Revistas
Revista:
GLIA
ISSN:
0894-1491
Año:
2023
Vol.:
71
N°:
3
Págs.:
571 - 587
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 alpha-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 alpha-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.
Revista:
NEUROBIOLOGY OF DISEASE
ISSN:
0969-9961
Año:
2023
Vol.:
183
Págs.:
106166
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.
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2022
Vol.:
77
N°:
S1
Págs.:
S257 - S258
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2022
Vol.:
77
N°:
Suppl. 1
Págs.:
S248 - S248
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2021
Vol.:
32
N°:
19-20
Págs.:
A16
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2021
Vol.:
75
N°:
Supl. 2
Págs.:
S694 - S694
Revista:
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
ISSN:
1932-6254
Año:
2020
Vol.:
14
N°:
1
Págs.:
123 - 134
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.
Revista:
GENES
ISSN:
2073-4425
Año:
2019
Vol.:
10
N°:
12
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 El/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.
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2019
Vol.:
70
Págs.:
632A - 632A
Revista:
HEPATOLOGY
ISSN:
0270-9139
Año:
2019
Vol.:
70
Págs.:
640A - 640A
Revista:
MOLECULAR THERAPY
ISSN:
1525-0016
Año:
2019
Vol.:
27
N°:
4
Págs.:
368 - 368
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2019
Vol.:
30
N°:
11
Págs.:
A2 - A2
Autores:
Suarez-Amaran, L.; Usai, C.; Di Scala, M.; et al.
Revista:
JOURNAL OF HEPATOLOGY
ISSN:
1600-0641
Año:
2018
Vol.:
69
N°:
1
Págs.:
262 - 264
Revista:
MOLECULAR THERAPY
ISSN:
1525-0016
Año:
2018
Vol.:
26
N°:
5
Págs.:
384 - 385
Revista:
NATURE COMMUNICATIONS
ISSN:
2041-1723
Año:
2018
Vol.:
9
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.
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2018
Vol.:
29
N°:
12
Págs.:
A108 - A108
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2017
Vol.:
67
N°:
4
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.
Revista:
JOURNAL OF HEPATOLOGY (ONLINE)
ISSN:
0168-8278
Año:
2017
Vol.:
66
N°:
1
Págs.:
S483 - S483
Revista:
MOLECULAR THERAPY
ISSN:
1525-0016
Año:
2017
Vol.:
25
N°:
5 Supl.1
Págs.:
79 - 80
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2017
Vol.:
28
N°:
12
Págs.:
A11 - A12
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2016
Vol.:
27
N°:
11
Págs.:
A139 - A140
Revista:
HUMAN GENE THERAPY
ISSN:
1043-0342
Año:
2016
Vol.:
27
N°:
11
Págs.:
A130
Revista:
JOURNAL OF INNATE IMMUNITY
ISSN:
1662-811X
Año:
2015
Vol.:
7
N°:
5
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.
Revista:
HAEMATOLOGICA
ISSN:
1138-0381
Año:
2015
Vol.:
100
N°:
8
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.
Revista:
PLOS ONE
ISSN:
1932-6203
Año:
2013
Vol.:
8
N°:
7
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.
Revista:
Hepatology
ISSN:
0270-9139
Año:
2012
Vol.:
56
N°:
2
Págs.:
474 - 483
Revista:
VACCINE
ISSN:
0264-410X
Año:
2010
Vol.:
28
N°:
32
Págs.:
5323 - 5331