RESUMEN
CRISPR/Cas9 technology allows facile modification of the genome in virtually any desired way through the use of easily designed plasmid constructs that express a gRNA targeting a genomic site-of-interest and Cas9. Hydrodynamic tail vein injection, on the other hand, is a simple method to deliver "naked" plasmid DNA to 5-40% of the hepatocytes of the liver of adult mice. Here, we describe how these two techniques can be combined to create a workflow for fast, easy, and cost-efficient in vivo genome editing of the adult mouse liver. Using this method, large cohorts of mice with genetically modified livers can be established within 3 weeks to generate models for gene function in normal physiology and diseases of the liver.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Animales , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones , Plásmidos/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. RESULTS: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Edición Génica/métodos , Fusión Génica , Proteínas del Choque Térmico HSP40/genética , Neoplasias Hepáticas/genética , Animales , Biomarcadores de Tumor/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas del Choque Térmico HSP40/metabolismo , Neoplasias Hepáticas/metabolismo , Ratones , Fenotipo , Factores de TiempoRESUMEN
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator. Second, FACS enrichment of cells expressing nucleases linked to fluorescent proteins can be used to maximize knockout or knock-in editing efficiencies or to balance editing efficiency and toxic/off-target effects. The two methods can be combined to form a pipeline for cell-line editing that facilitates the testing of new nuclease reagents and the generation of edited cell pools or clonal cell lines, reducing the number of clones that need to be generated and increasing the ease with which they are screened. The pipeline shortens the time line, but it most prominently reduces the workload of cell-line editing, which may be completed within 4 weeks.
Asunto(s)
Análisis Mutacional de ADN/métodos , Desoxirribonucleasas/metabolismo , Citometría de Flujo/métodos , Edición Génica/métodos , Genómica/métodos , Mutación INDEL , Animales , Células CHO , Cricetinae , Cricetulus , Técnicas de Sustitución del Gen , Técnicas de Inactivación de GenesRESUMEN
Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Neocórtex/embriología , Neocórtex/metabolismo , Proteínas de Unión al ARN/genética , Animales , Apoptosis/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Eliminación de Gen , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Neocórtex/patología , Proteínas del Tejido Nervioso , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Interferencia de ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Dominio T Box , Factores de Transcripción/metabolismoRESUMEN
Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment.
Asunto(s)
Endolina/genética , Animales , Secuencia de Bases , Línea Celular , Codón sin Sentido/genética , Sordera/genética , Dinamarca , Familia , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Repeticiones de Microsatélite/genética , Órgano Espiral/metabolismo , Linaje , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADNRESUMEN
The nuclease-based gene editing tools are rapidly transforming capabilities for altering the genome of cells and organisms with great precision and in high throughput studies. A major limitation in application of precise gene editing lies in lack of sensitive and fast methods to detect and characterize the induced DNA changes. Precise gene editing induces double-stranded DNA breaks that are repaired by error-prone non-homologous end joining leading to introduction of insertions and deletions (indels) at the target site. These indels are often small and difficult and laborious to detect by traditional methods. Here we present a method for fast, sensitive and simple indel detection that accurately defines indel sizes down to ±1 bp. The method coined IDAA for Indel Detection by Amplicon Analysis is based on tri-primer amplicon labelling and DNA capillary electrophoresis detection, and IDAA is amenable for high throughput analysis.
Asunto(s)
Análisis Mutacional de ADN/métodos , Mutación INDEL , Animales , Células CHO , Sistemas CRISPR-Cas , Línea Celular , Cricetulus , Electroforesis Capilar , Marcación de Gen , Cobayas , Humanos , Ratones , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.
Asunto(s)
Neoplasias Encefálicas/genética , Genómica , Glioblastoma/genética , Cateninas/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Factores de Transcripción/genética , Catenina deltaRESUMEN
Mesenchymal transformation is a hallmark of aggressive glioblastoma (GBM). Here, we report the development of an unbiased method for computational integration of copy number variation, expression, and mutation data from large datasets. Using this method, we identified rhophilin 2 (RHPN2) as a central genetic determinant of the mesenchymal phenotype of human GBM. Notably, amplification of the human RHPN2 gene on chromosome 19 correlates with a dramatic decrease in the survival of patients with glioma. Ectopic expression of RHPN2 in neural stem cells and astrocytes triggered the expression of mesenchymal genes and promoted an invasive phenotype without impacting cell proliferation. Mechanistically, these effects were implemented through RHPN2-mediated activation of RhoA, a master regulator of cell migration and invasion. Our results define RHPN2 amplification as a central genetic determinant of a highly aggressive phenotype that directs the worst clinical outcomes in patients with GBM.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Transformación Celular Neoplásica/patología , Glioblastoma/patología , Células Madre Mesenquimatosas/patología , Proteína de Unión al GTP rhoA/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/metabolismo , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Células HEK293 , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Mutación , Invasividad Neoplásica , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Fenotipo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
High-grade gliomas (HGGs) are incurable brain tumors that are characterized by the presence of glioma-initiating cells (GICs). GICs are essential to tumor aggressiveness and retain the capacity for self-renewal and multilineage differentiation as long as they reside in the perivascular niche. ID proteins are master regulators of stemness and anchorage to the extracellular niche microenvironment, suggesting that they may play a role in maintaining GICs. Here, we modeled the probable therapeutic impact of ID inactivation in HGG by selective ablation of Id in tumor cells and after tumor initiation in a new mouse model of human mesenchymal HGG. Deletion of 3 Id genes induced rapid release of GICs from the perivascular niche, followed by tumor regression. GIC displacement was mediated by derepression of Rap1gap and subsequent inhibition of RAP1, a master regulator of cell adhesion. We identified a signature module of 5 genes in the ID pathway, including RAP1GAP, which segregated 2 subgroups of glioma patients with markedly different clinical outcomes. The model-informed survival analysis together with genetic and functional studies establish that ID activity is required for the maintenance of mesenchymal HGG and suggest that pharmacological inactivation of ID proteins could serve as a therapeutic strategy.
Asunto(s)
Glioma/metabolismo , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Modelos Biológicos , Proteínas de Neoplasias/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Animales , Línea Celular Tumoral , Supervivencia sin Enfermedad , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Eliminación de Gen , Glioma/genética , Glioma/mortalidad , Glioma/terapia , Células HEK293 , Humanos , Proteína 1 Inhibidora de la Diferenciación/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Complejo Shelterina , Tasa de Supervivencia , Proteínas de Unión a Telómeros/genéticaRESUMEN
The brain tumor glioblastoma multiforme (GBM) is among the most lethal forms of human cancer. Here, we report that a small subset of GBMs (3.1%; 3 of 97 tumors examined) harbors oncogenic chromosomal translocations that fuse in-frame the tyrosine kinase coding domains of fibroblast growth factor receptor (FGFR) genes (FGFR1 or FGFR3) to the transforming acidic coiled-coil (TACC) coding domains of TACC1 or TACC3, respectively. The FGFR-TACC fusion protein displays oncogenic activity when introduced into astrocytes or stereotactically transduced in the mouse brain. The fusion protein, which localizes to mitotic spindle poles, has constitutive kinase activity and induces mitotic and chromosomal segregation defects and triggers aneuploidy. Inhibition of FGFR kinase corrects the aneuploidy, and oral administration of an FGFR inhibitor prolongs survival of mice harboring intracranial FGFR3-TACC3-initiated glioma. FGFR-TACC fusions could potentially identify a subset of GBM patients who would benefit from targeted FGFR kinase inhibition.
Asunto(s)
Transformación Celular Neoplásica , Proteínas Fetales/genética , Glioblastoma/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Aneuploidia , Animales , Antineoplásicos/farmacología , Benzamidas/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Inestabilidad Cromosómica , Inhibidores Enzimáticos/farmacología , Proteínas Fetales/química , Proteínas Fetales/metabolismo , Glioblastoma/metabolismo , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis , Trasplante de Neoplasias , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fusión de Oncogenes , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Piperazinas/farmacología , Estructura Terciaria de Proteína , Pirazoles/farmacología , Pirimidinas/farmacología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/química , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Huso Acromático/metabolismo , Translocación Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Stem-cell functions require activation of stem-cell-intrinsic transcriptional programs and extracellular interaction with a niche microenvironment. How the transcriptional machinery controls residency of stem cells in the niche is unknown. Here we show that Id proteins coordinate stem-cell activities with anchorage of neural stem cells (NSCs) to the niche. Conditional inactivation of three Id genes in NSCs triggered detachment of embryonic and postnatal NSCs from the ventricular and vascular niche, respectively. The interrogation of the gene modules directly targeted by Id deletion in NSCs revealed that Id proteins repress bHLH-mediated activation of Rap1GAP, thus serving to maintain the GTPase activity of RAP1, a key mediator of cell adhesion. Preventing the elevation of the Rap1GAP level countered the consequences of Id loss on NSC-niche interaction and stem-cell identity. Thus, by preserving anchorage of NSCs to the extracellular environment, Id activity synchronizes NSC functions to residency in the specialized niche.
Asunto(s)
Antígenos de Neoplasias/fisiología , Adhesión Celular/fisiología , Células-Madre Neurales/citología , Animales , Antígenos de Neoplasias/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Activadoras de GTPasa/genética , RatonesRESUMEN
Since it was suggested that cobalt chloride (CoCl(2)) could mimic the O(2) sensing role of mitochondria by increasing reactive oxygen species (ROS) generation during normoxia, we studied the correlation between CoCl(2)-generation of free radicals and the induction of a hypoxic cellular response in myogenic cell lines. In both L6C5 and C2C12 cell lines, exposure to CoCl(2) induced an increase of intracellular oxidants, the accumulation of HIF-1alpha protein, and the expression of vascular endothelial growth factor (VEGF) and/or iNOS genes. On the other hand, only ascorbic acid, but not trolox, was effective in lowering the CoCl(2) gene up-regulation. Neither the cytotoxicity nor the apoptosis induced by CoCl(2) in skeletal muscle cells were modified by culture supplementation with either ascorbic acid or trolox. Thus, CoCl(2) treatment of myogenic cell lines may represent a useful and convenient in vitro model to study gene modulation induced by hypoxia in skeletal muscle, although cellular loss induced by this metal may involve mechanisms other than HIF-1alpha stabilization. It is unlikely, however, that ROS would represent the main mediators of CoCl(2) effects on muscle cells.
Asunto(s)
Apoptosis , Cobalto/farmacología , Radicales Libres , Hipoxia , Músculo Esquelético/patología , Regulación hacia Arriba , Animales , Gatos , Relación Dosis-Respuesta a Droga , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Oxígeno/metabolismo , Ratas , Especies Reactivas de Oxígeno , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Angiogenesis is required for the development and biologic progression of glioblastoma multiform (GBM), which is the most malignant infiltrative astrocytoma. Vascular endothelial growth factor (VEGF) plays a predominant role in the increased vascularity and endothelial cell proliferation in GBMs driven by the expression of pro-angiogenic cytokines. In this study, we employed a vector-encoded VEGF siRNA to impair VEGF secretion from U87 human glioblastoma cells. The direct intra-tumor injection of a siRNA-encoding plasmid complexed with linear polyethylenimine (PEI) efficiently reduced the vascularization of treated tumors in xenografts established in SCID mice by subcutaneous inoculation of U87 cells, but was not able to reduce tumor growth. We then sought to strengthen the in vivo action of our siRNA by coupling it to a well known direct antiangiogenic agent, mouse interleukin 4 (mIL4). We infected U87 cells with a retroviral vector coexpressing the VEGF siRNA and mIL4 and produced stable cell lines that we used for an in vivo experiment of subcutaneous injection in SCID mice. In this setting, the concomitant expression of mIL4 and siRNA totally abolished the growth of subcutaneous tumors. These results suggest that our retroviral vector might be employed as a potential tool in future antiangiogenic gene therapy trials for glioblastoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Terapia Genética , Glioblastoma/terapia , Interleucina-4/uso terapéutico , Neovascularización Patológica/terapia , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/tratamiento farmacológico , Terapia Combinada , Regulación hacia Abajo , Glioblastoma/irrigación sanguínea , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Plásmidos/administración & dosificación , Plásmidos/genética , ARN Interferente Pequeño/genética , Retroviridae/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer.
Asunto(s)
Vectores Genéticos/genética , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/patología , Interferencia de ARN , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Humanos , Masculino , Ratones , Ratones SCID , Datos de Secuencia Molecular , Plásmidos/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Ratas , Alineación de Secuencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis, where it functions as one of the major angiogenic factors sustaining growth and draining catabolites. In this study, we developed an anti-VEGF ribozyme targeted to the 5' part of human VEGF mRNA. We endowed this ribozyme with an additional feature expected to improve its activity in vivo, by cloning it into a VAI transcriptional cassette. VAI is originally part of the adenovirus genome, and is characterized by high transcription rates, good stability due to its strong secondary structure and cytoplasmic localization. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secreted in the extracellular medium, indicating a good biological activity of the ribozyme. Moreover, this reduction in VEGF secretion had the important functional consequence of drastically diminishing the formation of tube-like structures of human umbilical vascular endothelial cells in a Matrigel in vitro angiogenesis assay. In conclusion, our VAI-embedded anti-VEGF ribozyme is a good inhibitor of angiogenesis in vitro, in a glioblastoma cell context. Thus, it may represent a useful tool for future applications in vivo, for antiangiogenic gene therapy of glioblastoma and of highly vascularized tumors.