Pañeda, A. (Astrid)

Search Results

Now showing 1 - 5 of 5
  • Thumbnail Image
    Treatment of chronic viral hepatitis in woodchucks by prolonged intrahepatic expression of interleukin-12
    (American Society for Microbiology, 2009) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Benito-Boilos, A. (Alberto); Rodriguez-Madoz, J.R. (Juan Roberto); Pañeda, A. (Astrid); Kochanek, S. (Stefan); Otano, I. (Itziar); Astudillo, A. (Aurora); Menne, S. (Stephan); Ochoa, L. (Laura); Crettaz, J. (Julien); Aurrekoetxea, I. (Igor); Prieto, J. (Jesús); Kreppel, F. (Florian); Ruiz, J. (Juan)
    Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.
  • Thumbnail Image
    Development of a liver-specific Tet-on inducible system for AAV vectors and its application in the treatment of liver cancer
    (Nature, 2011) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Gil-Fariña, I. (Irene); Baldim, V. (Victor); Pañeda, A. (Astrid); Otano, I. (Itziar); Tenenbaum, L. (Lilianne); Beattie, S.G. (Stuart G.); Prieto, J. (Jesús); Vanrell, L. (Lucía); Chtarto, A. (Abdelwahed); Scala, M. (Marianna) Di; Blanco-Fernández, L. (Laura)
    Recombinant adeno-associated virus (rAAV) are effective gene delivery vehicles that can mediate long-lasting transgene expression. However, tight regulation and tissue-specific transgene expression is required for certain therapeutic applications. For regulatable expression from the liver we designed a hepatospecific bidirectional and autoregulatory tetracycline (Tet)-On system (Tet(bidir)Alb) flanked by AAV inverted terminal repeats (ITRs). We characterized the inducible hepatospecific system in comparison with an inducible ubiquitous expression system (Tet(bidir)CMV) using luciferase (luc). Although the ubiquitous system led to luc expression throughout the mouse, luc expression derived from the hepatospecific system was restricted to the liver. Interestingly, the induction rate of the Tet(bidir)Alb was significantly higher than that of Tet(bidir)CMV, whereas leakage of Tet(bidir)Alb was significantly lower. To evaluate the therapeutic potential of this vector, an AAV-Tet(bidir)-Alb-expressing interleukin-12 (IL-12) was tested in a murine model for hepatic colorectal metastasis. The vector induced dose-dependent levels of IL-12 and interferon-γ (IFN-γ), showing no significant toxicity. AAV-Tet(bidir)-Alb-IL-12 was highly efficient in preventing establishment of metastasis in the liver and induced an efficient T-cell memory response to tumor cells. Thus, we have demonstrated persistent, and inducible in vivo expression of a gene from a liver-specific Tet-On inducible construct delivered via an AAV vector and proved to be an efficient tool for treating liver cancer.
  • Thumbnail Image
    Adeno-associated virus liver transduction efficiency measured by in vivo [18F]FHBG positron emission tomography imaging in rodents and nonhuman primates
    (Mary Ann Liebert, 2011) González-Aseguinolaza, G. (Gloria); Benito-Boilos, A. (Alberto); Snapper, J. (Jolanda); Collantes, M. (María); Lanciego, J.L. (José Luis); Peñuelas-Sanchez, I. (Ivan); Timmermans, E. (Eric); Pañeda, A. (Astrid); Rodriguez-Pena, M.S. (María Sol); Otano, I. (Itziar); Guembe, L. (L.); Beattie, S.G. (Stuart G.); Prieto, J. (Jesús)
    Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.
  • Thumbnail Image
    Intrahepatic injection of recombinant adeno-associated virus serotype 2 overcomes gender-related differences in liver transduction
    (Mary Ann Liebert, 2006) Berraondo, P. (Pedro); González-Aseguinolaza, G. (Gloria); Pañeda, A. (Astrid); Troconiz, I.F. (Iñaki F.); Ochoa, L. (Laura); Crettaz, J. (Julien); Prieto, J. (Jesús)
    The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was associated with the fastest and strongest onset of luciferase expression. Moreover, no gender differences in liver transduction were observed and luciferase expression was confined to the site of injection. Thus, direct intrahepatic injection of rAAV offers specific advantages, which support the potential of this route of administration for future clinical applications.
  • PET imaging of thymidine kinase gene expression in the liver of non-human primates following systemic delivery of an adenoviral vector
    (Nature Publishing Group, 2008-06-30) Collantes, M. (María); Peñuelas-Sanchez, I. (Ivan); Pañeda, A. (Astrid); Quincoces, G. (Gemma); Azpilicueta, A. (Arantza); Hervas-Stubbs, S. (Sandra); Melero, I. (Ignacio); Prieto, J. (Jesús); Fontanellas-Romá, A. (Antonio); Sampedro, A. (Ana); Mauleon, I. (Itsaso)
    Non-invasive in vivo imaging of transgene expression is currently providing very important means to optimize gene therapy regimes. Results in non-human primates are considered the most predictive models for the outcome in patients. In this study, we have documented that tumour and primary cell lines from human and non-human primates are comparably gene-transduced in vitro by serotype 5 adenovirus expressing HSV1-thymidine kinase. Transgene expression can be quantified in human and monkey cultured cells by positron emission tomography (PET) imaging when transduced cells are incubated with a fluoride-18 labelled penciclovir analogue. In our hands, PET images of cell cultures estimate the number of transduced cells rather than intensity of transgene expression once a threshold of TK per cell is reached. Interestingly, in vivo systemic administration of a clinical grade recombinant adenovirus expressing TK into macaques gives rise to an intense retention of the radiotracer in the liver parenchyma, providing an experimental system to visualize transgene expression that ought to be similar in human and macaques. Such imaging methodology might contribute to improve strategies based on adenoviral vectors.