RESUMEN
Myocarditis can lead to autoimmune disease, dilated cardiomyopathy, and heart failure, which is modeled in the mouse by cardiac myosin immunization (experimental autoimmune myocarditis [EAM]). Signal transducer and activator of transcription 3 (STAT3) systemic inhibition exerts both preventive and therapeutic effects in EAM, and STAT3 constitutive activation elicits immune-mediated myocarditis dependent on complement C3 and correlating with activation of the STAT3-interleukin 6 (IL-6) axis in the liver. Thus, liver-specific STAT3 inhibition may represent a therapeutic option, allowing to bypass the heart toxicity, predicted by systemic STAT3 inhibition. We therefore decided to explore the effectiveness of silencing liver Stat3 and C3 in preventing EAM onset and/or the recovery of cardiac functions. We first show that complement C3 and C5 genetic depletion significantly prevents the onset of spontaneous myocarditis, supporting the complement cascade as a viable target. In order to interfere with complement production and STAT3 activity specifically in the liver, we took advantage of liver-specific Stat3 or C3 small interfering (si)RNA nanoparticles, demonstrating that both siRNAs can significantly prevent myocarditis onset and improve the recovery of heart functions in EAM. Our data demonstrate that liver-specific Stat3/C3 siRNAs may represent a therapeutic option for autoimmune myocarditis and suggest that complement levels and activation might be predictive of progression to dilated cardiomyopathy.
RESUMEN
Translation is an essential biological process, and dysregulation is associated with a range of diseases including ribosomopathies, diabetes, and cancer. Here, we examine translation dysregulation in vivo using RNAi to knock down the m-subunit of the translation initiation factor eIF3 in the mouse liver. Transcriptome sequencing, ribosome profiling, whole proteome, and phosphoproteome analyses show that eIF3m deficiency leads to the transcriptional response and changes in cellular translation that yield few detectable differences in the translation of particular mRNAs. The transcriptional response fell into two main categories: ribosome biogenesis (increased transcription of ribosomal proteins) and cell metabolism (alterations in lipid, amino acid, nucleic acid, and drug metabolism). Analysis of ribosome biogenesis reveals inhibition of rRNA processing, highlighting decoupling of rRNA synthesis and ribosomal protein gene transcription in response to eIF3m knockdown. Interestingly, a similar reduction in eIF3m protein levels is associated with induction of the mTOR pathway in vitro but not in vivo. Overall, this work highlights the utility of a RNAi-based in vivo approach for studying the regulation of mammalian translation in vivo.
RESUMEN
Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images of mouse liver tissue and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells, since silencing Integrin-ß1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.
Asunto(s)
Polaridad Celular , Hepatocitos/citología , Cristales Líquidos/química , Hígado/citología , Algoritmos , Animales , Capilares/química , Capilares/citología , Capilares/metabolismo , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo , Femenino , Hepatocitos/química , Hepatocitos/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Hígado/irrigación sanguínea , Hígado/química , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal , Interferencia de ARNRESUMEN
CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9-sgRNA complex as a guide, the majority of the 3' end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3'-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA-RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells.
Asunto(s)
Sistemas CRISPR-Cas , ADN/genética , Edición Génica , ARN Guía de Kinetoplastida/genética , Alelos , Línea Celular Tumoral , Separación Celular , Citometría de Flujo , Proteínas Fluorescentes Verdes/química , Células HEK293 , Humanos , Células Jurkat , Nucleótidos/genética , Oligonucleótidos/genéticaRESUMEN
Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.
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Edición Génica/métodos , Técnicas de Transferencia de Gen , Hígado/metabolismo , ARN Guía de Kinetoplastida/genética , Animales , Sistemas CRISPR-Cas/genética , Ratones , Nanopartículas/química , Conformación de Ácido Nucleico , Proproteína Convertasa 9/genéticaRESUMEN
By performing nine genome-wide microRNA (miRNA) screens, we recently uncovered a new class of miRNAs, which target multiple cyclins and cyclin-dependent kinases (CDKs). Systemic delivery of selected cell cycle-targeting miRNAs to mouse xenograft models resulted in potent anti-tumorigenic effects without affecting animals' health. Here, we provide an in-depth description of our miRNA screening methodology, analyses of selected cell cycle-targeting miRNAs, and discuss why miRNA therapy might be a viable therapeutic option for cancer patients.
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Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Genoma , MicroARNs/metabolismo , Regiones no Traducidas 3' , Tejido Adiposo/metabolismo , Animales , Ciclo Celular , Humanos , Ratones , Neoplasias/genética , Neoplasias/patologíaRESUMEN
The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Regeneración Hepática , Proteómica/métodos , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Endocitosis/efectos de los fármacos , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/efectos de los fármacos , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Regeneración Hepática/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Mitógenos/farmacología , Ubiquitina-Proteína Ligasas Nedd4 , Poliubiquitina/metabolismo , Proteoma/metabolismo , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous human tumors. To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell-cycle proteins. We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation. By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of tumors to cell-cycle-targeting miRNAs. Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues. Our results highlight the utility of using cell-cycle-targeting miRNAs for treatment of refractory cancer types.
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Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Regiones no Traducidas 3' , Algoritmos , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Ratones Endogámicos , MicroARNs/administración & dosificación , MicroARNs/farmacología , Mutación , Nanopartículas , Proteínas Proto-Oncogénicas p21(ras)/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.
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Sistemas CRISPR-Cas/genética , Genoma Humano , Edición de ARN , Tirosinemias/terapia , Animales , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Vectores Genéticos , Humanos , Lípidos/química , Ratones , Mutación , Nanopartículas/química , Tirosinemias/genética , Virus/genéticaRESUMEN
BACKGROUND & AIMS: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models. METHODS: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2). RESULTS: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth. CONCLUSION: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver.
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Genómica/métodos , Hígado/crecimiento & desarrollo , Nanopartículas , Interferencia de ARN , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Proteínas de Ciclo Celular , Expresión Génica , Genes de la Neurofibromatosis 2 , Factor de Crecimiento de Hepatocito/genética , Hepatomegalia/etiología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Tamaño de los Órganos , Fosfoproteínas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Serina-Treonina Quinasa 3 , Proteína p53 Supresora de Tumor/fisiología , Proteínas Señalizadoras YAPRESUMEN
The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke's disease, a glucose-6-phosphatase (G6Pase) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.
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Endosomas/enzimología , Hígado/enzimología , Proteínas de Unión al GTP rab5/metabolismo , Animales , Simulación por Computador , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Técnicas de Silenciamiento del Gen , Gluconeogénesis/genética , Glucosa/metabolismo , Glucosa-6-Fosfatasa/metabolismo , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Hepatomegalia/enzimología , Hepatomegalia/patología , Hiperglucemia/enzimología , Hiperglucemia/patología , Hipoglucemia/enzimología , Hipoglucemia/patología , Insulina/metabolismo , Metabolismo de los Lípidos , Ratones Noqueados , Modelos Biológicos , Proteómica , Transducción de Señal/genéticaRESUMEN
PRLR(I146L) is the first identified gain-of-function variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7 (luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR(I146D)) had minimal impact on cell proliferation and cell differentiation status.
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Neoplasias de la Mama/metabolismo , Análisis Mutacional de ADN/métodos , Receptores de Prolactina/química , Receptores de Prolactina/genética , Sustitución de Aminoácidos , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Dicroismo Circular , Femenino , Células HEK293 , Humanos , Células MCF-7 , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Pliegue de Proteína , Receptores de Prolactina/metabolismoRESUMEN
OBJECTIVE: Fibroblast growth factors (Fgfs) are key orchestrators of development, and a role of Fgfs in tissue repair is emerging. Here we studied the consequences of inducible loss of Fgf receptor (Fgfr) 4, the major Fgf receptor (Fgfr) on hepatocytes, alone or in combination with Fgfr1 and Fgfr2, for liver regeneration after PH. DESIGN: We used siRNA delivered via nanoparticles combined with liver-specific gene knockout to study Fgfr function in liver regeneration. Liver or blood samples were analysed using histology, immunohistochemistry,real-time RT-PCR, western blotting and ELISA. RESULTS: siRNA-mediated knockdown of Fgfr4 severely affected liver regeneration due to impairment of hepatocyte proliferation combined with liver necrosis.Mechanistically, the proliferation defect resulted from inhibition of an Fgf15-Fgfr4-Stat3 signalling pathway,which is required for injury-induced expression of the Foxm1 transcription factor and subsequent cell cycle progression, while elevated levels of intrahepatic toxicbile acids were identified as the likely cause of the necrotic damage. Failure of liver mass restoration in Fgfr4 knockdown mice was prevented at least in part by compensatory hypertrophy of hepatocytes. Most importantly, our data revealed partially redundant functions of Fgf receptors in the liver, since knock down of Fgfr4 in mice lacking Fgfr1 and Fgfr2 in hepatocytes caused liver failure after PH due to severe liver necrosis and a defect in regeneration. CONCLUSIONS: These results demonstrate that Fgfr signalling in hepatocytes is essential for liver regeneration and suggest activation of Fgfr signalling asa promising approach for the improvement of the liver's regenerative capacity.
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Proliferación Celular , Regeneración Hepática/fisiología , Hígado/patología , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Animales , Western Blotting , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Hepatectomía/métodos , Hepatocitos/metabolismo , Hepatocitos/fisiología , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , ARN Interferente Pequeño/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Estadísticas no ParamétricasRESUMEN
A library of dendrimers was synthesized and optimized for targeted small interfering RNA (siRNA) delivery to different cell subpopulations within the liver. Using a combinatorial approach, a library of these nanoparticle-forming materials was produced wherein the free amines on multigenerational poly(amido amine) and poly(propylenimine) dendrimers were substituted with alkyl chains of increasing length, and evaluated for their ability to deliver siRNA to liver cell subpopulations. Interestingly, two lead delivery materials could be formulated in a manner to alter their tissue tropism within the liver-with formulations from the same material capable of preferentially delivering siRNA to 1)â endothelial cells, 2)â endothelial cells and hepatocytes, or 3)â endothelial cells, hepatocytes, and tumor cells inâ vivo. The ability to broaden or narrow the cellular destination of siRNA within the liver may provide a useful tool to address a range of liver diseases.
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Aminas/química , Dendrímeros/química , ARN Interferente Pequeño/metabolismo , Línea Celular Tumoral , Células Endoteliales/citología , Células Endoteliales/metabolismo , Factor VII/antagonistas & inhibidores , Factor VII/genética , Factor VII/metabolismo , Células HeLa , Humanos , Hígado/citología , Nanoestructuras/química , Interferencia de ARN , Transfección , alfa-Fetoproteínas/antagonistas & inhibidores , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMEN
The liver is unique in its capacity to regenerate after injury, during which hepatocytes actively divide and establish cell-cell contacts through cell adhesion complexes. Here, we demonstrate that the loss of α-catenin, a well-established adhesion component, dramatically disrupts liver regeneration. Using a partial hepatectomy model, we show that regenerated livers from α-catenin knockdown mice are grossly larger than control regenerated livers, with an increase in cell size and proliferation. This increased proliferation correlated with increased YAP activation, implicating α-catenin in the Hippo/YAP pathway. Additionally, α-catenin knockdown mice exhibited a phenotype reminiscent of clinical cholestasis, with drastically altered bile canaliculi, elevated levels of bile components and signs of jaundice and inflammation. The disrupted regenerative capacity is a result of actin cytoskeletal disorganisation, leading to a loss of apical microvilli, dilated lumens in the bile canaliculi, and leaky tight junctions. This study illuminates a novel, essential role for α-catenin in liver regeneration.
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Colestasis/genética , Regeneración Hepática/fisiología , alfa Catenina/genética , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Canalículos Biliares/patología , Canalículos Biliares/ultraestructura , Proteínas de Ciclo Celular , Proliferación Celular , Colestasis/sangre , Femenino , Hepatocitos/fisiología , Ratones , Ratones Noqueados , Microvellosidades/ultraestructura , Modelos Animales , Fosfoproteínas/metabolismo , Proteínas Señalizadoras YAP , alfa Catenina/deficienciaRESUMEN
Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.
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Proteínas Argonautas/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Fibroblastos/citología , Hígado/metabolismo , ARN Mensajero/genética , Animales , Proteínas Argonautas/genética , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Fibroblastos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Interferencia de ARNRESUMEN
The liver has a unique regenerative capability, which involves extensive remodelling of cell-cell and cell-matrix contacts. Here we study the role of integrins in mouse liver regeneration using Cre/loxP-mediated gene deletion or intravenous delivery of ß1-integrin siRNA formulated into nanoparticles that predominantly target hepatocytes. We show that although short-term loss of ß1-integrin has no obvious consequences for normal livers, partial hepatectomy leads to severe liver necrosis and reduced hepatocyte proliferation. Mechanistically, loss of ß1-integrin in hepatocytes impairs ligand-induced phosphorylation of the epidermal growth factor and hepatocyte growth factor receptors, thereby attenuating downstream receptor signalling in vitro and in vivo. These results identify a crucial role and novel mechanism of action of ß1-integrins in liver regeneration and demonstrate that protein depletion by nanoparticle-based delivery of specific siRNA is a powerful strategy to study gene function in the regenerating liver.
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Proliferación Celular/genética , Hepatocitos/metabolismo , Integrina beta1/genética , Regeneración Hepática/genética , Hígado/metabolismo , Animales , Receptores ErbB/metabolismo , Técnicas de Silenciamiento del Gen , Hepatectomía , Factor de Crecimiento de Hepatocito , Integrina beta1/metabolismo , Hígado/cirugía , Ratones , Ratones Noqueados , Transducción de Señal/genéticaRESUMEN
Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.
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Células Endoteliales/metabolismo , Nanopartículas/química , Polímeros/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular , Humanos , Ratones , Nanopartículas/ultraestructura , Neoplasias/genética , Neoplasias/terapia , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Integrins play an important role during development, regulating cell differentiation, proliferation and survival. Here we show that knockdown of integrin subunits slows down the progression of hepatocellular carcinoma (HCC). Using nanoparticulate delivery of short interfering RNAs targeting ß1 and αv integrin subunits, we downregulate all integrin receptors in hepatocytes. Short-term integrin knockdown (2 weeks) does not cause apparent structural or functional perturbations of normal liver tissue. Alterations in liver morphology accumulate on sustained integrin downregulation (7 weeks). The integrin knockdown leads to significant retardation of HCC progression, reducing proliferation and increasing tumour cell death. This tumour retardation is accompanied by reduced activation of the MET oncogene as well as expression of its mature form on the cell surface. Our data suggest that transformed proliferating cells from HCC are more sensitive to knockdown of integrins than normal quiescent hepatocytes, highlighting the potential of small interfering RNA-mediated inhibition of integrins as an anti-cancer therapeutic approach.
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Carcinoma Hepatocelular/genética , Hepatocitos/metabolismo , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/genética , Neoplasias Hepáticas Experimentales/genética , Nanopartículas , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Interferente Pequeño , Animales , Proliferación Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , RatonesRESUMEN
We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in â¼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.