI Listing
May 2010

Dengue virus inhibits the production of type I interferon in primary human dendritic cells.

J Virol 2010 May 17;84(9):4845-50. Epub 2010 Feb 17.

Department of Microbiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1124, New York, NY 10029, USA.

Dengue virus (DENV) infects human immune cells in vitro and likely infects dendritic cells (DCs) in vivo. DENV-2 productive infection induces activation and release of high levels of chemokines and proinflammatory cytokines in monocyte-derived DCs (moDCs), with the notable exception of alpha/beta interferon (IFN-alpha/beta). Interestingly, DENV-2-infected moDCs fail to prime T cells, most likely due to the lack of IFN-alpha/beta released by moDCs, since this effect was reversed by addition of exogenous IFN-beta. Together, our data show that inhibition of IFN-alpha/beta production by DENV in primary human moDCs is a novel mechanism of immune evasion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02514-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863727PMC
May 2010

Semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma.

J Virol 2009 Dec 9;83(23):12266-78. Epub 2009 Sep 9.

Division of Gene Therapy, School of Medicine, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

A vector based on Semliki Forest virus (SFV) expressing high levels of interleukin-12 (SFV-enhIL-12) has previously demonstrated potent antitumoral efficacy in small rodents with hepatocellular carcinoma (HCC) induced by transplantation of tumor cells. In the present study, the infectivity and antitumoral/antiviral effects of SFV vectors were evaluated in the clinically more relevant woodchuck model, in which primary HCC is induced by chronic infection with woodchuck hepatitis virus (WHV). Intratumoral injection of SFV vectors expressing luciferase or IL-12 resulted in high reporter gene activity within tumors and cytokine secretion into serum, respectively, demonstrating that SFV vectors infect woodchuck tumor cells. For evaluating antitumoral efficacy, woodchuck tumors were injected with increasing doses of SFV-enhIL-12, and tumor size was measured by ultrasonography following treatment. In five (83%) of six woodchucks, a dose-dependent, partial tumor remission was observed, with reductions in tumor volume of up to 80%, but tumor growth was restored thereafter. Intratumoral treatment further produced transient changes in WHV viremia and antigenemia, with >or=1.5-log(10) reductions in serum WHV DNA in half of the woodchucks. Antitumoral and antiviral effects were associated with T-cell responses to tumor and WHV antigens and with expression of CD4 and CD8 markers, gamma interferon, and tumor necrosis factor alpha in peripheral blood mononuclear cells, suggesting that immune responses against WHV and HCC had been induced. These experimental observations suggest that intratumoral administration of SFV-enhIL-12 may represent a strategy for treatment of chronic HBV infection and associated HCC in humans but indicate that this approach could benefit from further improvements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01597-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2786746PMC
December 2009

Development of a new noncytopathic Semliki Forest virus vector providing high expression levels and stability.

Virology 2008 Jun 28;376(1):242-51. Epub 2008 Apr 28.

Division of Gene Therapy, School of Medicine, Center for Applied Medical Research (CIMA),University of Navarra, Av. Pio XII 55, 31008 Pamplona, Spain.

Alphavirus vectors express high levels of recombinant proteins in mammalian cells, but their cytopathic nature makes this expression transient. In order to generate a Semliki Forest virus (SFV) noncytopathic vector we introduced mutations previously described to turn Sindbis virus noncytopathic into a conserved position in an SFV vector expressing LacZ. Interestingly, mutant P718T in replicase nsp2 subunit was able to replicate in only a small percentage of BHK cells, producing beta-gal-expressing colonies without selection. Puromycin N-acetyl-transferase (pac) gene was used to replace LacZ in this mutant allowing selection of an SFV noncytopathic replicon containing a second mutation in nsp2 nuclear localization signal (R649H). This latter mutation did not confer a noncytopathic phenotype by itself and did not alter nsp2 nuclear translocation. Replicase synthesis was diminished in the SFV double mutant, leading to genomic and subgenomic RNA levels that were 125-fold and 66-fold lower than in wild-type vector, respectively. Interestingly, this mutant expressed beta-gal levels similar to parental vector. By coexpressing pac and LacZ from independent subgenomic promoters this vector was able to generate stable cell lines maintaining high expression levels during at least 10 passages, indicating that it could be used as a powerful system for protein production in mammalian cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virol.2008.03.016DOI Listing
June 2008

Biodistribution and tumor infectivity of semliki forest virus vectors in mice: effects of re-administration.

Mol Ther 2007 Dec 31;15(12):2164-71. Epub 2007 Jul 31.

Division of Gene Therapy, School of Medicine, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.

Semliki Forest virus (SFV)-based vectors have broad tropism, with the ability to infect cells from various origins, including those from tumors. These vectors express cytokines after intra-tumoral (IT) injection, and have therefore been used for inducing efficient anti-tumoral responses in several tumor models. We were interested in studying whether SFV vectors could escape from tumors after IT injection and whether they could target tumors if administered systemically. We analyzed the biodistribution of an SFV vector expressing luciferase (SFV-Luc) after intravenous (IV), intraperitoneal (IP), and IT administration in immunocompetent mice. SFV-Luc systemic inoculation led to high infectivity in heart and lung, and moderate levels of infectivity in spleen, kidney, and gonads, without gender being a factor in the outcome. Tumor-specific infection, without the vector spreading to other tissues, was achieved only by IT inoculation. We also investigated the effect of SFV pre-inoculation on subsequent vector administrations. Systemic inoculation with one dose of 10(7) vp (viral particles), or two doses of 10(6) vp of SFV-LacZ given with a 20-day interval between the doses, was able to strongly inhibit luciferase expression in animals re-inoculated systemically with SFV-Luc, correlating with high sera neutralizing antibodies titers. However, IT pre-inoculation with 10(8) vp of SFV-LacZ impaired tumor re-infection only moderately, thereby indicating that tumors can be treated with several doses of SFV vectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/sj.mt.6300274DOI Listing
December 2007

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

Cancer Res 2006 Feb;66(3):1620-9

Division of Hepatology and Gene Therapy, School of Medicine, Centro de Investigación Médica Aplicada, University of Navarra, 31008 Pamplona, Spain.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-05-0877DOI Listing
February 2006

Semliki forest virus vectors engineered to express higher IL-12 levels induce efficient elimination of murine colon adenocarcinomas.

Mol Ther 2005 Jul;12(1):153-63

Division of Gene Therapy, School of Medicine, Center for Applied Medical Research, University of Navarra, Avenida Pio XII 55, 31008 Pamplona, Spain.

To evaluate the use of alphavirus vectors for tumor treatment we have constructed and compared two Semliki Forest virus (SFV) vectors expressing different levels of IL-12. SFV-IL-12 expresses both IL-12 subunits from a single subgenomic promoter, while in SFV-enhIL-12 each IL-12 subunit is expressed from an independent subgenomic promoter fused to the SFV capsid translation enhancer. This latter strategy provided an eightfold increase of IL-12 expression. We chose the poorly immunogenic MC38 colon adenocarcinoma model to evaluate the therapeutic potential of SFV vectors. A single intratumoral injection of 10(8) viral particles of SFV-IL-12 or SFV-enh-IL-12 induced>or=80% complete tumor regressions with long-term tumor-free survival. However, lower doses of SFV-enhIL-12 were more efficient than SFV-IL-12 in inducing antitumoral responses, indicating a positive correlation between the IL-12 expression level and the therapeutic effect. Moreover, repeated intratumoral injections of suboptimal doses of SFV-enhIL-12 increased the antitumoral response. In all cases SFV vectors were more efficient at eliminating tumors than a first-generation adenovirus vector expressing IL-12. In addition, the antitumoral effect of SFV vectors was only moderately affected by preimmunization of animals with high doses of SFV vectors. This antitumoral effect was produced, at least partially, by a potent CTL-mediated immune response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2005.02.011DOI Listing
July 2005