Nuestros investigadores

Gloria González Aseguinolaza

Departamento
Terapia Génica y Regulación de la Expresión Génica
Centro de Investigación Médica Aplicada. Fundación para la Investigación Médica Aplicada
Líneas de investigación
Terapia génica, Enfermedades hepáticas, Vectores virales

Publicaciones científicas más recientes (desde 2010)

Autores: Godoy, C. ; Tabernero, D., (Autor de correspondencia); Sopena, S.; et al.
Revista: WORLD JOURNAL OF GASTROENTEROLOGY
ISSN 1007-9327  Vol. 25  Nº 13  2019  págs. 1566 - 1579
BACKGROUND Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses. AIM To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients. METHODS Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity. RESULTS CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype. CONCLUSION The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.
Autores: Kwikkers, K. L.; Zabaleta, Nerea; et al.
Revista: BLOOD ADVANCES
ISSN 2473-9529  Vol. 3  Nº 17  2019  págs. 2632 - 2641
Adeno-associated virus (AAV)-based liver gene therapy has been shown to be clinically successful. However, the presence of circulating neutralizing antibodies (NABs) against AAV vector capsids remains a major challenge as it may prevent successful transduction of the target cells. Therefore, there is a need to develop strategies that would enable AAV-mediated gene delivery to patients with preexisting anti-AAV NABs. In the current study, the feasibility of using an immunoadsorption (IA) procedure for repeated, liver-targeted gene delivery in nonhuman primates was explored. The animals were administered IV with recombinant AAV5 (rAAV5) carrying the reporter gene human secreted embryonic alkaline phosphatase (hSEAP). Seven weeks after the first rAAV treatment, all of the animals were readministered with rAAV5 carrying the therapeutic hemophilia B gene human factor IX (hFIX). Half of the animals administered with rAAV5-hSEAP underwent IA prior to the second rAAV5 exposure. The transduction efficacies of rAAV5-hSEAP and rAAV5-hFIX were assessed by measuring the levels of hSEAP and hFIX proteins. Although no hFIX was detected after rAAV5-hFIX readministration without prior IA, all animals submitted to IA showed therapeutic levels of hFIX expression, and a threshold of anti-AAV5 NAB levels compatible with successful readministration was demonstrated. In summary, our data demonstrate that the use of a clinically applicable IA procedure enables successful readministration of an rAAV5-based gene transfer in a clinically relevant animal model. Finally, the analysis of anti-AAV NAB levels in human subjects submitted to IA confirmed the safety and efficacy of the procedure to reduce anti-AAV NABs. Furthermore, clinical translation was assessed using an immunoglobulin G assay as surrogate.
Autores:  et al.
Revista: HEPATOLOGY
ISSN 0270-9139  Vol. 70  Nº 1  2019  págs. 108 - 126
Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8¿ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal¿binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (delta57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper.
Autores: Camps, G.; Usai, C.; et al.
Revista: HEPATOLOGY
ISSN 0270-9139  Vol. 70  2019  págs. 640A - 640A
Autores: Weber, N. D.; Odriozola, L.; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 203 - 203
Autores: Ganan, I. R.; Salas, D.; Rodriguez, E.; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A115 - A115
Autores: Benichou, B.; Collantes M; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 197 - 198
Autores:  et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A2 - A2
Autores: Zabaleta, Nerea; Torella, L.; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 27  Nº 4  2019  págs. 368 - 368
Autores: Zabaleta, Nerea; Gil-Farina, I.; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 30  Nº 11  2019  págs. A219 - A220
Autores: Suarez-Amaran, L.; Usai, C.; Di Scala, M.; et al.
Revista: JOURNAL OF HEPATOLOGY
ISSN 1600-0641  Vol. 69  Nº 1  2018  págs. 262 - 264
Autores: Zabaleta, Nerea; Barberia, M.; Martin-Higueras, C. ; et al.
Revista: NATURE COMMUNICATIONS
ISSN 2041-1723  Vol. 9  2018  págs. 5454
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.
Autores: Rodríguez-García, E. ; Ríus-Rocabert, S. ; et al.
Revista: IMMUNOHORIZONS
ISSN 2573-7732  Vol. 2  Nº 11  2018  págs. 363 - 376
The innate immune system provides a primary line of defense against pathogens. Stimulator of IFN genes (STING), encoded by the TMEM173 gene, is a critical protein involved in IFN-ß induction in response to infection by different pathogens. In this study, we describe the expression of three different alternative-spliced human (h) TMEM173 mRNAs producing STING truncated isoforms 1, 2, and 3 in addition to the full-length wild-type (wt) hSTING. All of the truncated isoforms lack exon 7 and share the N-terminal transmembrane region with wt hSTING. Overexpression of the three STING truncated isoforms failed to induce IFN-ß, and they acted as selective pathway inhibitors of wt hSTING even in combination with upstream inducer cyclic-di-GMP-AMP synthase. Truncated isoforms alter the stability of wt hSTING, reducing protein t1/2 to some extent by the induction of proteasome-dependent degradation. Knocking down expression of truncated isoforms increased production of IFN-ß by THP1 monocytes in response to intracellular cytosolic DNA or HSV-1 infection. At early stages of infection, viruses like HSV-1 or vesicular stomatitis virus reduced the ratio of full-length wt hSTING/truncated STING isoforms, suggesting the skewing of alternative splicing of STING toward truncated forms as a tactic to evade antiviral responses. Finally, in silico analysis revealed that the human intron¿exon gene architecture of TMEM173 (splice sites included) is preserved in other mammal species, predominantly primates, stressing the relevance of alternative splicing in regulating STING antiviral biology.
Autores: Torralba, D.; Baixauli, F. ; Villarroya-Beltri, C.; et al.
Revista: NATURE COMMUNICATIONS
ISSN 2041-1723  Vol. 9  2018 
Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate antipathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.
Autores:  et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 68  Nº 5  2018  págs. 1088 - 1090
Autores: Maestro, S.; Cortese, M. F.; Gonzalez, C. ; et al.
Revista: HEPATOLOGY
ISSN 0270-9139  Vol. 68  Nº Supl. 1  2018  págs. 239A - 240A
Autores: Zabaleta, Nerea; Barberia, M.; Martin-Higueras, C. ; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 26  Nº 5  2018  págs. 384 - 385
Autores: González-Aseguinolaza, Gloria; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 26  Nº 5  2018  págs. 388 - 388
Autores: González-Aseguinolaza, Gloria; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 26  Nº 5  2018  págs. 249 - 250
Autores:  et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 68  Nº Supl. 1  2018  págs. S83 - S83
Autores: Zabaleta, Nerea; Torella, L. ; Rodríguez, Saray; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 29  Nº 12  2018  págs. A108 - A108
Autores: Godoy, C.; Ni, Y.; Hommel, Mirja; et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 67  Nº 4  2017  págs. 669 - 679
BACKGROUND & AIMS: Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. METHODS: HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. RESULTS: AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. CONCLUSION: The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments.
Autores: Martinez-Florensa, M.; Consuegra-Fernandez, M.; Aranda, F.; et al.
Revista: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
ISSN 0066-4804  Vol. 61  Nº 1  2017  págs. e01391-16
Sepsis still constitutes an unmet clinical need, which could benefit from novel adjunctive strategies to conventional antibiotic therapy. The soluble form of the scavenger-like human CD6 lymphocyte receptor (shCD6) binds to key pathogenic components from Gram-positive and-negative bacteria and shows time-and dose-dependent efficacy in mouse models of monobacterial sepsis. The objective of the present work was to demonstrate the effectiveness of infusing mouse and human sCD6 by different systemic routes, either alone or as adjunctive therapy to gold standard antibiotics, in a lethal model of polymicrobial sepsis. To this end, C57BL/6 mice undergoing high-grade septic shock induced by cecal ligation and puncture (CLP; >= 90% lethality) were infused via the intraperitoneal (i.p.) or intravenous (i.v.) route with shCD6 at different doses and time points, either alone or in combination with imipenem/cilastatin (I/C) at a dose of 33 mg/kg of body weight every 8 h. Significantly reduced mortality and proinflammatory cytokine levels were observed by i.p. infusion of a single shCD6 dose (1.25 mg/kg) 1 h pre-or post-CLP. When using the i.v. route, mice survival was significantly extended by starting shCD6 infusion at later time points post-CLP (up to 6 h after CLP). Significant adjunctive effects on mouse survival were observed by i.p. or i.v. infusion of shCD6 in combination with i.p. I/C post-CLP. Similar results were obtained in mice expressing high sustained levels (5 to 10 mu g/ml) of mouse sCD6 in serum by means of transduction with hepatotropic adeno-associated virus (AAV). Taken together, the data support the conserved antibacterial effects of human and mouse sCD6 and their use as adjunctive therapy in experimental models of complex and severe polymicrobial sepsis.
Autores: Pignataro, J. D.; Sucunza, D.; Vanrell, L.; et al.
Revista: FRONTIERS IN NEUROANATOMY
ISSN 1662-5129  Vol. 11  2017  págs. 2
Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor ß2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.
Autores: Gil-Farina, I.; Dietrich, K.; Ceiler, J.; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 28  Nº 12  2017  págs. A13
Autores: Rodriguez-Madoz, Juan Roberto; Garcia Barvo, M.; Zapata, Natalia María; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 25  Nº 5 Supl.1  2017  págs. 116
Autores: Godoy, C. ; Ni, Y. ; Hommel, Mirja; et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 66  Nº 1  2017  págs. S483 - S483
Autores: Gil Farina, I. ; Dietrich, K. ; Ceiler, J.; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 25  Nº 5, Supl.1  2017  págs. 1 - 1
Autores: Kwikkers, K. ; Majowicz, A. ; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 25  Nº 5 - Supl.1  2017  págs. 183 - 183
Autores: Zabaleta, Nerea; Barberia, M.; Zapata, Natalia María; et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 25  Nº 5 Supl.1  2017  págs. 79 - 80
Autores:  et al.
Revista: MOLECULAR THERAPY
ISSN 1525-0016  Vol. 25  Nº 5 - Supl.1  2017  págs. 188 - 188
Autores:  et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 66  Nº 1  2017  págs. S5 - S6
Autores: Zabaleta, Nerea; Rodriguez, S.; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 28  Nº 12  2017  págs. A11 - A12
Autores:  et al.
Revista: JOURNAL OF HEPATOLOGY
ISSN 1600-0641  Vol. 64  Nº 2  2016  págs. 419-26
Our data demonstrate that AAV8-AAT-ATP7B-mediated gene therapy provides long-term correction of copper metabolism in a clinically relevant animal model of WD providing support for future translational studies.
Autores: D'Avola, Delia; Sangro, Bruno; Grossios, N.; et al.
Revista: JOURNAL OF HEPATOLOGY
ISSN 0168-8278  Vol. 65  Nº 4   2016  págs. 776 - 783
BACKGROUND & AIMS: Acute intermittent porphyria (AIP) results from porphobilinogen deaminase (PBGD) haploinsufficiency, which leads to hepatic over-production of the neurotoxic heme precursors porphobilinogen (PBG) and delta-aminolevulinic acid (ALA) and the occurrence of neurovisceral attacks. Severe AIP is a devastating disease that can only be corrected by liver transplantation. Gene therapy represents a promising curative option. The objective of this study was to investigate the safety of a recombinant adeno-associated vector expressing PBGD (rAAV2/5-PBGD) administered for the first time in humans for the treatment of AIP. METHODS: In this phase I, open label, dose-escalation, multicenter clinical trial, four cohorts of 2 patients each received a single intravenous injection of the vector ranging from 5×10(11) to 1.8×10(13) genome copies/kg. Adverse events and changes in urinary PBG and ALA and in the clinical course of the disease were periodically evaluated prior and after treatment. Viral shedding, immune response against the vector and vector persistence in the liver were investigated. RESULTS: Treatment was safe in all cases. All patients developed anti-AAV5 neutralizing antibodies but no cellular responses against AAV5 or PBGD were observed. There was a trend towards a reduction of hospitalizations and heme treatments, although ALA and PBG levels remained unchanged. Vector genomes and transgene expression could be detected in the liver one year after therapy. CONCLUSIONS: rAAV2/5-PBGD administration is safe but AIP metabolic correction was not achieved at the doses tested in this trial. Notwithstanding, the treatment had a positive impact in clinical outcomes in most patients. LAY SUMMARY: Studies in an acute intermittent porphyria (AIP) animal model have shown that gene delivery of PBGD to hepatocytes using an adeno-associated virus vector (rAAV2/5-PBG) prevent mice from suffering porphyria acute attacks. In this phase I, open label, dose-escalation, multicenter clinical trial we show that the administration of rAAV2/5-PBGD to patients with severe AIP is safe but metabolic correction was not achieved at the doses tested; the treatment, however, had a positive but heterogeneous impact on clinical outcomes among treated patients and 2 out of 8 patients have stopped hematin treatment. CLINICAL TRIAL NUMBER: The observational phase was registered at Clinicaltrial.gov as NCT 02076763. The interventional phase study was registered at EudraCT as n° 2011-005590-23 and at Clinicaltrial.gov as NCT02082860.
Autores: Paredes, V.; et al.
Revista: ONCOIMMUNOLOGY
ISSN 2162-402X  Vol. 5  Nº 8  2016  págs. e1196309
Scavenger receptor class B type I (SR-B1) binds pathogen-associated molecular patterns participating in the regulation of the inflammatory reaction but there is no information regarding potential interactions between SR-B1 and the interferon system. Herein, we report that SR-B1 ligands strongly regulate the transcriptional response to interferon ¿ (IFN¿) and enhance its antiviral and antitumor activity. This effect was mediated by the activation of TLR2 and TLR4 as it was annulled by the addition of anti-TLR2 or anti-TLR4 blocking antibodies. In vivo, we maximized the antitumor activity of IFN¿ co-expressing in the liver a SR-B1 ligand and IFN¿ by adeno-associated viruses. This gene therapy strategy eradicated liver metastases from colon cancer with reduced toxicity. On the other hand, genetic and pharmacological inhibition of SR-B1 blocks the clathrin-dependent interferon receptor recycling pathway with a concomitant reduction in IFN¿ signaling and bioactivity. This effect can be applied to enhance cancer immunotherapy with oncolytic viruses. Indeed, SR-B1 antagonists facilitate replication of oncolytic viruses amplifying their tumoricidal potential. In conclusion, SR-B1 agonists behave as IFN¿ enhancers while SR-B1 inhibitors dampen IFN¿ activity. These results demonstrate that SR-B1 is a suitable pharmacology target to enhance cancer immunotherapy based on IFN¿ and oncolytic viruses.
Autores: Salido, E.; Blasi, M.; Merino, J; et al.
Revista: JOURNAL OF IMMUNOLOGY
ISSN 0022-1767  Vol. 197  Nº 6  2016  págs. 2145 - 2156
The etiopathogenesis of autoimmune hepatitis (AIH) remains poorly understood. In this study, we sought to develop an animal model of human AIH to gain insight into the immunological mechanisms driving this condition. C57BL/6 mice were i.v. injected with adeno-associated viral vectors encodingmurine IL-12 or luciferase under the control of a liver-specific promoter. Organ histology, response to immunosuppressive therapy, and biochemical and immunological parameters, including Ag-specific humoral and cellular response, were analyzed. Mechanistic studies were carried out using genetically modified mice and depletion of lymphocyte subpopulations. Adeno-associated virus IL-12-treated mice developed histological, biochemical, and immunological changes resembling type 1 AIH, including marked and persistent liver mononuclear cell infiltration, hepatic fibrosis, hypergammaglobulinemia, antinuclear and anti-smooth muscle actin Abs, and disease remission with immunosuppressive drugs. Interestingly, transgenic IL-12 was short-lived, but endogenous IL-12 expression was induced, and both IL-12 and IFN-g remained elevated during the entire study period. IFN-g was identified as an essential mediator of liver damage, and CD4 and CD8 T cells but not NK, NKT, or B cells were essential executors of hepatic injury. Furthermore, both MHC class I and MHC class II expression was upregulated at the hepatocellular membrane, and induction of autoreactive liver-specific T cells was detected. Remarkably, although immunoregulatory mechanisms were activated, they only partially mitigated liver damage. Thus, low and transient expression of transgenic IL-12 in hepatocytes causes loss of tolerance to hepatocellular Ags, leading to chronic hepatitis resembling human AIH type 1. This model provides a practical tool to explore AIH pathogenesis and novel therapies.
Autores: Zapata, Natalia María; Rodríguez, Saray; Salido, E.; et al.
Revista: STEM CELL RESEARCH
ISSN 1873-5061  Vol. 16  Nº 1  2016  págs. 116 - 119
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
Autores: Carte, Beatriz; Prieto, Jesús María; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 11  Nº 3  2016  págs. e0152031
Development of reporter systems for in vivo examination of IFN-ß induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-ß induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.
Autores: Amaran, L. S. ; Ni, Y. ; Urban, S.; et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 64  Nº 2, supl.  2016  págs. S385 - S386
Autores: Zabaleta, Nerea; Kappel, C.; et al.
Revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN 0168-8278  Vol. 64  Nº 2, supl.  2016  págs. S303
Background and Aims: Recombinant AAV transduction of postmitotic cells, such as hepatocytes, leads mainly to formation of episomal monomeric and concatameric circles or linear episomes, which assimilate into chromatin with a typical nucleosomal pattern. However in proliferating cells, nonintegrated viral genomes are instable and are lost soon upon proliferation of transduced cells. Integration of AAV vectors can occur in presence of a transcriptionally active or damaged genome. Therefore, the objective of this project was to analyze the integration of AAV vectors in presence or absence of genotoxic damage. Methods: To perform this study we have compare the integration profile of AAV genomes in the liver of mice after the administration into adult mice of an AAV vector expressing PBGD (the therapeutic vector for acute intermittent porphyria) or an AAV vector expressing Thymidine Kinase (TK) followed by Gancyclovir administration, which causes genotoxicity. Integration analysis was performed by the method called LAM-PCR. Results: Here we showed that when hepatocyte proliferation is induced by a genotoxic injury, AAV genomes are efficiently integrated. We found that while the percentage of AAV genome integration in the liver of AAV-PBGD treated mice is lower than 1% and 99% of the viral genomes remain episomal, in the case of the animals receiving AAV-TK in combination with Gancyclovir the situation is completely the opposite, with a percentage of integration higher than 99%. We analyzed the relative sequence count (which might act as marker for clonal outgrowth) of each exact mappable integration sites (IS) in relation to all sequences resembling concatemeric or proviral structures and we detected 13 IS with a high relative sequence count of >10%. The genes which is located nearby the IS with a high relative sequence count were determined by ingenuity pathway analyzing tools and found that they regulate different cellular functions. Conclusions: AAV genomes stay mainly forming episomal structures in absence of injury, but the 99% of these genomes integrate in the cellular DNA in presence of genotoxic damage. We are also able to determine the exact IS of the AAV genome.
Autores: Zabaleta, N.; Barberia, M.; Zapata, Natalia María; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 27  Nº 11  2016  págs. A130
Autores: Zabaleta, Nerea; Barberia, M.; Zapata, Natalia María; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 27  Nº 11  2016  págs. A139 - A140
Autores: Aldabe, R; González-Aseguinolaza, Gloria;
Revista: PATHOGENS
ISSN 2076-0817  Vol. 4  Nº 1  2015  págs. 46 - 65
Hepatitis delta virus (HDV) is a defective RNA virus that has an absolute requirement for a virus belonging to the hepadnaviridae family like hepatitis B virus (HBV) for its replication and formation of new virions. HDV infection is usually associated with a worsening of HBV-induced liver pathogenesis, which leads to more frequent cirrhosis, increased risk of hepatocellular carcinoma (HCC), and fulminant hepatitis. Importantly, no selective therapies are available for HDV infection. The mainstay of treatment for HDV infection is pegylated interferon alpha; however, response rates to this therapy are poor. A better knowledge of HDV-host cell interaction will help with the identification of novel therapeutic targets, which are urgently needed. Animal models like hepadnavirus-infected chimpanzees or the eastern woodchuck have been of great value for the characterization of HDV chronic infection. Recently, more practical animal models in which to perform a deeper study of host virus interactions and to evaluate new therapeutic strategies have been developed. Therefore, the main focus of this review is to discuss the current knowledge about HDV host interactions obtained from cell culture and animal models.
Autores: Berraondo, Pedro; Korolowicz, K. ; Thampi, L. M. ; et al.
Revista: JOURNAL OF HEPATOLOGY
ISSN 1600-0641  Vol. 63  Nº 2  2015  págs. 329-336
BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-¿ therapy is associated with considerable toxicity. Previously, we showed that linking IFN¿ to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFN¿. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFN¿ fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFN¿ (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.
Autores: Bravo-Pérez, C.; Carte, Beatriz; et al.
Revista: MOLECULAR CANCER
ISSN 1476-4598  Vol. 14  2015  págs. 210
Background: The limited efficacy of current treatments against pancreatic cancer has prompted the search of new alternatives such as virotherapy. Activation of the immune response against cancer cells is emerging as one of the main mechanisms of action of oncolytic viruses (OV). Direct oncolysis releases tumor antigens, and viral replication within the tumor microenvironment is a potent danger signal. Arming OV with immunostimulatory transgenes further enhances their therapeutic effect. However, standard virotherapy protocols do not take full advantage of OV as cancer vaccines because repeated viral administrations may polarize immune responses against strong viral antigens, and the rapid onset of neutralizing antibodies limits the efficacy of redosing. An alternative paradigm based on sequential combination of antigenically distinct OV has been recently proposed. Methods: We have developed a protocol consisting of sequential intratumor administrations of new Adenovirus (Ad) and Newcastle Disease Virus (NDV)-based OV encoding the immunostimulatory cytokine oncostatin M (OSM). Transgene expression, toxicity and antitumor effect were evaluated using an aggressive orthotopic pancreatic cancer model in Syrian hamsters, which are sensitive to OSM and permissive for replication of both OVs. Results: NDV-OSM was more cytolytic, whereas Ad-OSM caused higher OSM expression in vivo. Both viruses achieved only a marginal antitumor effect in monotherapy. In addition, strong secretion of OSM in serum limited the maximal tolerated dose of Ad-OSM. In contrast, moderate doses of Ad-OSM followed one week later by NDV-OSM were safe, showed a significant antitumor effect and stimulated immune responses against cancer cells. Similar efficacy was observed when the order of virus administrations was reversed. Conclusion: Sequential administration of oncolytic Ad and NDV encoding OSM is a promising approach against pancreatic cancer.
Autores:  et al.
Revista: JOURNAL OF INNATE IMMUNITY
ISSN 1662-811X  Vol. 7  Nº 5  2015  págs. 466 - 481
RIG-I-like receptors (RLRs) are cellular sensor proteins that detect certain RNA species produced during viral infections. RLRs activate a signaling cascade that results in the production of IFN-ß as well as several other cytokines with antiviral and proinflammatory activities. We explored the potential of different constructs based on RLRs to induce the IFN-ß pathway and create an antiviral state in type I IFN-unresponsive models. A chimeric construct composed of RIG-I 2CARD and the first 200 amino acids of MAVS (2CARD-MAVS200) showed an enhanced ability to induce IFN-ß when compared to other stimulatory constructs. Furthermore, this human chimeric construct showed a superior ability to activate IFN-ß expression in cells from various species. This construct was found to overcome the restrictions of blocking IFN-ß induction or signaling by a number of viral IFN-antagonist proteins. Additionally, the antiviral activity of this chimera was demonstrated in influenza virus and HBV infection mouse models using adeno-associated virus (AAV) vectors as a delivery vehicle. We propose that AAV vectors expressing 2CARD-MAVS200 chimeric protein can reconstitute IFN-ß induction and recover a partial antiviral state in different models that do not respond to recombinant IFN-ß treatment.
Autores: Guidotti, L. G.; Inverso, D.; Sironi, L.; et al.
Revista: CELL
ISSN 0092-8674  Vol. 161  Nº 3  2015  págs. 486 - 500
Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.
Autores: Moreno-Cugnon L; Esparza-Baquer A; Larruskain A; et al.
Revista: MOLECULAR IMMUNOLOGY
ISSN 0161-5890  Vol. 63  Nº 2  2015  págs. 505-512
The major histocompatibility complex (MHC)-containing genes are among the most polymorphic in vertebrates. MHC genes code for proteins that are critical in the immune system response. In this study, the polymorphism of the second exon of the MHC class II DRB gene was characterized in the Eastern woodchuck (Marmota monax). Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) represent the best available animal model for the study of chronic hepatitis B infection in humans. In the genotyped animals we found fifteen alleles, which were expressed in two independent loci and that were named DRB1A and DRB1B in this work. The 15 alleles investigated showed an elevated divergence. A significant excess of non-synonymous substitutions was detected, which could indicate that a historical positive selection is acting in the woodchuck DRB1 genes. This hypothesis was confirmed in our study by the high variability in or near the antigen binding sites (ABS) and by the results obtained in sequence variability analyses. This analysis identified the presence of a microsatellite sequence that is located at the start of the second intron, which could further allow the development of a fast and cheap semiautomatic sequencing method.
Autores: Sánchez-Bayona, Rodrigo; Alignani, D. ; et al.
Revista: HAEMATOLOGICA
ISSN 1138-0381  Vol. 100  Nº 8  2015  págs. 1014-1022
Interferon-¿ is a potent antiviral agent and a vigorous adjuvant in the induction of T-cell responses but its use is limited by hematologic toxicity. Interferon-¿ alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-¿ exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-¿ to achieve sustained high serum levels of the cytokine. The bone marrow of these animals showed diminished long-term and short-term hematopoietic stem cells, reduction of multipotent progenitor cells, and marked decrease of B cells, but significant increase in the proportion of CD8(+) and CD4(+)CD8(+) T cells. Upon adoptive transfer to RAG(-/-) mice, bone marrow cells from interferon-¿-treated animals generated CD4(+) and CD8(+) T cells while CD19(+), CD11b(+) and NK1.1(+) lineages failed to develop. These effects are associated with the transcriptional downregulation of transcription factors involved in B-cell differentiation and modulation of key factors for T-cell development. Thus, sustained interferon-¿ exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.
Autores: Del Campo AB; Carretero J; Muñoz, J. A. ; et al.
Revista: CANCER GENE THERAPY
ISSN 0929-1903  Vol. 21  Nº 8  2014  págs. 317-332
Optimal tumor cell surface expression of human leukocyte antigen (HLA) class I molecules is essential for the presentation of tumor-associated peptides to T-lymphocytes. However, a hallmark of many types of tumor is the loss or downregulation of HLA class I expression associated with ineffective tumor antigen presentation to T cells. Frequently, HLA loss can be caused by structural alterations in genes coding for HLA class I complex, including the light chain of the complex, ß2-microglobulin (ß2m). Its best-characterized function is to interact with HLA heavy chain and stabilize the complex leading to a formation of antigen-binding cleft recognized by T-cell receptor on CD8+ T cells. Our previous study demonstrated that alterations in the ß2m gene are frequently associated with cancer immune escape leading to metastatic progression and resistance to immunotherapy. These types of defects require genetic transfer strategies to recover normal expression of HLA genes. Here we characterize a replication-deficient adenoviral vector carrying human ß2m gene, which is efficient in recovering proper tumor cell surface HLA class I expression in ß2m-negative tumor cells without compromising the antigen presentation machinery. Tumor cells transduced with ß2m induced strong activation of T cells in a peptide-specific HLA-restricted manner. Gene therapy using recombinant adenoviral vectors encoding HLA genes increases tumor antigen presentation and represents a powerful tool for modulation of tumor cell immunogenicity by restoration of missing or altered HLA genes. It should be considered as part of cancer treatment in combination with immunotherapy.
Autores: Sandra Hervas-Stubbs; González-Aseguinolaza, Gloria; et al.
Revista: LIVER INTERNATIONAL
ISSN 1478-3223  Vol. 35  Nº 4  2014  págs. 1274 - 1289
BACKGROUND & AIMS: Adenoviral (Ad) vectors are currently one of the most efficient tools for in vivo gene transfer to the liver. However, anti-Ad immune responses limit the safety and efficacy of these vectors. The initial inflammatory reaction is a concern in terms of toxicity, and it favours the development of cellular and humoral responses leading to short transgene persistence and inefficient vector re-administrations. Therefore, safe and simple ways to interfere with these processes are needed. Study ways to deplete specific immune cell populations and their impact on liver-directed gene transfer. METHODS: First-generation Ad vectors encoding reporter genes (luciferase or ß-galactosidase) were injected intravenously into Balb/c mice. Kupffer cells and splenic macrophages were depleted by intravenous administration of clodronate liposomes. B lymphocytes, CD4(+) , CD8(+) T lymphocytes or NK cells were depleted by intraperitoneal injection of anti-M plus anti-D, anti-CD4, anti-CD8 or anti-asialo-GM1 antibodies respectively. Long-term evolution of luciferase expression in the liver was monitored by bioluminescence imaging. RESULTS: The anti-CD4 monoclonal antibody impaired cellular and humoral immune responses, leading to efficient vector re-administration. Clodronate liposomes had no impact on humoral responses but caused a 100-1000 fold increase in liver transduction, stabilized transgene expression, reduced the concentration of inflammatory cytokines, and inhibited lymphocyte activation. CONCLUSIONS: Transient CD4(+) T-cell depletion using antibodies is a clinically feasible procedure that allows efficient Ad redosing. Systemic administration of clodronate liposomes may further increase the safety and efficacy of vectors.
Autores: Del Campo AB; Kyte JA; Carretero J; et al.
Revista: INTERNATIONAL JOURNAL OF CANCER
ISSN 0020-7136  Vol. 134  Nº 1  2014  págs. 102-113
Cancer cells escape T-cell-mediated destruction by losing human leukocyte antigen (HLA) class I expression via various mechanisms, including loss of beta2-microglobulin (ß2m). Our study illustrates the immune escape of HLA class I-negative tumor cells and chronological sequence of appearance of tumor ß2m gene mutation in successive lesions obtained from a patient with metastatic melanoma. We observed a gradual decrease in HLA expression in consecutive lesions with few HLA-negative nodules in the primary tumor and the emergence of a totally negative lesion at later stages of the disease. We detected loss of ß2m in ß2m-negative nests of the primary tumor caused by a combination of two alterations: (i) a mutation (G to T substitution) in codon 67 in exon 2 of ß2m gene, producing a stop codon and (ii) loss of the second gene copy by loss of heterozygosity (LOH) in chromosome 15. The same ß2m mutation was found in a homogeneously ß2m-negative metastasis 10 months later and in a cell line established from a biopsy of a postvaccination lymph node. Microsatellite analysis revealed the presence of LOH in chromosomes 6 and 15 in tumor samples, showing an accumulation of chromosomal loss at specific short tandem repeats in successive metastases during disease progression. HLA loss correlated with decreased tumor CD8+ T-cell infiltration. Early incidence of ß2m defects can cause an immune selection and expansion of highly aggressive melanoma clones with irreversible genetic defects causing total loss of HLA class I expression and should be taken into consideration as a therapeutic target in the development of cancer immunotherapy protocols.
Autores: Viana-da-Silva, S.; González-Aseguinolaza, Gloria; Mulle, C.; et al.
Revista: EMBO MOLECULAR MEDICINE
ISSN 1757-4676  Vol. 6  Nº 10  2014  págs. 1246 - 1262
Insulin-like growth factor 2 (IGF2) was recently found to play a critical role in memory consolidation in rats and mice, and hippocampal or systemic administration of recombinant IGF2 enhances memory. Here, using a gene therapy-based approach with adeno-associated virus (AAV), we show that IGF2 overexpression in the hippocampus of aged wild-type mice enhances memory and promotes dendritic spine formation. Furthermore, we report that IGF2 expression decreases in the hippocampus of patients with Alzheimer's disease, and this leads us to hypothesize that increased IGF2 levels may be beneficial for treating the disease. Thus, we used the AAV system to deliver IGF2 or IGF1 into the hippocampus of the APP mouse model Tg2576 and demonstrate that IGF2 and insulin-like growth factor 1 (IGF1) rescue behavioural deficits, promote dendritic spine formation and restore normal hippocampal excitatory synaptic transmission. The brains of Tg2576 mice that overexpress IGF2 but not IGF1 also show a significant reduction in amyloid levels. This reduction probably occurs through an interaction with the IGF2 receptor (IGF2R). Hence, IGF2 and, to a lesser extent, IGF1 may be effective treatments for Alzheimer's disease.
Autores: Sack, B. K. ; Miller, J. L. ; Vaughan, A. M. ; et al.
Revista: INFECTION AND IMMUNITY
ISSN 0019-9567  Vol. 82  Nº 2  2014  págs. 808-817
Evidence from clinical trials of malaria vaccine candidates suggests that both cell-mediated and humoral immunity to pre-erythrocytic parasite stages can provide protection against infection. Novel pre-erythrocytic antibody (Ab) targets could be key to improving vaccine formulations, which are currently based on targeting antigens such as the circumsporozoite protein (CSP). However, methods to assess the effects of sporozoite-specific Abs on pre-erythrocytic infection in vivo remain underdeveloped. Here, we combined passive transfer of monoclonal Abs (MAbs) or immune serum with a luciferase-expressing Plasmodium yoelii sporozoite challenge to assess Ab-mediated inhibition of liver infection in mice. Passive transfer of a P. yoelii CSP MAb showed inhibition of liver infection when mice were challenged with sporozoites either intravenously or by infectious mosquito bite. However, inhibition was most potent for the mosquito bite challenge, leading to a more significant reduction of liver-stage burden and even a lack of progression to blood-stage parasitemia. This suggests that Abs provide effective protection against a natural infection. Inhibition of liver infection was also achieved by passive transfer of immune serum from whole-parasite-immunized mice. Furthermore, we demonstrated that passive transfer of a MAb against P. falciparum CSP inhibited liver-stage infection in a humanized mouse/P. falciparum challenge model. Together, these models constitute unique and sensitive in vivo methods to assess serum-transferable protection against Plasmodium sporozoite challenge.
Autores: Hernández, Rubén; Sangro, Bruno; Berraondo, Pedro; et al.
Revista: EXPERT OPINION ON INVESTIGATIONAL DRUGS
ISSN 1354-3784  Vol. 22  Nº 7  2013  págs. 827 - 841
INTRODUCTION: Cytokines are key mediators of the immune system and have been proposed as therapeutic agents against cancer, either as recombinant proteins, or as transgenes in gene therapy approaches. Stimulation of immune responses against cancer cells is an appealing method to treat tumors with high risk of relapse and systemic dissemination. AREAS COVERED: We provide a critical overview of clinical trials involving the use of cytokines for the treatment of liver, colon and pancreatic cancers. Special attention has been paid to advances in the field of gene therapy and oncolytic viruses. The potential of new developments still in a pre-clinical stage is also discussed. We have revised public sources of information (PubMed, US National Institutes of Health clinical trials database) up to January 2013. EXPERT OPINION: The complexity of the immune system and the unfavorable pharmacokinetic properties of cytokines limit the efficacy of these molecules as single agents for the treatment of cancer. Expression from gene therapy vectors, together with new methods of targeting and stabilization, may overcome these hurdles. We believe cytokines will play a crucial role as part of combined approaches, enhancing the action of adoptive cell immunotherapy, oncolytic viruses or biological therapies.
Autores: Kaeppel, C.; Gil, Ana Gloria; D'Avola, Delia; et al.
Revista: HUMAN GENE THERAPY
ISSN 1043-0342  Vol. 24  Nº 12  2013  págs. 1007 - 1017
Acute intermittent porphyria (AIP) results from haplo-insufficient activity of porphobilinogen deaminase (PBGD) and is characterized clinically by life-threatening, acute neurovisceral attacks. To date, liver transplantation is the only curative option for AIP. The aim of the present preclinical nonhuman primate study was to determine the safety and transduction efficacy of an adeno-associated viral vector encoding PBGD (recombinant AAV serotype 5-codon-optimized human porphobilinogen deaminase, rAAV5-cohPBGD) administered intravenously as part of a safety program to start a clinical study in patients with AIP. Macaques injected with either 1 × 10(13) or 5 × 10(13) vector genomes/kg of clinical-grade rAAV5-cohPBGD were monitored by standardized clinical parameters, and vector shedding was analyzed. Liver transduction efficacy, biodistribution, vector integration, and histopathology at day 30 postvector administration were determined. There was no evidence of acute toxicity, and no adverse effects were observed. The vector achieved efficient and homogenous hepatocellular transduction, reaching transgenic PBGD expression levels equivalent to 50% of the naturally expressed PBGD mRNA. No cellular immune response was detected against the human PBGD or AAV capsid proteins. Integration site analysis in transduced liver cells revealed an almost random integration pattern supporting the good safety profile of rAAV5-cohPBGD. Together, data obtained in nonhuman primates indicate that rAAV5-cohPBGD represents a safe therapy to correct the metabolic defect present in AIP patients.
Autores: Montenegro-Miranda PS; Pañeda A; Ten Bloemendaal L; et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 12  2013  págs. e82597
Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.
Autores:  et al.
Revista: PLOS ONE
ISSN 1932-6203  Vol. 8  Nº 7  2013  págs. e67748
Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-¿ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents.
Autores: Hafalla JC; Bauza K; Friesen J; et al.
Revista: PLOS PATHOGENS
ISSN 1553-7374  Vol. 9  Nº 5  2013  págs. e1003303
CD8¿ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8¿ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20¿¿¿ and PbTRAP¿¿¿ respectively, as targets of CD8¿ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20¿¿¿ and PbTRAP¿¿¿ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP¿¿¿-specific CD8¿ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP¿¿¿-specific, but not PbS20¿¿¿-specific, CD8¿ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8¿ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8¿ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8¿ T cells will allow interrogation of the development of immune responses against malaria liver stages.
Autores: Dotor, J.; Crettaz, J.; et al.
Revista: Hepatology
ISSN 0270-9139  Vol. 56  Nº 2  2012  págs. 474 - 483
Autores: Rodríguez, D.; González-Aseguinolaza, Gloria; Rodríguez, J. R.; et al.
Revista: PLoS One
ISSN 1932-6203  Vol. 7  Nº 4  2012  págs.  e34445
With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.
Autores: Ferrage F; Dutta K; Patel JR; et al.
Revista: STRUCTURE
ISSN 0969-2126  Vol. 20  Nº 12  2012  págs. 2048-2061
RIG-I is a cytosolic sensor of viral RNA, comprised of two N-terminal CARDs followed by helicase and C-terminal regulatory domains (helicase-CTD). Viral RNA binds to the helicase-CTD and "exposes" the CARDs for downstream signaling. The role of the second CARD (CARD2) is essential as RIG-I activation requires dephosphorylation of Thr170 followed by ubiquitination at Lys172. Here, we present the solution structure and dynamics of human RIG-I CARD2. Surprisingly, we find that Thr170 is mostly buried. Parallel studies on the phosphomimetic T170E mutant suggest that the loss of function upon Thr170 phosphorylation is likely associated with changes in the CARD1-CARD2 interface that may prevent Lys172 ubiquitination and/or binding to free K63-linked polyubiquitin. We also demonstrate a strong interaction between CARD2 and the helicase-CTD, and show that mutations at the interface result in constitutive activation of RIG-I. Collectively, our data suggests a close interplay between phosphorylation, ubiquitination, and activation of human RIG-I, all mediated by CARD2.
Autores: Collantes M; Beattie, SG; et al.
Revista: Human Gene Therapy (Print)
ISSN 1043-0342  Vol. 22  Nº 8  2011  págs.  999-1009
Autores: Liu, K. H.; Ascenzi, M. A.; Bellezza, C. A.; et al.
Revista: JOURNAL OF VIROLOGY
ISSN 0022-538X  Vol. 85  Nº 10  2011  págs. 4853-4862
Autores: Larrea, Esther; et al.
Revista: Hepatology
ISSN 0270-9139  Vol. 53  Nº 6  2011  págs. 1864 - 1873
Interferon alpha (IFN alpha) is widely used for the treatment of viral hepatitis but substantial toxicity hampers its clinical use. In this work, we aimed at improving the efficacy of IFN alpha therapy by increasing the IFN alpha half-life and providing l
Autores: Benito, Alberto; et al.
Revista: JOURNAL OF MEDICAL VIROLOGY
ISSN 0146-6615  Vol. 83  Nº 7  2011  págs. 1221 - 1229
Autores: Vanrell, Lucía; Blanco, Laura; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Vol. 19  Nº 7  2011  págs. 1245 - 1253
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.
Autores:  et al.
Revista: GENE THERAPY
ISSN 0969-7128  Vol. 18  Nº 11  2011  págs. 1025-1033
Autores: González-Aseguinolaza, Gloria; Prieto, Jesús María;
Revista: CLINICS AND RESEARCH IN HEPATOLOGY AND GASTROENTEROLOGY
ISSN 2210-7401  Vol. 35  Nº 11  2011  págs. 699 - 708
Liver diseases including inherited metabolic disorders, chronic viral hepatitis, liver cirrhosis and primary and metastatic liver cancer constitute a formidable health problem because of their high prevalence and the important limitations of current therapies. Gene therapy, a procedure based on the transfer of therapeutic genes to tissues, has been used since the 1990s as a new approach to treating a number of incurable conditions. After a period of lights and shades recent success in treating several devastating diseases like inherited immune deficiency disorders, beta-thalassemia, or inherited blindness appear to herald a new era where gene therapy can be listed among standard therapy options for a wide variety of human conditions. In this review, we provide information illustrating the potentiality of gene therapy in the management of liver diseases lacking other effective therapies.
Autores: Parra-Guillen, ZP; González-Aseguinolaza, Gloria; Berraondo, Pedro; et al.
Revista: Pharmaceutical research
ISSN 1573-904X  Vol. 27  Nº 8  2010  págs. 1487 - 1497
Autores: Rodríguez, Carlos Manuel; Romero-Trevejo, JL; et al.
Revista: Hepatology
ISSN 0270-9139  Vol. 51  Nº 3  2010  págs. 912 - 921
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 ¿-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 (TGFß), 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 (HNF4¿) (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.
Autores:  et al.
Revista: Journal of Hepatology
ISSN 0168-8278  Vol. 52  Nº 3  2010  págs. 417 - 424
Background & Aims: Acute intermittent porphyria (AIP) is characterized by hepatic porphobilinogen deaminase (PBGD) deficiency resulting in a marked overproduction of presumably toxic porphyrin precursors. Our study aimed to assess the protective effects of bone marrow transplantation or PBGD gene transfer into the liver against phenotypic manifestations of acute porphyria attack induced in an AIP murine model. Methods: Lethally irradiated AIP mice were intravenously injected with 5×106 nucleated bone marrow cells from wild type or AIP donor mice. To achieve liver gene transfer, AIP mice received via hydrodynamic injection plasmids expressing human PBGD or luciferase, driven by a liver-specific promoter. Results: Erythrocyte PBGD activity increased 2.4-fold in AIP mice receiving bone marrow cells from normal animals. Nevertheless, phenobarbital administration in these mice reproduced key features of acute attacks, such as massively increased urinary porphyrin precursor excretion and decreased motor coordination. Hepatic PBGD activity increased 2.2-fold after hydrodynamic injection of therapeutic plasmid. Mice injected with the luciferase control plasmid showed a high excretion of porphyrin precursors after phenobarbital administration whereas just a small increase was observed in AIP mice injected with the PBGD plasmid. Furthermore, motor disturbance was almost completely abolished in AIP mice treated with the therapeutic plasmid. Conclusions: PBGD deficiency in erythroid tissue is not associated with phenotypic manifestations of acute porphyria. In contrast, PBGD over-expression in hepatocytes, albeit in a low proportion, reduced precursor accumulation, which is the hallmark of acute porphyric attacks. Liver-directed gene therapy might offer an alternative to liver transplantation applicable in patients with severe and recurrent manifestations.
Autores: Smerdou, Cristian; Ochoa, María del Carmen; Quetglas, José Ignacio; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Nº 18  2010  págs. 456 - 459
Autores: Rodrigo, Manuel; Berraondo, Pedro; Ochoa, L.; et al.
Revista: Cancer Gene Therapy (Print)
ISSN 0929-1903  Vol. 1  Nº 17  2010  págs. 20 - 7
Autores:  et al.
Revista: VACCINE
ISSN 0264-410X  Vol. 28  Nº 32  2010  págs. 5323 - 5331
Autores: González-Aseguinolaza, Gloria; Prieto, Jesús María;
Revista: GASTROENTEROLOGY
ISSN 0016-5085  Vol. 139  Nº 3  2010  págs. 726 - 729
Autores: Smerdou, Cristian; Menne, S.; Hernández, Rubén; et al.
Revista: Current Opinion in Investigational Drugs
ISSN 1472-4472  Vol. 11  Nº 12  2010  págs. 1368 - 1377
Autores: Alegre, Manuel; et al.
Revista: Molecular Therapy
ISSN 1525-0016  Vol. 19  Nº 2  2010  págs. 243 - 250
Acute intermittent porphyria (AIP) is characterized by a hereditary deficiency of hepatic porphobilinogen deaminase (PBGD) activity. Clinical features are acute neurovisceral attacks accompanied by overproduction of porphyrin precursors in the liver. Recurrent life-threatening attacks can be cured only by liver transplantation. We developed recombinant adeno-associated virus (rAAV) vectors expressing human PBGD protein driven by a liver-specific promoter to provide sustained protection against induced attacks in a predictive model for AIP. Phenobarbital injections in AIP mice induced porphyrin precursor accumulation, functional block of nerve conduction, and progressive loss of large-caliber axons in the sciatic nerve. Hepatocyte transduction showed no gender variation after rAAV2/8 injection, while rAAV2/5 showed lower transduction efficiency in females than males. Full protection against induced phenobarbital-attacks was achieved in animals showing over 10% of hepatocytes expressing high amounts of PBGD. More importantly, sustained hepatic expression of hPBGD protected against loss of large-caliber axons in the sciatic nerve and disturbances in nerve conduction velocity as induced by recurrent phenobarbital administrations. These data show for the first time that porphyrin precursors generated in the liver interfere with motor function. rAAV2/5-hPBGD vector can be produced in sufficient quantity for an intended gene therapy trial in patients with recurrent life-threatening porphyria attacks.
Autores: Tros de Ilarduya, María de la Concepción; González-Aseguinolaza, Gloria;
Libro:  Nanomedicine and cancer
2012  págs. 26 - 45