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1.
Sci Rep ; 9(1): 14010, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570767

RESUMO

Chronic Kidney Disease is a growing problem across the world and can lead to end-stage kidney disease and cardiovascular disease. Fibrosis is the underlying mechanism that leads to organ dysfunction, but as yet we have no therapeutics that can influence this process. Ras monomeric GTPases are master regulators that direct many of the cytokines known to drive fibrosis to downstream effector cascades. We have previously shown that K-Ras is a key isoform that drives fibrosis in the kidney. Here we demonstrate that K-Ras expression and activation are increased in rodent models of CKD. By knocking down expression of K-Ras using antisense oligonucleotides in a mouse model of chronic folic acid nephropathy we can reduce fibrosis by 50% and prevent the loss of renal function over 3 months. In addition, we have demonstrated in vitro and in vivo that reduction of K-Ras expression is associated with a reduction in Jag1 expression; we hypothesise this is the mechanism by which targeting K-Ras has therapeutic benefit. In conclusion, targeting K-Ras expression with antisense oligonucleotides in a mouse model of CKD prevents fibrosis and protects against renal dysfunction.


Assuntos
Ácido Fólico/toxicidade , Rim/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Insuficiência Renal Crônica/prevenção & controle , Injúria Renal Aguda/complicações , Animais , Modelos Animais de Doenças , Fibrose , Técnicas de Silenciamento de Genes , Proteína Jagged-1/metabolismo , Masculino , Camundongos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/patologia
2.
Sci Transl Med ; 10(465)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30381411

RESUMO

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant neurodegenerative disorder characterized by cerebellar and retinal degeneration, and is caused by a CAG-polyglutamine repeat expansion in the ATAXIN-7 gene. Patients with SCA7 develop progressive cone-rod dystrophy, typically resulting in blindness. Antisense oligonucleotides (ASOs) are single-stranded chemically modified nucleic acids designed to mediate the destruction, prevent the translation, or modify the processing of targeted RNAs. Here, we evaluated ASOs as treatments for SCA7 retinal degeneration in representative mouse models of the disease after injection into the vitreous humor of the eye. Using Ataxin-7 aggregation, visual function, retinal histopathology, gene expression, and epigenetic dysregulation as outcome measures, we found that ASO-mediated Ataxin-7 knockdown yielded improvements in treated SCA7 mice. In SCA7 mice with retinal disease, intravitreal injection of Ataxin-7 ASOs also improved visual function despite initiating treatment after symptom onset. Using color fundus photography and autofluorescence imaging, we also determined the nature of retinal degeneration in human SCA7 patients. We observed variable disease severity and cataloged rapidly progressive retinal degeneration. Given the accessibility of neural retina, availability of objective, quantitative readouts for monitoring therapeutic response, and the rapid disease progression in SCA7, ASOs targeting ATAXIN-7 might represent a viable treatment for SCA7 retinal degeneration.


Assuntos
Ataxina-7/metabolismo , Proteínas Mutantes/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ataxias Espinocerebelares/fisiopatologia , Visão Ocular/efeitos dos fármacos , Animais , Ataxina-7/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Peptídeos/metabolismo , Fenótipo , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Agregados Proteicos/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/complicações , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Ataxias Espinocerebelares/complicações , Ataxias Espinocerebelares/patologia
3.
Nucleic Acid Ther ; 27(5): 272-284, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28605247

RESUMO

Antisense oligonucleotides (ASOs) are widely accepted therapeutic agents that suppress RNA transcription. While the majority of ASOs are well tolerated in vivo, few sequences trigger inflammatory responses in absence of conventional CpG motifs. In this study, we identified non-CpG oligodeoxy-nucleotide (ODN) capable of triggering an inflammatory response resulting in B cell and macrophage activation in a MyD88- and TLR9-dependent manner. In addition, we found the receptor for advance glycation end product (RAGE) receptor to be involved in the initiation of inflammatory response to suboptimal concentrations of both CpG- and non-CpG-containing ODNs. In contrast, dosing RAGE KO mice with high doses of CpG or non-CpG ODNs lead to a stronger inflammatory response than observed in wild-type mice. Together, our data provide a previously uncharacterized in vivo mechanism contingent on ODN-administered dose, where TLR9 governs the primary response and RAGE plays a distinct and cooperative function in providing a pivotal role in balancing the immune response.


Assuntos
Imunidade Celular/imunologia , Inflamação/imunologia , Oligonucleotídeos Antissenso/uso terapêutico , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Linfócitos B/imunologia , Citocinas/sangue , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Cultura Primária de Células , RNA/genética , RNA/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Análise de Célula Única , Receptor Toll-Like 9/genética , Transcrição Gênica
4.
Cell Rep ; 17(1): 125-136, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27681426

RESUMO

Spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder, is caused by a CAG/glutamine expansion in the androgen receptor (polyQ AR). Recent studies demonstrate that skeletal muscle is an important site of toxicity that contributes to the SBMA phenotype. Here, we sought to identify critical pathways altered in muscle that underlie disease manifestations in AR113Q mice. This led to the unanticipated identification of gene expression changes affecting regulators of carbohydrate metabolism, similar to those triggered by denervation. AR113Q muscle exhibits diminished glycolysis, altered mitochondria, and an impaired response to exercise. Strikingly, the expression of genes regulating muscle energy metabolism is rescued following peripheral polyQ AR gene silencing by antisense oligonucleotides (ASO), a therapeutic strategy that alleviates disease. Our data establish the occurrence of a metabolic imbalance in SBMA muscle triggered by peripheral expression of the polyQ AR and indicate that alterations in energy utilization contribute to non-neuronal disease manifestations.


Assuntos
Inativação Gênica , Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/farmacologia , Receptores Androgênicos/genética , Animais , Metabolismo dos Carboidratos/genética , Ciclo do Ácido Cítrico/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Glicólise/genética , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Músculo Esquelético , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Peptídeos/química , Peptídeos/metabolismo , Condicionamento Físico Animal , Receptores Androgênicos/metabolismo
5.
Nucleic Acid Ther ; 26(4): 236-49, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27140858

RESUMO

The effects of repeated complement activation in cynomolgus monkeys after chronic antisense oligonucleotide (ASO) treatment were evaluated by using ISIS 104838, a representative 2'-O-methoxyethyl (2'-MOE) modified ASO. The treatment was up to 9 months with a total weekly dose of 30 mg/kg, given either as daily [4.3 mg/kg/day, subcutaneous (s.c.) injection] or once weekly [30 mg/kg, either as s.c. injection or 30-min intravenous (i.v.) infusion]. Acute elevations of complement split products (Bb and C3a) and a transient decrease in C3 occurred after the first dose and were drug plasma concentration dependent. However, with repeated complement activation after chronic ASO treatment, there were progressive increases in basal (predose) levels of Bb and C3a, and a sustained C3 reduction in all treated groups. There was also a progressive increase in C3d-bound circulating immune complex (CIC) that was considered secondary to the C3 depletion. Evidence of vascular inflammation was observed, mostly in the liver, kidney, and heart, and correlated with severe C3 depletion and increases in plasma IgG and IgM. Vascular inflammation was accompanied by increased C3 and IgM immunereactivity in the affected vasculatures and endothelial activation markers in serum. In summary, repeated complement activations in monkeys lead to a sustained decrease in circulating C3 over time. The concomitantly increased inflammatory signals and decreased CIC clearance due to impairment of complement function may lead to vascular inflammation after chronic ASO treatment in monkeys. However, based on the known sensitivity of monkeys to ASO-induced complement activation, these findings have limited relevance to humans.


Assuntos
Ativação do Complemento/efeitos dos fármacos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Fosforotioatos/administração & dosagem , Fator de Necrose Tumoral alfa/genética , Animais , Relação Dose-Resposta a Droga , Coração/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Macaca fascicularis , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Fosforotioatos/química , Fator de Necrose Tumoral alfa/antagonistas & inibidores
6.
Neuron ; 82(2): 295-307, 2014 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-24742458

RESUMO

X-linked spinal and bulbar muscular atrophy (SBMA) is characterized by adult-onset muscle weakness and lower motor neuron degeneration. SBMA is caused by CAG-polyglutamine (polyQ) repeat expansions in the androgen receptor (AR) gene. Pathological findings include motor neuron loss, with polyQ-AR accumulation in intranuclear inclusions. SBMA patients exhibit myopathic features, suggesting a role for muscle in disease pathogenesis. To determine the contribution of muscle, we developed a BAC mouse model featuring a floxed first exon to permit cell-type-specific excision of human AR121Q. BAC fxAR121 mice develop systemic and neuromuscular phenotypes, including shortened survival. After validating termination of AR121 expression and full rescue with ubiquitous Cre, we crossed BAC fxAR121 mice with Human Skeletal Actin-Cre mice. Muscle-specific excision prevented weight loss, motor phenotypes, muscle pathology, and motor neuronopathy and dramatically extended survival. Our results reveal a crucial role for muscle expression of polyQ-AR in SBMA and suggest muscle-directed therapies as effective treatments.


Assuntos
Encéfalo/patologia , Músculo Esquelético/metabolismo , Transtornos Musculares Atróficos/genética , Transtornos Musculares Atróficos/patologia , Peptídeos/genética , Receptores Androgênicos/genética , Actinas/genética , Actinas/metabolismo , Fatores Etários , Animais , Peso Corporal/genética , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Força Muscular/genética , Músculo Esquelético/patologia , Transtornos Musculares Atróficos/complicações , Fenótipo , Receptores Androgênicos/metabolismo
7.
J Clin Invest ; 124(1): 251-61, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24355919

RESUMO

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.


Assuntos
Oligonucleotídeos Antissenso/genética , Deficiência de alfa 1-Antitripsina/terapia , Animais , Feminino , Técnicas de Silenciamento de Genes , Células Hep G2 , Hepatócitos/enzimologia , Humanos , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática/enzimologia , Cirrose Hepática/genética , Cirrose Hepática/terapia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/enzimologia , Deficiência de alfa 1-Antitripsina/genética
8.
Nucleic Acid Ther ; 23(6): 369-78, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24161045

RESUMO

Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.


Assuntos
Técnicas de Silenciamento de Genes , Oligonucleotídeos Antissenso/administração & dosagem , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Animais , Células Cultivadas , Hibridização In Situ , Injeções Subcutâneas , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Sensibilidade e Especificidade
9.
J Huntingtons Dis ; 2(2): 217-28, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-25063516

RESUMO

BACKGROUND: Huntington's disease (HD) is a neurological disorder caused by mutations in the huntingtin (HTT) gene, the product of which leads to selective and progressive neuronal cell death in the striatum and cortex. Transcriptional dysregulation has emerged as a core pathologic feature in the CNS of human and animal models of HD. It is still unclear whether perturbations in gene expression are a consequence of the disease or importantly, contribute to the pathogenesis of HD. OBJECTIVE: To examine if transcriptional dysregulation can be ameliorated with antisense oligonucleotides that reduce levels of mutant Htt and provide therapeutic benefit in the YAC128 mouse model of HD. METHODS: Quantitative real-time PCR analysis was used to evaluate dysregulation of a subset of striatal genes in the YAC128 mouse model. Transcripts were then evaluated following ICV delivery of antisense oligonucleotides (ASO). Rota rod and Porsolt swim tests were used to evaluate phenotypic deficits in these mice following ASO treatment. RESULTS: Transcriptional dysregulation was detected in the YAC128 mouse model and appears to progress with age. ICV delivery of ASOs directed against mutant Htt resulted in reduction in mutant Htt levels and amelioration in behavioral deficits in the YAC128 mouse model. These improvements were correlated with improvements in the levels of several dysregulated striatal transcripts. CONCLUSIONS: The role of transcriptional dysregulation in the pathogenesis of Huntington's disease is not well understood, however, a wealth of evidence now strongly suggests that changes in transcriptional signatures are a prominent feature in the brains of both HD patients and animal models of the disease. Our study is the first to show that a therapeutic agent capable of improving an HD disease phenotype is concomitantly correlated with normalization of a subset of dysregulated striatal transcripts. Our data suggests that correction of these disease-altered transcripts may underlie, at least in part, the therapeutic efficacy shown associated with ASO-mediated correction of HD phenotypes and may provide a novel set of early biomarkers for evaluating future therapeutic concepts for HD.


Assuntos
Comportamento Animal/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Doença de Huntington/genética , Destreza Motora/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas Nucleares/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Animais , Modelos Animais de Doenças , Fosfoproteína 32 Regulada por cAMP e Dopamina/efeitos dos fármacos , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Encefalinas/efeitos dos fármacos , Encefalinas/genética , Proteína Huntingtina , Hipoxantina Fosforribosiltransferase/efeitos dos fármacos , Hipoxantina Fosforribosiltransferase/genética , Infusões Intraventriculares , Camundongos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor CB1 de Canabinoide/efeitos dos fármacos , Receptor CB1 de Canabinoide/genética , Receptores de Dopamina D1/efeitos dos fármacos , Receptores de Dopamina D1/genética , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/genética
10.
Cancer Res ; 73(3): 1180-9, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23243023

RESUMO

The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), also known as MALAT-1 or NEAT2 (nuclear-enriched abundant transcript 2), is a highly conserved nuclear noncoding RNA (ncRNA) and a predictive marker for metastasis development in lung cancer. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements using zinc finger nucleases. The achieved 1,000-fold MALAT1 silencing provides a unique loss-of-function model. Proposed mechanisms of action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but actively regulates gene expression including a set of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft. Antisense oligonucleotides (ASO) blocking MALAT1 prevent metastasis formation after tumor implantation. Thus, targeting MALAT1 with ASOs provides a potential therapeutic approach to prevent lung cancer metastasis with this ncRNA serving as both predictive marker and therapeutic target. Finally, regulating gene expression, but not alternative splicing, is the critical function of MALAT1 in lung cancer metastasis. In summary, 10 years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unravels the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis.


Assuntos
Neoplasias Pulmonares/secundário , RNA Longo não Codificante/fisiologia , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Movimento Celular , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , RNA Longo não Codificante/antagonistas & inibidores
11.
Cell Rep ; 2(1): 111-23, 2012 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-22840402

RESUMO

Genome-wide studies have identified thousands of long noncoding RNAs (lncRNAs) lacking protein-coding capacity. However, most lncRNAs are expressed at a very low level, and in most cases there is no genetic evidence to support their in vivo function. Malat1 (metastasis associated lung adenocarcinoma transcript 1) is among the most abundant and highly conserved lncRNAs, and it exhibits an uncommon 3'-end processing mechanism. In addition, its specific nuclear localization, developmental regulation, and dysregulation in cancer are suggestive of it having a critical biological function. We have characterized a Malat1 loss-of-function genetic model that indicates that Malat1 is not essential for mouse pre- and postnatal development. Furthermore, depletion of Malat1 does not affect global gene expression, splicing factor level and phosphorylation status, or alternative pre-mRNA splicing. However, among a small number of genes that were dysregulated in adult Malat1 knockout mice, many were Malat1 neighboring genes, thus indicating a potential cis-regulatory role of Malat1 gene transcription.


Assuntos
Crescimento e Desenvolvimento/genética , RNA Longo não Codificante/fisiologia , Sequências Reguladoras de Ácido Ribonucleico/fisiologia , Transcrição Gênica , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Modelos Biológicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Transcrição Gênica/genética
12.
J Pharmacol Exp Ther ; 342(1): 150-62, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22505629

RESUMO

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.


Assuntos
RNA Helicases DEAD-box/imunologia , DNA/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Oligonucleotídeos Antissenso/imunologia , Ribose/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Alanina Transaminase/genética , Alanina Transaminase/imunologia , Alanina Transaminase/metabolismo , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA/genética , DNA/metabolismo , Hepatócitos/imunologia , Hepatócitos/metabolismo , Imunidade Inata/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Oligonucleotídeos Antissenso/genética , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Ribose/genética , Ribose/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo
13.
PLoS One ; 7(2): e29429, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22347364

RESUMO

BACKGROUND: Wig-1 is a transcription factor regulated by p53 that can interact with hnRNP A2/B1, RNA Helicase A, and dsRNAs, which plays an important role in RNA and protein stabilization. in vitro studies have shown that wig-1 binds p53 mRNA and stabilizes it by protecting it from deadenylation. Furthermore, p53 has been implicated as a causal factor in neurodegenerative diseases based in part on its selective regulatory function on gene expression, including genes which, in turn, also possess regulatory functions on gene expression. In this study we focused on the wig-1 transcription factor as a downstream p53 regulated gene and characterized the effects of wig-1 down regulation on gene expression in mouse liver and brain. METHODS AND RESULTS: Antisense oligonucleotides (ASOs) were identified that specifically target mouse wig-1 mRNA and produce a dose-dependent reduction in wig-1 mRNA levels in cell culture. These wig-1 ASOs produced marked reductions in wig-1 levels in liver following intraperitoneal administration and in brain tissue following ASO administration through a single striatal bolus injection in FVB and BACHD mice. Wig-1 suppression was well tolerated and resulted in the reduction of mutant Htt protein levels in BACHD mouse brain but had no effect on normal Htt protein levels nor p53 mRNA or protein levels. Expression microarray analysis was employed to determine the effects of wig-1 suppression on genome-wide expression in mouse liver and brain. Reduction of wig-1 caused both down regulation and up regulation of several genes, and a number of wig-1 regulated genes were identified that potentially links wig-1 various signaling pathways and diseases. CONCLUSION: Antisense oligonucleotides can effectively reduce wig-1 levels in mouse liver and brain, which results in specific changes in gene expression for pathways relevant to both the nervous system and cancer.


Assuntos
Proteínas de Transporte/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Oligonucleotídeos Antissenso/farmacologia , Transdução de Sinais/genética , Animais , Encéfalo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Genômica , Fígado , Camundongos , Neoplasias , Sistema Nervoso , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteína Supressora de Tumor p53
14.
Amyloid ; 17(2): 43-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20462362

RESUMO

Leptomeningeal amyloidosis associated with mutations in transthyretin (TTR) is a rare but fatal form of amyloidosis. Dementia and intracerebral haemorrhage are prominent features of this disease for which no specific therapy is known. In previous studies, we have shown that antisense oligonucleotides (ASOs) specific for human TTR could inhibit hepatic synthesis of TTR in mice transgenic for a human amyloid-associated TTR and may offer a medical means of treating systemic TTR amyloidosis. Parenteral administration of TTR-specific ASO, however, had no effect on the expression of TTR by the choroid plexus, which is believed to be the source of the amyloid protein in patients who have leptomeningeal amyloidosis. In the present study, mice transgenic for the human TTR amyloid-associated mutation Ile84Ser were treated by administration of TTR-specific ASO (50 microg or 75 microg per day) via an osmotic pump into the cerebral ventricular system over a 4-week period. Intraventricular administration of TTR-specific ASO significantly reduced choroid human TTR mRNA levels, and these findings correlated with decreased TTR in choroid plexus epithelial cells as demonstrated by immunohistochemistry. Suppression of choroid TTR expression by intraventricular administered ASO may offer a medical means of treating leptomeningeal amyloidosis.


Assuntos
Plexo Corióideo/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Pré-Albumina/biossíntese , Neuropatias Amiloides Familiares/tratamento farmacológico , Animais , Plexo Corióideo/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Pré-Albumina/genética
15.
Proc Natl Acad Sci U S A ; 103(12): 4463-8, 2006 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-16537381

RESUMO

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.


Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Neurofibromina 2/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Mutação , Neurofibromina 2/genética , Ratos , Proteínas Supressoras de Tumor/genética , MAP Quinase Quinase Quinase 11 Ativada por Mitógeno
16.
Int J Oncol ; 23(6): 1493-500, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14612918

RESUMO

The loss of NF2 gene function leads to vestibular nerve schwannoma formation in humans. The NF2 gene product, Merlin/Schwannomin, has recently been found to interact with the two PDZ domains containing protein EBP50/NHE-RF, which is itself known to interact with the PDGF receptor (PDGFR) in several cell types. In this study, an up-regulation of both PDGFR and EBP50/NHE-RF, and an interaction of both proteins were found in primary human schwannoma tissue. Furthermore, using an adenoviral vector mediated gene transfer technique, changes in the phenotypic characteristics after NF2 gene restoration in a newly established NF2 gene-mutated human schwannoma cell line (HEI 193) were investigated. The overexpression of Merlin/Schwannomin in HEI 193 led to an inhibition of cell proliferation under serum-free conditions. Upon PDGF stimulation in culture, Merlin/Schwannomin appeared to inhibit the activation of the MAPK and PI3K signaling pathways, impinging on the phosphorylation of Erk 1/2 and Akt, respectively. The data also show that PDGFR is more rapidly internalized by the schwannoma cells overexpressing NF2. Therefore, this process is suggested as a model for a mechanism of Merlin/Schwannomin tumor suppressor function, which intermediates acceleration of the cell surface growth factor degradation.


Assuntos
Genes da Neurofibromatose 2 , Neurilemoma/tratamento farmacológico , Neurilemoma/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Adenoviridae/genética , Western Blotting , Divisão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Meios de Cultura Livres de Soro/farmacologia , Regulação para Baixo , Ativação Enzimática , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde , Humanos , Ligantes , Proteínas Luminescentes/metabolismo , Sistema de Sinalização das MAP Quinases , Modelos Biológicos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/biossíntese , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Trocadores de Sódio-Hidrogênio , Fatores de Tempo , Regulação para Cima
17.
J Virol ; 77(10): 5712-20, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12719564

RESUMO

The colony formation assay is the most commonly used titration method for defining the concentration of replication-incompetent murine leukemia virus-derived retroviral vectors. However, titer varies with target cell type and number, transduction time, and concentration of polycation (e.g., Polybrene). Moreover, because most of the viruses cannot encounter target cells due to Brownian motion, their short half-lives, and the requirement for target cell division for activity, the actual infectious retrovirus concentration in the collected supernatant is higher than the viral titer. Here we correlate the physical viral particle concentration with the infectious virus concentration and colony formation titer with the help of a mathematical model. Ecotropic murine leukemia retrovirus supernatant, collected from the GP+E86/LNCX retroviral vector producer cell line, was concentrated by centrifugation and further purified by a sucrose density gradient. The physical concentration of purified viral vectors was determined by direct particle counting with an electron microscope. The concentrations of fresh and concentrated supernatant were determined by a quantitative reverse transcriptase activity assay. Titration of all supernatants by neomycin-resistant colony formation assay was also performed. There were 767 +/- 517 physical viral particles per infectious CFU in the crude viral supernatant. However, the infectious viral concentration determined by mathematical simulation was 143 viral particles per infectious unit, which is more consistent with the concentration determined by particle counting in purified viral solution. Our results suggest that the mathematical model can be used to extract a more accurate and reliable concentration of infectious retrovirus.


Assuntos
Vetores Genéticos , Modelos Biológicos , Vírus da Leucemia Murina de Moloney/isolamento & purificação , Vírus da Leucemia Murina de Moloney/patogenicidade , Virologia/métodos , Células 3T3 , Animais , Imunofluorescência , Congelamento , Camundongos , Microscopia Eletrônica , Vírus da Leucemia Murina de Moloney/fisiologia , DNA Polimerase Dirigida por RNA/metabolismo , Transdução Genética , Vírion/metabolismo
18.
Laryngoscope ; 112(6): 948-50, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12160289

RESUMO

OBJECTIVE: To evaluate the proliferation potential of recurrent acoustic schwannoma following gamma knife radiosurgery (GKR) versus microsurgery. STUDY DESIGN: Retrospective study. METHODS: A review of surgical records of the House Ear Clinic revealed 8 patients who had undergone GKR and 15 patients who had undergone microsurgery who had unilateral acoustic schwannoma recurrences. Immunohistochemical studies were performed to evaluate the expression of proliferating cell nuclear antigen (PCNA) on archival paraffin-embedded blocks. RESULTS: All 8 GKR and 15 microsurgical tumors had positive staining for PCNA. The recurrent GKR tumors had significantly lower proliferation levels than in the microsurgical group (P = .03). Two GKR tumors had high proliferation levels. CONCLUSIONS: Our study indicates that recurrent vestibular schwannomas treated with GKR have lower proliferation potential as assessed by PCNA compared with recurrences following microsurgery. Radiation-induced apoptosis is thought to contribute to the lower tumor cell proliferation in GKR tumor. The two GKR tumors with high proliferation potential could be a result of radiation-induced sporadic mutation, resulting in high tumor cell proliferation.


Assuntos
Microcirurgia , Neuroma Acústico/patologia , Neuroma Acústico/cirurgia , Radiocirurgia , Apoptose/efeitos da radiação , Humanos , Recidiva Local de Neoplasia , Antígeno Nuclear de Célula em Proliferação/análise , Estudos Retrospectivos
19.
Glia ; 38(4): 363-70, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12007148

RESUMO

Differentiation of primary human vestibular nerve schwannomas (VS) caused by mutations of the NF2 gene was evaluated by examining the expression patterns of genes that are specifically expressed in different stages of Schwann cell lineage. In schwannoma cells that are not in contact with an axon, the expression levels of the major myelin sheath proteins, such as protein zero glycoprotein (P0), myelin basic protein (MBP), and peripheral myelin protein 22 (PMP22), were greatly reduced. However, high expression levels of nerve growth factor receptor (NGFR), neural cell adhesion molecule (N-CAM), and cell adhesion molecule L1 (L1) were observed. In addition, expression of transcription factors Krox20, Krox24, and SCIP/Oct6 was also detected in the tumor cells. These results suggest that loss of the NF2 gene was responsible for the transformation of the Schwann cells into a neoplastic stage that has a similar genetic profile to the pro-myelinating stage. Finally, the primary human vestibular schwannoma cells failed to be regulated and redifferentiated by a regenerating axon, when the human tumors were transplanted into sciatic nerve of nude rat. These results suggest that the NF2 gene might be involved in the differentiation of Schwann cells.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Transformação Celular Neoplásica/genética , Proteínas Imediatamente Precoces , Neurofibromina 2/genética , Neuroma Acústico/genética , Células de Schwann/metabolismo , Células-Tronco/metabolismo , Adulto , Fatores Etários , Animais , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Proteína 2 de Resposta de Crescimento Precoce , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Complexo Antígeno L1 Leucocitário , Glicoproteínas de Membrana/metabolismo , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Regeneração Nervosa/fisiologia , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurofibromina 2/metabolismo , Neuroma Acústico/metabolismo , Neuroma Acústico/patologia , Fator 6 de Transcrição de Octâmero , Ratos , Ratos Nus , Receptor de Fator de Crescimento Neural/metabolismo , Células de Schwann/citologia , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/transplante , Regulação para Cima/fisiologia , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Degeneração Walleriana/patologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-12021506

RESUMO

Histopathological examination of seven temporal bones from patients who underwent a removal of vestibular nerve schwannomas by the translabyrithine or middle fossa approaches has demonstrated small tumor remnants that failed to grow as long as 25 years after surgery. In spite of the high incidence of residual tumors, the clinical recurrence rate of tumors operated at our institution by the translabyrinthine or middle fossa approaches is low (0.3%). Immunohistochemical labeling of dividing cells demonstrated that segments of tumor adjacent to the vestibular nerve and ganglion contained more dividing cells than were present in areas of the tumor at a distance from them.


Assuntos
Neuroma Acústico/patologia , Neuroma Acústico/cirurgia , Gânglio Espiral da Cóclea/patologia , Gânglio Espiral da Cóclea/cirurgia , Nervo Vestibular/patologia , Nervo Vestibular/cirurgia , Idoso , Idoso de 80 Anos ou mais , Divisão Celular , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67/imunologia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasia Residual , Neuroma Acústico/imunologia , Período Pós-Operatório , Antígeno Nuclear de Célula em Proliferação/imunologia , Gânglio Espiral da Cóclea/imunologia , Osso Temporal/imunologia , Osso Temporal/patologia , Nervo Vestibular/imunologia
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