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1.
PLoS Pathog ; 20(5): e1011749, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38739648

RESUMEN

Hepatitis delta virus (HDV) infection represents the most severe form of human viral hepatitis; however, the mechanisms underlying its pathology remain incompletely understood. We recently developed an HDV mouse model by injecting adeno-associated viral vectors (AAV) containing replication-competent HBV and HDV genomes. This model replicates many features of human infection, including liver injury. Notably, the extent of liver damage can be diminished with anti-TNF-α treatment. Here, we found that TNF-α is mainly produced by macrophages. Downstream of the TNF-α receptor (TNFR), the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) serves as a cell fate regulator, playing roles in both cell survival and death pathways. In this study, we explored the function of RIPK1 and other host factors in HDV-induced cell death. We determined that the scaffolding function of RIPK1, and not its kinase activity, offers partial protection against HDV-induced apoptosis. A reduction in RIPK1 expression in hepatocytes through CRISPR-Cas9-mediated gene editing significantly intensifies HDV-induced damage. Contrary to our expectations, the protective effect of RIPK1 was not linked to TNF-α or macrophage activation, as their absence did not alter the extent of damage. Intriguingly, in the absence of RIPK1, macrophages confer a protective role. However, in animals unresponsive to type-I IFNs, RIPK1 downregulation did not exacerbate the damage, suggesting RIPK1's role in shielding hepatocytes from type-I IFN-induced cell death. Interestingly, while the damage extent is similar between IFNα/ßR KO and wild type mice in terms of transaminase elevation, their cell death mechanisms differ. In conclusion, our findings reveal that HDV-induced type-I IFN production is central to inducing hepatocyte death, and RIPK1's scaffolding function offers protective benefits. Thus, type-I IFN together with TNF-α, contribute to HDV-induced liver damage. These insights may guide the development of novel therapeutic strategies to mitigate HDV-induced liver damage and halt disease progression.


Asunto(s)
Citocinas , Virus de la Hepatitis Delta , Hepatocitos , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Animales , Ratones , Hepatocitos/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Citocinas/metabolismo , Virus de la Hepatitis Delta/fisiología , Hepatitis D/metabolismo , Muerte Celular , Ratones Endogámicos C57BL , Apoptosis , Ratones Noqueados , Humanos , Factor de Necrosis Tumoral alfa/metabolismo , Modelos Animales de Enfermedad
2.
Glia ; 71(3): 571-587, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36353934

RESUMEN

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.


Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Ratones , Animales , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Microglía/metabolismo , Astrocitos/metabolismo , Mesencéfalo/metabolismo , Inflamación
3.
Neurobiol Dis ; 183: 106166, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37245833

RESUMEN

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.


Asunto(s)
Sinucleinopatías , alfa-Sinucleína , Ratones , Humanos , Animales , alfa-Sinucleína/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Células HEK293 , Ratones Transgénicos , Neuronas Dopaminérgicas/metabolismo
4.
J Hepatol ; 67(4): 669-679, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28527664

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Hepatitis D/inmunología , Interferón beta/biosíntesis , Inmunidad Adaptativa , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Coinfección/inmunología , Coinfección/patología , Coinfección/virología , Dependovirus/genética , Modelos Animales de Enfermedad , Genoma Viral , Hepatitis B/complicaciones , Hepatitis B/inmunología , Hepatitis B/virología , Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis D/complicaciones , Hepatitis D/virología , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/inmunología , Virus de la Hepatitis Delta/fisiología , Antígenos de Hepatitis delta/metabolismo , Humanos , Inmunidad Innata , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Transducción de Señal/inmunología , Replicación Viral
5.
J Virol ; 90(19): 8563-74, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27440883

RESUMEN

UNLABELLED: In chronic hepatitis B (CHB), failure to control hepatitis B virus (HBV) is associated with T cell dysfunction. HBV transgenic mice mirror many features of the human disease, including T cell unresponsiveness, and thus represent an appropriate model in which to test novel therapeutic strategies. To date, the tolerant state of CD8(+) T cells in these animals could be altered only by strong immunogens or by immunization with HBV antigen-pulsed dendritic cells; however, the effectors induced were unable to suppress viral gene expression or replication. Because of the known stimulatory properties of alpha interferon (IFN-α) and interleukin-15 (IL-15), this study explored the therapeutic potential of liver-directed gene transfer of these cytokines in a murine model of CHB using adeno-associated virus (AAV) delivery. This combination not only resulted in a reduction in the viral load in the liver and the induction of an antibody response but also gave rise to functional and specific CD8(+) immunity. Furthermore, when splenic and intrahepatic lymphocytes from IFN-α- and IL-15-treated animals were transferred to new HBV carriers, partial antiviral immunity was achieved. In contrast to previous observations made using either cytokine alone, markedly attenuated PD-L1 induction in hepatic tissue was observed upon coadministration. An initial study with CHB patient samples also gave promising results. Hence, we demonstrated synergy between two stimulating cytokines, IL-15 and IFN-α, which, given together, constitute a potent approach to significantly enhance the CD8(+) T cell response in a state of immune hyporesponsiveness. Such an approach may be useful for treating chronic viral infections and neoplastic conditions. IMPORTANCE: With 350 million people affected worldwide and 600,000 annual deaths due to HBV-induced liver cirrhosis and/or hepatocellular carcinoma, chronic hepatitis B (CHB) is a major health problem. However, current treatment options are costly and not very effective and/or need to be administered for life. The unprecedented efficacy of the strategy described in our paper may offer an alternative and is relevant for a broad spectrum of readers because of its clear translational importance to other chronic viral infections in which a hyporesponsive antigen-specific T cell repertoire prevents clearance of the pathogen.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Interferón-alfa/administración & dosificación , Interleucina-15/administración & dosificación , Adenoviridae/genética , Animales , Modelos Animales de Enfermedad , Portadores de Fármacos , Terapia Genética , Anticuerpos contra la Hepatitis B/sangre , Interferón-alfa/genética , Interleucina-15/genética , Hígado/virología , Ratones Transgénicos , Resultado del Tratamiento , Carga Viral
6.
Haematologica ; 100(8): 1014-22, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25715405

RESUMEN

Interferon-α 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-α alters hematopoietic stem cell dormancy and impairs myelocytic and erythrocytic/megakaryocytic differentiation from hematopoietic progenitors. However, the effect of chronic interferon-α exposure on hematopoietic precursors has still not been well characterized. Here, we transduced the liver of mice with an adenoassociated vector encoding interferon-α 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-α-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-α exposure causes hematopoietic stem cells exhaustion and drives common lymphoid progenitors towards T-cell generation.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Interferón-alfa/farmacología , Linfopoyesis/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Interferón-alfa/genética , Recuento de Leucocitos , Leucocitos/citología , Leucocitos/metabolismo , Linfopoyesis/genética , Masculino , Ratones , Ratones Noqueados , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
Viruses ; 16(3)2024 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-38543745

RESUMEN

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.


Asunto(s)
Virus de la Hepatitis Delta , ARN Viral , Animales , Ratones , Antígenos de Hepatitis delta/genética , Antígenos de Hepatitis delta/metabolismo , ARN Viral/metabolismo , Ratones Endogámicos C57BL , Replicación Viral/genética , Procesamiento Proteico-Postraduccional , Hígado/metabolismo
8.
EMBO Mol Med ; 16(1): 112-131, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38182795

RESUMEN

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.


Asunto(s)
Sistemas CRISPR-Cas , Hiperoxaluria Primaria , Humanos , Animales , Ratones , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Edición Génica , Hiperoxaluria Primaria/genética , Hiperoxaluria Primaria/terapia
10.
Pharmaceutics ; 15(10)2023 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-37896219

RESUMEN

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

11.
Cells ; 12(18)2023 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-37759522

RESUMEN

Oxidative stress-induced myocardial apoptosis and necrosis are critically involved in ischemic infarction, and several sources of extracellular vesicles appear to be enriched in therapeutic activities. The central objective was to identify and validate the differential exosome miRNA repertoire in human cardiac progenitor cells (CPC). CPC exosomes were first analyzed by LC-MS/MS and compared by RNAseq with exomes of human mesenchymal stromal cells and human fibroblasts to define their differential exosome miRNA repertoire (exo-miRSEL). Proteomics demonstrated a highly significant representation of cardiovascular development functions and angiogenesis in CPC exosomes, and RNAseq analysis yielded about 350 different miRNAs; among the exo-miRSEL population, miR-935 was confirmed as the miRNA most significantly up-regulated; interestingly, miR-935 was also found to be preferentially expressed in mouse primary cardiac Bmi1+high CPC, a population highly enriched in progenitors. Furthermore, it was found that transfection of an miR-935 antagomiR combined with oxidative stress treatment provoked a significant increment both in apoptotic and necrotic populations, whereas transfection of a miR-935 mimic did not modify the response. Conclusion. miR-935 is a highly differentially expressed miRNA in exo-miRSEL, and its expression reduction promotes oxidative stress-associated apoptosis. MiR-935, together with other exosomal miRNA members, could counteract oxidative stress-related apoptosis, at least in CPC surroundings.

12.
Viruses ; 13(5)2021 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-33925087

RESUMEN

Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, but little is known about the molecular mechanisms involved. We have recently developed an HDV mouse model based on the delivery of HDV replication-competent genomes using adeno-associated vectors (AAV), which developed a liver pathology very similar to the human disease and allowed us to perform mechanistic studies. We have generated different AAV-HDV mutants to eliminate the expression of HDV antigens (HDAgs), and we have characterized them both in vitro and in vivo. We confirmed that S-HDAg is essential for HDV replication and cannot be replaced by L-HDAg or host cellular proteins, and that L-HDAg is essential to produce the HDV infectious particle and inhibits its replication. We have also found that lack of L-HDAg resulted in the increase of S-HDAg expression levels and the exacerbation of liver damage, which was associated with an increment in liver inflammation but did not require T cells. Interestingly, early expression of L-HDAg significantly ameliorated the liver damage induced by the mutant expressing only S-HDAg. In summary, the use of AAV-HDV represents a very attractive platform to interrogate in vivo the role of viral components in the HDV life cycle and to better understand the mechanism of HDV-induced liver pathology.


Asunto(s)
Dependovirus/genética , Vectores Genéticos/genética , Hepatitis D/virología , Virus de la Hepatitis Delta/fisiología , Replicación Viral , Animales , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Ingeniería Genética , Hepatitis D/patología , Humanos , Técnicas In Vitro , Hígado/metabolismo , Hígado/patología , Hígado/virología , Ratones , Mutación
13.
JHEP Rep ; 2(3): 100098, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32382723

RESUMEN

BACKGROUND & AIMS: HDV infection induces the most severe form of human viral hepatitis. However, the specific reasons for the severity of the disease remain unknown. Recently, we developed an HDV replication mouse model in which, for the first time, liver damage was detected. METHODS: HDV and HBV replication-competent genomes and HDV antigens were delivered to mouse hepatocytes using adeno-associated vectors (AAVs). Aminotransferase elevation, liver histopathology, and hepatocyte death were evaluated and the immune infiltrate was characterized. Liver transcriptomic analysis was performed. Mice deficient for different cellular and molecular components of the immune system, as well as depletion and inhibition studies, were employed to elucidate the causes of HDV-mediated liver damage. RESULTS: AAV-mediated HBV/HDV coinfection caused hepatocyte necrosis and apoptosis. Activated T lymphocytes, natural killer cells, and proinflammatory macrophages accounted for the majority of the inflammatory infiltrate. However, depletion studies and the use of different knockout mice indicated that neither T cells, natural killer cells nor macrophages were necessary for HDV-induced liver damage. Transcriptomic analysis revealed a strong activation of type I and II interferon (IFN) and tumor necrosis factor (TNF)-α pathways in HBV/HDV-coinfected mice. While the absence of IFN signaling had no effect, the use of a TNF-α antagonist resulted in a significant reduction of HDV-associated liver injury. Furthermore, hepatic expression of HDAg resulted in the induction of severe liver damage, which was T cell- and TNF-α-independent. CONCLUSIONS: Both host (TNF-α) and viral (HDV antigens) factors play a relevant role in HDV-induced liver damage. Importantly, pharmacological inhibition of TNF-α may offer an attractive strategy to aid control of HDV-induced acute liver damage. LAY SUMMARY: Chronic hepatitis delta constitutes the most severe form of viral hepatitis. There is limited data on the mechanism involved in hepatitis delta virus (HDV)-induced liver pathology. Our data indicate that a cytokine (TNF-α) and HDV antigens play a relevant role in HDV-induced liver damage.

14.
J Tissue Eng Regen Med ; 14(1): 123-134, 2020 01.
Artículo en Inglés | MEDLINE | ID: mdl-31677236

RESUMEN

Adeno-associated viruses (AAV) have become one of the most promising tools for gene transfer in clinics. Among all the serotypes, AAV9 has been described as the most efficient for cardiac transduction. In order to achieve optimal therapeutic delivery in heart disease, we have explored AAV9 transduction efficiency in an infarcted heart using different routes of administration and promoters, including a cardiac-specific one. AAV9 vectors carrying luciferase or green fluorescence protein under the control of the ubiquitous elongation-factor-1-alpha or the cardiac-specific troponin-T (TnT) promoters were administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. The transduction efficacy and specificity, the time-course expression, and the safety of each vector were tested. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the elongation-factor-1-alpha-promoter, independently of the administration route and heart damage. Moreover, tissue-specific green fluorescence protein expression was found in cardiomyocytes with the TnT vector, whereas minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that myocardial infarction greatly increased the transcriptional activity of AAV genomes. Our findings show that the use of cardiac promoters allows for specific and stable cardiac gene expression, which is optimal and robust when intramyocardially injected. Furthermore, our data indicate that the pathological status of the tissue can alter the transcriptional activity of AAV genomes, an aspect that should be carefully evaluated for clinical applications.


Asunto(s)
Dependovirus/genética , Isquemia Miocárdica/patología , Animales , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos , Genoma Viral , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Corazón/fisiología , Humanos , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/patología , Miocardio/patología , Miocitos Cardíacos/citología , Factor 1 de Elongación Peptídica/metabolismo , Regiones Promotoras Genéticas , Distribución Tisular , Transducción Genética , Transgenes , Troponina T/metabolismo
15.
Genes (Basel) ; 10(12)2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31861246

RESUMEN

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.


Asunto(s)
Adenoviridae/genética , Vectores Genéticos/metabolismo , Pez Cebra/genética , Animales , Encéfalo/metabolismo , Embrión no Mamífero/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente , Modelos Animales , Pez Cebra/crecimiento & desarrollo
16.
J Gene Med ; 10(10): 1092-101, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18642400

RESUMEN

BACKGROUND: High-capacity adenoviruses (HC-Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large-scale production, and the absence of standardized methods for production and titration. In the present study, we compare the different methods found in the literature for characterizing HC-Ad production. METHODS: Two productions of the HC-Ad carrying murine IL-12 gene were obtained. The viral titer and adenovirus-helper contamination as well as viral particle concentration of both productions were determined using different methods: (i) quantification of total viral particles by spectrophotometry and plaque assay to estimate first-generation (FG)-helper-Ad contamination; (ii) quantification of HC-Ad and FG-helper-Ad genomes by the quantitative polymerase chain reaction (qPCR) directly from viral stock; (iii) quantification of viral genomes after cell infection by the slot-blot hybridization assay and (iv) qPCR. RESULTS: Dramatic differences with respect to viral titer were found depending on the method used. The first method overestimates HC-Ad titer and underestimates FG-helper-Ad contamination and no information on the infectivity of the HC-Ad is obtained. qPCR analysis of viral stock is more sensitive and accurate, but information about infectivity remains unknown and FG-helper-Ad contamination is overestimated. Quantification of HC-Ad and FG-helper-Ad infectious units by-slot blot DNA hybridization and qPCR assay are found to be equally sensitive and accurate. CONCLUSIONS: The results of the present study demonstrate that a standardized method should be developed for HC-Ad characterization for future clinical applications of this vector. Quantification of HC-Ad production by qPCR is a fast, safe and reliable method for determining HC-Ad and FG-helper-Ad particles and infectious units.


Asunto(s)
Adenoviridae/genética , Reacción en Cadena de la Polimerasa/normas , Adenoviridae/metabolismo , Animales , ADN Viral/análisis , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Genoma Viral , Células HeLa , Humanos , Interleucina-12/genética , Interleucina-12/metabolismo , Ratones
17.
Nat Commun ; 9(1): 5454, 2018 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-30575740

RESUMEN

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.


Asunto(s)
Oxidorreductasas de Alcohol/antagonistas & inhibidores , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperoxaluria Primaria/terapia , Oxalatos/orina , Oxidorreductasas de Alcohol/genética , Animales , Modelos Animales de Enfermedad , Edición Génica , Células HEK293 , Humanos , Masculino , Ratones , Nefrocalcinosis/prevención & control
18.
Methods Mol Biol ; 1506: 179-192, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27830553

RESUMEN

Hepatocyte transplantation is the best approach to maintain and propagate differentiated hepatocytes from different species. Host liver has to be adapted for transplanted hepatocytes productive engraftment and proliferation being required a chronic liver injury to eliminate host hepatocytes and provide a proliferative advantage to the transplanted hepatocytes. Most valuable mouse models for xenograft hepatocyte transplantation are based on genetically modified animals to cause a chronic liver damage and to limit host hepatocyte regeneration potential. We present a methodology that generates a chronic liver damage and can be applied to any host mouse strain and animal species based on the inoculation of a recombinant adenovirus to express herpes simplex thymidine kinase in host hepatocytes sensitizing them to ganciclovir treatment. This causes a prolonged liver damage that allows hepatocyte transplantation and generation of regenerative nodules in recipient mouse liver integrated by transplanted cells and host sinusoidal. Obtained chimeric animals maintain functional chimeric nodules for several weeks, ready to be used in any study.


Asunto(s)
Adenoviridae/genética , Trasplante de Células/métodos , Hepatocitos/trasplante , Regeneración Hepática/efectos de los fármacos , Hígado/fisiología , Acondicionamiento Pretrasplante/métodos , Animales , Separación Celular/métodos , Trasplante de Células/efectos adversos , Trasplante de Células/instrumentación , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Modelos Animales de Enfermedad , Ganciclovir/toxicidad , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Simplexvirus/genética , Timidina Quinasa/genética , Transducción Genética/métodos , Quimera por Trasplante/fisiología , Quimera por Trasplante/cirugía , Trasplante Heterólogo/efectos adversos , Trasplante Heterólogo/métodos , Proteínas no Estructurales Virales/genética
19.
Front Neuroanat ; 11: 2, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28239341

RESUMEN

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.

20.
Oncotarget ; 7(31): 49008-49026, 2016 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-27356750

RESUMEN

Interleukin-15 (IL-15) is a cell growth-factor that regulates lymphocyte function and homeostasis. Its strong immunostimulatory activity coupled with an apparent lack of toxicity makes IL-15 an exciting candidate for cancer therapy, somehow limited by its short half-life in circulation. To increase IL-15 bioavailability we constructed a recombinant adeno-associated vector expressing murine IL-15 (AAV-mIL15) in the liver. Mice injected with AAV-mIL15 showed sustained and vector dose-dependent levels of IL-15/IL-15Rα complexes in serum, production of IFN-γ and activation of CD8+ T-cells and macrophages. The antitumoral efficacy of AAV-mIL15 was tested in a mouse model of metastatic colorectal cancer established by injection of MC38 cells. AAV-mIL15 treatment slightly inhibits MC38 tumor-growth and significantly increases the survival of mice. However, mIL-15 sustained expression was associated with development of side effects like hepatosplenomegaly, liver damage and the development of haematological stress, which results in the expansion of hematopoietic precursors in the bone marrow. To elucidate the mechanism, we treated IFN-γ receptor-, RAG1-, CD1d- and µMT-deficient mice and performed adoptive transfer of bone marrow cells from WT mice to RAG1-defcient mice. We demonstrated that the side effects of murine IL-15 administration were mainly mediated by IFN-γ-producing T-cells. CONCLUSIONS: IL-15 induces the activation and survival of effector immune cells that are necessary for its antitumoral activity; but, long-term exposure to IL-15 is associated with the development of important side effects mainly mediated by IFN-γ-producing T-cells. Strategies to modulate T-cell activation should be combined with IL-15 administration to reduce secondary adverse events while maintaining its antitumoral effect.


Asunto(s)
Interferón gamma/metabolismo , Interleucina-15/farmacología , Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Antígenos CD1d/metabolismo , Células de la Médula Ósea/citología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Neoplasias Colorrectales/terapia , Dependovirus , Progresión de la Enfermedad , Femenino , Regulación de la Expresión Génica , Terapia Genética/efectos adversos , Terapia Genética/métodos , Vectores Genéticos , Proteínas de Homeodominio/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Hígado/metabolismo , Activación de Linfocitos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados
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