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1.
PLoS Pathog ; 15(5): e1007467, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31075158

RESUMO

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5' genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5' end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5' end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood.


Assuntos
Nucleotídeos de Citosina/genética , Genoma Viral , Hepacivirus/genética , Hepatite C/virologia , MicroRNAs/metabolismo , Mutação , RNA Viral/genética , Sítios de Ligação , Hepatite C/genética , Hepatite C/metabolismo , Interações Hospedeiro-Patógeno , Humanos , MicroRNAs/genética , Replicação Viral
2.
Lancet ; 389(10070): 709-717, 2017 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-28087069

RESUMO

BACKGROUND: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. METHODS: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1). We enrolled men and postmenopausal or hysterectomised women (aged 18-65 years) with chronic HCV genotype 1, 3, or 4 infection diagnosed at least 24 weeks before screening who were either treatment naive to or relapsed after interferon-α based therapy. Patients with co-infection (hepatitis B virus or HIV infection), evidence of decompensated liver disease, or a history of hepatocellular carcinoma were excluded. Randomisation was done by an independent, unblinded, statistician using the SAS procedure Proc Plan. The first cohort received one subcutaneous injection of 2 mg/kg RG-101 or placebo; the second cohort received one subcutaneous injection of 4 mg/kg or placebo. Patients were followed up for 8 weeks (all patients) and up to 76 weeks (patients with no viral rebound and excluding those who were randomised to the placebo group) after randomisation. The primary objective was safety and tolerability of RG-101. This trial was registered with EudraCT, number 2013-002978-49. FINDINGS: Between June 4, 2014, and Oct 27, 2014, we enrolled 32 patients with chronic HCV genotype 1 (n=16), 3 (n=10), or 4 (n=6) infections. In the first cohort, 14 patients were randomly assigned to receive 2 mg/kg RG-101 and two patients were randomly assigned to receive placebo, and in the second cohort, 14 patients were randomly assigned to receive 4 mg/kg RG-101 and two patients were randomly assigned to receive placebo. Overall, 26 of the 28 patients dosed with RG-101 reported at least one treatment-related adverse event. At week 4, the median viral load reduction from baseline was 4·42 (IQR 3·23-5·00) and 5·07 (4·19-5·35) log10 IU/mL in patients dosed with 2 mg/kg RG-101 or 4 mg/kg RG-101. Three patients had undetectable HCV RNA levels 76 weeks after a single dose of RG-101. Viral rebound at or before week 12 was associated with the appearance of resistance associated substitutions in miR-122 binding regions in the 5' UTR of the HCV genome. INTERPRETATION: This study showed that one administration of 2 mg/kg or 4 mg/kg RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated anti-miR-122 oligonucleotide, was well tolerated and resulted in substantial viral load reduction in all treated patients within 4 weeks, and sustained virological response in three patients for 76 weeks. FUNDING: Regulus Therapeutics, Inc.


Assuntos
Hepatite C Crônica/tratamento farmacológico , MicroRNAs/antagonistas & inibidores , MicroRNAs/uso terapêutico , Acetilgalactosamina , Estudos de Coortes , Método Duplo-Cego , Feminino , Humanos , Injeções Subcutâneas , Masculino , MicroRNAs/farmacocinética , Pessoa de Meia-Idade , Oligonucleotídeos , Carga Viral/efeitos dos fármacos
3.
Hepatology ; 66(1): 57-68, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28295463

RESUMO

MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity. Thirty-two chronic HCV patients infected with HCV genotypes 1, 3, and 4 received a single subcutaneous administration of RG-101 at 2 mg/kg (n = 14) or 4 mg/kg (n = 14) or received a placebo (n = 2/dosing group). Plasma and peripheral blood mononuclear cells were collected at multiple time points, and comprehensive immunological analyses were performed. Following RG-101 administration, HCV RNA declined in all patients (mean decline at week 2, 3.27 log10 IU/mL). At week 8 HCV RNA was undetectable in 15/28 patients. Plasma interferon-γ-induced protein 10 (IP-10) levels declined significantly upon dosing with RG-101. Furthermore, the frequency of natural killer (NK) cells increased, the proportion of NK cells expressing activating receptors normalized, and NK cell interferon-γ production decreased after RG-101 dosing. Functional HCV-specific interferon-γ T-cell responses did not significantly change in patients who had undetectable HCV RNA levels by week 8 post-RG-101 injection. No increase in the magnitude of HCV-specific T-cell responses was observed at later time points, including 3 patients who were HCV RNA-negative 76 weeks postdosing. CONCLUSION: Dosing with RG-101 is associated with a restoration of NK-cell proportions and a decrease of NK cells expressing activation receptors; however, the magnitude and functionality of ex vivo HCV-specific T-cell responses did not increase following RG-101 injection, suggesting that NK cells, but not HCV adaptive immunity, may contribute to HCV viral control following RG-101 therapy. (Hepatology 2017;66:57-68).


Assuntos
Hepatite C Crônica/tratamento farmacológico , Células Matadoras Naturais/efeitos dos fármacos , MicroRNAs/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , ELISPOT/métodos , Feminino , Citometria de Fluxo , Seguimentos , Hepatite C Crônica/diagnóstico , Humanos , Injeções Subcutâneas , Células Matadoras Naturais/imunologia , Masculino , MicroRNAs/administração & dosagem , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Países Baixos , Fenótipo , Linfócitos T/imunologia , Resultado do Tratamento
4.
Proc Natl Acad Sci U S A ; 108(51): 20455-60, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22158898

RESUMO

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human ß nerve growth factor (hßNGF). These antibodies, initially naïve to SHM, were subjected to AID-directed SHM in vitro and selected using the same mammalian cell display system, as illustrated by the maturation of one of the antibodies to low pM K(D). This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.


Assuntos
Anticorpos/química , Membrana Celular/metabolismo , Mutação , Hipermutação Somática de Imunoglobulina , Sequência de Aminoácidos , Linfócitos B/imunologia , Citometria de Fluxo/métodos , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina M/química , Cinética , Dados de Sequência Molecular , Fator de Crescimento Neural/química , Homologia de Sequência de Aminoácidos
5.
Nat Cell Biol ; 24(6): 954-967, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35637408

RESUMO

Epidemiological studies demonstrate an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. We show that BC-derived extracellular vesicles (EVs) suppress pancreatic insulin secretion to impair glucose homeostasis. EV-encapsulated miR-122 targets PKM in ß-cells to suppress glycolysis and ATP-dependent insulin exocytosis. Mice receiving high-miR-122 EVs or bearing BC tumours exhibit suppressed insulin secretion, enhanced endogenous glucose production, impaired glucose tolerance and fasting hyperglycaemia. These effects contribute to tumour growth and are abolished by inhibiting EV secretion or miR-122, restoring PKM in ß-cells or supplementing insulin. Compared with non-cancer controls, patients with BC have higher levels of circulating EV-encapsulated miR-122 and fasting glucose concentrations but lower fasting insulin; miR-122 levels are positively associated with glucose and negatively associated with insulin. Therefore, EV-mediated impairment of whole-body glycaemic control may contribute to tumour progression and incidence of type 2 diabetes in some patients with BC.


Assuntos
Neoplasias da Mama , Diabetes Mellitus Tipo 2 , Vesículas Extracelulares , MicroRNAs , Animais , Neoplasias da Mama/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Vesículas Extracelulares/metabolismo , Feminino , Glucose/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Secreção de Insulina , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo
6.
Nat Commun ; 10(1): 4148, 2019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31515477

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD. RGLS4326 is discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and de-represses multiple miR-17 mRNA targets including Pkd1 and Pkd2. Importantly, RGLS4326 demonstrates a favorable preclinical safety profile and attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. The preclinical characteristics of RGLS4326 support its clinical development as a disease-modifying treatment for ADPKD.


Assuntos
MicroRNAs/antagonistas & inibidores , Oligonucleotídeos/uso terapêutico , Doenças Renais Policísticas/tratamento farmacológico , Doenças Renais Policísticas/genética , Animais , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Redes Reguladoras de Genes/efeitos dos fármacos , Células HeLa , Hematopoese/efeitos dos fármacos , Humanos , Túbulos Renais/patologia , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Tecidual/efeitos dos fármacos
7.
J Clin Invest ; 112(2): 244-55, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12865412

RESUMO

Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid-binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription factors CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPdelta as well as SREBP-1c (ADD-1), but not PPARgamma. These changes in C/EBPalpha and C/EBPdelta were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPalpha, C/EBPdelta, SREBP-1, epidermal fatty acid-binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-2 protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Western Blotting , Proteína delta de Ligação ao Facilitador CCAAT , Colágeno/farmacologia , DNA/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Fator 7 de Crescimento de Fibroblastos , Immunoblotting , Hibridização In Situ , Laminina/farmacologia , Metabolismo dos Lipídeos , Receptores X do Fígado , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/química , Receptores Nucleares Órfãos , Fosfolipídeos/metabolismo , Isoformas de Proteínas , Proteoglicanas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/metabolismo
8.
Protein Eng Des Sel ; 26(2): 151-64, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23173178

RESUMO

Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro. By collectively employing these methods, we have engineered a highly stable, high-affinity, full-length antibody with a T(m) over 90°C that retains significant activity after heating to 90°C for 1 h, and has ~95-fold improved antigen-binding affinity. The stabilized IgG framework is compatible with affinity maturation, and should provide a broadly useful scaffold for grafting a variety of complementarity-determining region loops for the development of stable antibodies with desired specificities.


Assuntos
Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Técnicas de Visualização da Superfície Celular , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Simulação por Computador , Cistina/química , Cistina/genética , Evolução Molecular Direcionada , Células HEK293 , Temperatura Alta , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Cinética , Levivirus/imunologia , Camundongos , Modelos Moleculares , Método de Monte Carlo , Mutagênese Sítio-Dirigida , Ligação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Desdobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/genética , Temperatura de Transição
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