Revistas
Revista:
ACS NANO
ISSN:
1936-086X
Año:
2023
Vol.:
17
N°:
23
Págs.:
23331¿23346
Synthetic riboswitches are promising regulatory devices due to their small size, lack of immunogenicity, and ability to fine-tune gene expression in the absence of exogenous trans-acting factors. Based on a gene inhibitory system developed at our lab, termed U1snRNP interference (U1i), we developed tetracycline (TC)-inducible riboswitches that modulate mRNA polyadenylation through selective U1 snRNP recruitment. First, we engineered different TC-U1i riboswitches, which repress gene expression unless TC is added, leading to inductions of gene expression of 3-to-4-fold. Second, we developed a technique called Systematic Evolution of Riboswitches by Exponential Enrichment (SEREX), to isolate riboswitches with enhanced U1 snRNP binding capacity and activity, achieving inducibilities of up to 8-fold. Interestingly, by multiplexing riboswitches we increased inductions up to 37-fold. Finally, we demonstrated that U1i-based riboswitches are dose-dependent and reversible and can regulate the expression of reporter and endogenous genes in culture cells and mouse models, resulting in attractive systems for gene therapy applications. Our work probes SEREX as a much-needed technology for the in vitro identification of riboswitches capable of regulating gene expression in vivo.
Revista:
FRONTIERS IN IMMUNOLOGY
ISSN:
1664-3224
Año:
2022
Vol.:
13
Págs.:
829335
The study of the interferon (IFN) alpha-induced cell transcriptome has shown altered expression of several long non-coding RNAs (lncRNAs). ISR8/IRF1-AS1 (IFN stimulated RNA 8), located close to IFN regulatory factor 1 (IRF1) coding gene, transcribes a lncRNA induced at early times after IFN alpha treatment or IRF1 or NF-kappa B activation. Depletion or overexpression of ISR8 RNA does not lead to detected deregulation of the IFN response. Surprisingly, disruption of ISR8 locus with CRISPR-Cas9 genome editing results in cells that fail to induce several key ISGs and pro-inflammatory cytokines after a trigger with IFN alpha or overexpression of IRF1 or the NF-kappa B subunit RELA. This suggests that the ISR8 locus may play a relevant role in IFN alpha and NF-kappa B pathways. Interestingly, IFN alpha, IRFs and NF-kappa B-responding luciferase reporters are normally induced in ISR8-disrupted cells when expressed from a plasmid but not when integrated into the genome. Therefore, IFN alpha and NF-kappa B pathways are functional to induce the expression of exogenous episomic transcripts but fail to activate transcription from genomic promoters. Transcription from these promoters is not restored with silencing inhibitors, by decreasing the levels of several negative regulators or by overexpression of inducers. Transcriptome analyses indicate that ISR8-disrupted cells have a drastic increase in the levels of negative regulators such as XIST and Zinc finger proteins. Our results agree with ISR8 loci being an enhancer region that is fundamental for proper antiviral and proinflammatory responses. These results are relevant because several SNPs located in the ISR8 region are associated with chronic inflammatory and autoimmune diseases including Crohn's disease, inflammatory bowel disease, ulcerative colitis or asthma.
Revista:
MOLECULAR THERAPY - NUCLEIC ACIDS
ISSN:
2162-2531
Año:
2022
Vol.:
28
Págs.:
831 - 846
Here, we show that direct recruitment of U1A to target transcripts can increase gene expression. This is a new regulatory role, in addition to previous knowledge showing that U1A decreases the levels of U1A mRNA and other specific targets. In fact, genome-wide, U1A more often increases rather than represses gene expression and many U1A-upregulated transcripts are directly bound by U1A according to individual nucleotide resolution crosslinking and immunoprecipitation (iCLIP) studies. Interestingly, U1A-mediated positive regulation can be transferred to a heterologous system for biotechnological purposes. Finally, U1A-bound genes are enriched for those involved in cell cycle and adhesion. In agreement with this, higher U1A mRNA expression associates with lower disease-free survival and overall survival in many cancer types, and U1A mRNA levels positively correlate with those of some oncogenes involved in cell proliferation. Accordingly, U1A depletion leads to decreased expression of these genes and the migration-related gene CCN2/CTGF, which shows the strongest regulation by U1A. A decrease in U1A causes a strong drop in CCN2 expression and CTGF secretion and defects in the expression of CTGF EMT targets, cell migration, and proliferation. These results support U1A as a putative therapeutic target for cancer treatment. In addition, U1A-binding sequences should be considered in biotechnological applications.
Revista:
JOURNAL OF IMMUNOLOGY
ISSN:
0022-1767
Año:
2021
Vol.:
206
N°:
8
Págs.:
1932 - 1942
The cell has several mechanisms to sense and neutralize stress. Stress-related stimuli activate pathways that counteract danger, support cell survival, and activate the inflammatory response. We use human cells to show that these processes are modulated by EGOT, a long noncoding RNA highly induced by viral infection, whose inhibition results in increased levels of antiviral IFNstimulated genes (ISGs) and decreased viral replication. We now show that EGOT is induced in response to cell stress, viral replication, or the presence of pathogen-associated molecular patterns via the PI3K/AKT, MAPKs, and NF-kappa B pathways, which lead to cell survival and inflammation. Transcriptome analysis and validation experiments show that EGOT modulates PI3K/AKT and NF-kappa B responses. On the one hand, EGOT inhibition decreases expression of PI3K/AKT-induced cellular receptors and cell proliferation. In fact, EGOT levels are increased in several tumors. On the other hand, EGOT inhibition results in decreased levels of key NF-kappa B target genes, including those required for inflammation and ISGs in those cells that build an antiviral response. Mechanistically, EGOT depletion decreases the levels of the key coactivator TBLR1, essential for transcription by NF-kappa B. In summary, EGOT is induced in response to stress and may function as a switch that represses ISG transcription until a proper antiviral or stress response is initiated. EGOT then helps PI3K/AKT, MAPKs, and NF-kappa B pathways to activate the antiviral response, cell inflammation, and growth. We believe that modulation of EGOT levels could be used as a therapy for the treatment of certain viral infections, immune diseases, and cancer.
Revista:
CANCER RESEARCH
ISSN:
0008-5472
Año:
2021
Vol.:
81
N°:
19
Págs.:
4910 - 4925
Long noncoding RNAs (lncRNA) are emerging as key players in cancer as parts of poorly understood molecular mechanisms. Here, we investigated lncRNAs that play a role in hepatocellular carcinoma (HCC) and identified NIHCOLE, a novel lncRNA induced in HCC with oncogenic potential and a role in the ligation efficiency of DNA double-stranded breaks (DSB). NIHCOLE expression was associated with poor prognosis and survival of HCC patients. Depletion of NIHCOLE from HCC cells led to impaired proliferation and increased apoptosis. NIHCOLE deficiency led to accumulation of DNA damage due to a specific decrease in the activity of the nonhomologous end-joining (NHEJ) pathway of DSB repair. DNA damage induction in NIHCOLE-depleted cells further decreased HCC cell growth. NIHCOLE was associated with DSB markers and recruited several molecules of the Ku70/Ku80 heterodimer. Further, NIHCOLE putative structural domains supported stable multimeric complexes formed by several NHEJ factors including Ku70/80, APLF, XRCC4, and DNA ligase IV. NHEJ reconstitution assays showed that NIHCOLE promoted the ligation efficiency of bluntended DSBs. Collectively, these data show that NIHCOLE serves as a scaffold and facilitator of NHEJ machinery and confers an advantage to HCC cells, which could be exploited as a targetable vulnerability. Significance: This study characterizes the role of lncRNA NIHCOLE in DNA repair and cellular fitness in HCC, thus implicating it as a therapeutic target.
Revista:
JOURNAL OF CLINICAL INVESTIGATION
ISSN:
0021-9738
Año:
2020
Vol.:
130
N°:
4
Págs.:
1879 - 1895
Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found the miRNA cluster mir181ab1 as a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically-engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype underscoring their non-redundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in two highly aggressive and difficult to treat KRAS-mutated cancers.
Revista:
CELL DEATH AND DISEASE
ISSN:
2041-4889
Año:
2019
Vol.:
10
N°:
1
Págs.:
14
Liver cirrhosis results from chronic hepatic damage and is characterized by derangement of the organ architecture with increased liver fibrogenesis and defective hepatocellular function. It frequently evolves into progressive hepatic insufficiency associated with high mortality unless liver transplantation is performed. We have hypothesized that the deficiency of critical nutrients such as essential omega-3 fatty acids might play a role in the progression of liver cirrhosis. Here we evaluated by LC-MS/MS the liver content of omega-3 docosahexaenoic fatty acid (DHA) in cirrhotic patients and investigated the effect of DHA in a murine model of liver injury and in the response of hepatic stellate cells (HSCs) (the main producers of collagen in the liver) to pro-fibrogenic stimuli. We found that cirrhotic livers exhibit a marked depletion of DHA and that this alteration correlates with the progression of the disease. Administration of DHA exerts potent anti-fibrogenic effects in an acute model of liver damage. Studies with HSCs show that DHA inhibits fibrogenesis more intensely than other omega-3 fatty acids. Data from expression arrays revealed that DHA blocks TGF beta and NF-kappa B pathways. Mechanistically, DHA decreases late, but not early, SMAD3 nuclear accumulation and inhibits p65/RelA-S536 phosphorylation, which is required for HSC survival. Notably, DHA increases ADRP expression, leading to the formation of typical quiescence-associated perinuclear lipid droplets. In conclusion, a marked depletion of DHA is present in the liver of patients with advanced cirrhosis. DHA displays anti-fibrogenic activities on HSCs targeting NF-kappa B and TGF beta pathways and inducing ADPR expression and quiescence in these cells.
Revista:
ONCOTARGET
ISSN:
1949-2553
Año:
2018
Vol.:
9
N°:
16
Págs.:
12842 - 12852
Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.
Revista:
LIVER INTERNATIONAL
ISSN:
1478-3223
Año:
2014
Vol.:
35
N°:
4
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.
Revista:
GENE THERAPY
ISSN:
0969-7128
Año:
2012
Vol.:
19
N°:
4
Págs.:
411 - 417
In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.
Revista:
Nucleic acids research (print)
ISSN:
0305-1048
Año:
2012
Vol.:
40
N°:
1
Págs.:
e8
Inhibition of gene expression can be achieved with RNA interference (RNAi) or U1 small nuclear RNA-snRNA-interference (U1i). U1i is based on U1 inhibitors (U1in), U1 snRNA molecules modified to inhibit polyadenylation of a target pre-mRNA. In culture, we have shown that the combination of RNAi and U1i results in stronger inhibition of reporter or endogenous genes than that obtained using either of the techniques alone. We have now used these techniques to inhibit gene expression in mice. We show that U1ins can induce strong inhibition of the expression of target genes in vivo. Furthermore, combining U1i and RNAi results in synergistic inhibitions also in mice. This is shown for the inhibition of hepatitis B virus (HBV) sequences or endogenous Notch1. Surprisingly, inhibition obtained by combining a U1in and a RNAi mediator is higher than that obtained by combining two U1ins or two RNAi mediators. Our results suggest that RNAi and U1i cooperate by unknown mechanisms to result in synergistic inhibitions. Analysis of toxicity and specificity indicates that expression of U1i inhibitors is safe. Therefore, we believe that the combination of RNAi and U1i will be a good option to block damaging endogenous genes, HBV and other infectious agents in vivo.
Revista:
Nucleic acids research (print)
ISSN:
0305-1048
Año:
2010
Vol.:
38
N°:
3
Págs.:
750 - 763
Revista:
Nucleic acids research (print)
ISSN:
0305-1048
Año:
2010
Vol.:
38
N°:
13
Págs.:
e136
Revista:
Hepatology
ISSN:
0270-9139
Año:
2010
Vol.:
51
N°:
3
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.
Nacionales y Regionales
Título:
Inhibición de genes no codificantes con valor terapéutico en hepatocarcinoma. Análisis de monoterapias y terapias combinadas.
Código de expediente:
PI19/00742
Financiador:
INSTITUTO DE SALUD CARLOS III
Convocatoria:
2019 AES Proyectos de investigación
Fecha de inicio:
01/01/2020
Fecha fin:
31/12/2023
Importe concedido:
93.170,00€
Otros fondos:
Fondos FEDER
Título:
Estudio de la acción antifibrogénica de los ácidos grasos omega 3 : Aplicaciones terapéuticas
Código de expediente:
PI16/02081
Financiador:
INSTITUTO DE SALUD CARLOS III
Convocatoria:
2016 AES PROYECTOS DE INVESTIGACIÓN
Fecha de inicio:
01/01/2017
Fecha fin:
30/06/2021
Importe concedido:
139.755,00€
Otros fondos:
Fondos FEDER
