RESUMEN
One of the most promising therapeutic approaches for familial amyotrophic lateral sclerosis linked to superoxide dismutase 1 (SOD1) is the suppression of toxic mutant SOD1 in the affected tissues. Here, we report an innovative molecular strategy for inducing substantial, widespread, and sustained reduction of mutant human SOD1 (hSOD1) levels throughout the body of SOD1G93A mice, leading to therapeutic effects in animals. Adeno-associated virus serotype rh10 vectors (AAV10) were used to mediate exon skipping of the hSOD1 pre-mRNA by expression of exon-2-targeted antisense sequences embedded in a modified U7 small-nuclear RNA (AAV10-U7-hSOD). Skipping of hSOD1 exon 2 led to the generation of a premature termination codon, inducing production of a deleted transcript that was subsequently degraded by the activation of nonsense-mediated decay. Combined intravenous and intracerebroventricular delivery of AAV10-U7-hSOD increased the survival of SOD1G93A mice injected either at birth or at 50 days of age (by 92% and 58%, respectively) and prevented weight loss and the decline of neuromuscular function. This study reports the effectiveness of an exon-skipping approach in SOD1-ALS mice, supporting the translation of this technology to the treatment of this as yet incurable disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Dependovirus/genética , Terapia Genética , Vectores Genéticos/genética , Superóxido Dismutasa-1/genética , Edad de Inicio , Esclerosis Amiotrófica Lateral/mortalidad , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/terapia , Animales , Modelos Animales de Enfermedad , Exones , Orden Génico , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Ratones Transgénicos , Actividad Motora/genética , Oligonucleótidos Antisentido , Sitios de Empalme de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Recuperación de la Función , Superóxido Dismutasa-1/metabolismo , Tasa de Supervivencia , Transducción GenéticaRESUMEN
BACKGROUND: New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. METHODS: We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. RESULTS: EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFß maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. CONCLUSION: This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Núcleo Celular , Modelos Animales de Enfermedad , Detección Precoz del Cáncer , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Colágeno/metabolismoRESUMEN
Lung tumor-infiltrating neutrophils are known to support growth and dissemination of cancer cells and to suppress T cell responses. However, the precise impact of tissue neutrophils on programming and differentiation of anticancer CD8 T cells in vivo remains poorly understood. Here, we identified cancer cell-autonomous secretion of CXCL5 as sufficient to drive infiltration of mature, protumorigenic neutrophils in a mouse model of non-small cell lung cancer (NSCLC). Consistently, CXCL5 transcripts correlate with neutrophil density and poor prognosis in a large human lung adenocarcinoma compendium. CXCL5 genetic deletion, unlike antibody-mediated depletion, completely and selectively prevented neutrophils accumulation in lung tissues. Depletion of tumor-infiltrating neutrophils promoted expansion of tumor-specific CD8 T cells, differentiation into effector cells and acquisition of cytolytic functions. Transfer of effector CD8 T cells into neutrophil-rich tumors, inhibited IFN-Ï production, indicating active suppression of effector functions. Importantly, blocking neutrophils infiltration in the lung, overcame resistance to checkpoint blockade. Hence, this study demonstrates that neutrophils curb acquisition of cytolytic functions in lung tumor tissues and suggests targeting of CXCL5 as a strategy to restore anti-tumoral T cell functions.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , NeutrófilosRESUMEN
Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.
RESUMEN
AIMS: Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. METHODS AND RESULTS: We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. CONCLUSION: Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells.
Asunto(s)
Apelina/metabolismo , Linaje de la Célula , Vasos Coronarios/metabolismo , Células Endoteliales/metabolismo , Músculo Esquelético/irrigación sanguínea , Neoplasias/irrigación sanguínea , Neovascularización Patológica , Neovascularización Fisiológica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apelina/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Microambiente Celular , Vasos Coronarios/citología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Carga Tumoral , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Alternative splicing (AS) is a pervasive molecular process generating multiple protein isoforms, from a single gene. It plays fundamental roles during development, differentiation and maintenance of tissue homeostasis, while aberrant AS is considered a hallmark of multiple diseases, including cancer. Cancer-restricted AS isoforms represent either predictive biomarkers for diagnosis/prognosis or targets for anti-cancer therapies. Here, we discuss the contribution of AS regulation in cancer angiogenesis, a complex process supporting disease development and progression. We consider AS programs acting in a specific and non-redundant manner to influence morphological and functional changes involved in cancer angiogenesis. In particular, we describe relevant AS variants or splicing regulators controlling either secreted or membrane-bound angiogenic factors, which may represent attractive targets for therapeutic interventions in human cancer.
Asunto(s)
Empalme Alternativo , Proteínas Portadoras/metabolismo , Células Endoteliales/metabolismo , Neoplasias/irrigación sanguínea , Animales , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismoRESUMEN
Cardiac ischemia is the leading cause of morbidity and mortality in a worldwide epidemic. The progressive understanding of the mechanisms driving new blood vessel formation has led to numerous attempts to revascularize the ischemic heart in animal models and in humans. Here, we provide an overview of the current state of the art and discuss the major obstacles that have so far limited the clinical success of cardiac revascularization.