RESUMEN
miR-155, a microRNA associated with poor prognosis in lymphoma and leukaemia, has been implicated in the progression of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). In this study, we developed and tested cobomarsen (MRG-106), a locked nucleic acid-modified oligonucleotide inhibitor of miR-155. In MF and human lymphotropic virus type 1 (HTLV-1+) CTCL cell lines in vitro, inhibition of miR-155 with cobomarsen de-repressed direct miR-155 targets, decreased expression of multiple gene pathways associated with cell survival, reduced survival signalling, decreased cell proliferation and activated apoptosis. We identified a set of genes that are significantly regulated by cobomarsen, including direct and downstream targets of miR-155. Using clinical biopsies from MF patients, we demonstrated that expression of these pharmacodynamic biomarkers is dysregulated in MF and associated with miR-155 expression level and MF lesion severity. Further, we demonstrated that miR-155 simultaneously regulates multiple parallel survival pathways (including JAK/STAT, MAPK/ERK and PI3K/AKT) previously associated with the pathogenesis of MF, and that these survival pathways are inhibited by cobomarsen in vitro. A first-in-human phase 1 clinical trial of cobomarsen in patients with CTCL is currently underway, in which the panel of proposed biomarkers will be leveraged to assess pharmacodynamic response to cobomarsen therapy.
Asunto(s)
Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Linfoma Cutáneo de Células T , MicroARNs/antagonistas & inhibidores , Oligonucleótidos/farmacología , ARN Neoplásico/antagonistas & inhibidores , Línea Celular Tumoral , Supervivencia Celular , Ensayos Clínicos Fase I como Asunto , Supervivencia sin Enfermedad , Femenino , Infecciones por HTLV-I/tratamiento farmacológico , Infecciones por HTLV-I/metabolismo , Infecciones por HTLV-I/mortalidad , Infecciones por HTLV-I/patología , Humanos , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/metabolismo , Linfoma Cutáneo de Células T/mortalidad , Linfoma Cutáneo de Células T/patología , Masculino , MicroARNs/metabolismo , ARN Neoplásico/metabolismo , Tasa de SupervivenciaRESUMEN
MicroRNAs (miRNAs) are important regulators of biology and disease. Recent animal efficacy studies validate the therapeutic benefit of miRNA modulation and underscore the therapeutic value of miRNA-targeting oligonucleotides. However, whether disease conditions (stress) influence the pharmacological effects of an anti-miR is currently unknown. To study the effect of disease on target regulation after anti-miR treatment, we injected animals with anti-miR-208a, a synthetic oligonucleotide that inhibits the cardiomyocyte-specific miR-208a. Our data indicate that the presence of stress increases the number of regulated miR-208a targets, and that higher stress levels correlate with stronger target derepression. Additionally, the type of stress also influences which targets are regulated upon miR-208a inhibition. Studies in a large animal model indicate a similar stress-dependent anti-miR effect. Subsequent in vitro studies suggest that the influence of stress on anti-miR efficacy depends at least in part on increased cellular anti-miR uptake. These data indicate that the pharmacological effect of anti-miRs is stronger under disease conditions, and that both the type and severity of disease determine the therapeutic outcome. These facts will be important for assessing the therapeutic dose and predicting the therapeutic outcome when applying anti-miRs in a clinical setting.
Asunto(s)
Antagomirs/genética , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Estrés Fisiológico/genética , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Masculino , Interferencia de ARN , Ratas , PorcinosRESUMEN
RATIONALE: The pathogenesis of pulmonary arterial hypertension (PAH) remains unclear. The 4 microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. OBJECTIVE: To elucidate the transcriptional regulation of the miR-143/145 cluster and the role of miR-143 in PAH. METHODS AND RESULTS: We identified the promoter region that regulates miR-143/145 microRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signaling pathways, including estrogen receptor, liver X factor/retinoic X receptor, transforming growth factor-ß (Smads), and hypoxia (hypoxia response element), that regulated levels of all pri-miR stem loop transcription and resulting microRNA expression. We observed that miR-143-3p is selectively upregulated compared with miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMC-derived exosomes. Using assays with pulmonary arterial endothelial cells, we demonstrated a paracrine promigratory and proangiogenic effect of miR-143-3p-enriched exosomes from PASMC. Quantitative polymerase chain reaction and in situ hybridization showed elevated expression of miR-143 in calf models of PAH and in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role of miR-143 in experimental pulmonary hypertension in vivo in miR-143-/- and anti-miR-143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. CONCLUSIONS: MiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, whereas inhibition of miR-143-3p blocked experimental pulmonary hypertension. Taken together, these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.
Asunto(s)
Comunicación Celular , Células Endoteliales/metabolismo , Hipertensión Pulmonar/metabolismo , MicroARNs/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Presión Arterial , Sitios de Unión , Estudios de Casos y Controles , Bovinos , Movimiento Celular , Células Endoteliales/patología , Exosomas/metabolismo , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/prevención & control , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/patología , Regiones Promotoras Genéticas , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Remodelación Vascular , Función Ventricular Derecha , Presión VentricularRESUMEN
RATIONALE: Myocardial infarction (MI) is a leading cause of death worldwide. Because endogenous cardiac repair mechanisms are not sufficient for meaningful tissue regeneration, MI results in loss of cardiac tissue and detrimental remodeling events. MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in a sequence dependent manner. Our previous data indicate that miRNAs are dysregulated in response to ischemic injury of the heart and actively contribute to cardiac remodeling after MI. OBJECTIVE: This study was designed to determine whether miRNAs are dysregulated on ischemic damage in porcine cardiac tissues and whether locked nucleic acid (LNA)-modified anti-miR chemistries can target cardiac expressed miRNAs to therapeutically inhibit miR-15 on ischemic injury. METHODS AND RESULTS: Our data indicate that the miR-15 family, which includes 6 closely related miRNAs, is regulated in the infarcted region of the heart in response to ischemia-reperfusion injury in mice and pigs. LNA-modified chemistries can effectively silence miR-15 family members in vitro and render cardiomyocytes resistant to hypoxia-induced cardiomyocyte cell death. Correspondingly, systemic delivery of miR-15 anti-miRs dose-dependently represses miR-15 in cardiac tissue of both mice and pigs, whereas therapeutic targeting of miR-15 in mice reduces infarct size and cardiac remodeling and enhances cardiac function in response to MI. CONCLUSIONS: Oligonucleotide-based therapies using LNA-modified chemistries for modulating cardiac miRNAs in the setting of heart disease are efficacious and validate miR-15 as a potential therapeutic target for the manipulation of cardiac remodeling and function in the setting of ischemic injury.
Asunto(s)
MicroARNs/antagonistas & inhibidores , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Animales , Supervivencia Celular/efectos de los fármacos , Femenino , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/efectos de los fármacos , Modelos Animales , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Oligonucleótidos/farmacología , Oligonucleótidos/uso terapéutico , PorcinosRESUMEN
BACKGROUND: Diastolic dysfunction in response to hypertrophy is a major clinical syndrome with few therapeutic options. MicroRNAs act as negative regulators of gene expression by inhibiting translation or promoting degradation of target mRNAs. Previously, we reported that genetic deletion of the cardiac-specific miR-208a prevents pathological cardiac remodeling and upregulation of Myh7 in response to pressure overload. Whether this miRNA might contribute to diastolic dysfunction or other forms of heart disease is currently unknown. METHODS AND RESULTS: Here, we show that systemic delivery of an antisense oligonucleotide induces potent and sustained silencing of miR-208a in the heart. Therapeutic inhibition of miR-208a by subcutaneous delivery of antimiR-208a during hypertension-induced heart failure in Dahl hypertensive rats dose-dependently prevents pathological myosin switching and cardiac remodeling while improving cardiac function, overall health, and survival. Transcriptional profiling indicates that antimiR-208a evokes prominent effects on cardiac gene expression; plasma analysis indicates significant changes in circulating levels of miRNAs on antimiR-208a treatment. CONCLUSIONS: These studies indicate the potential of oligonucleotide-based therapies for modulating cardiac miRNAs and validate miR-208 as a potent therapeutic target for the modulation of cardiac function and remodeling during heart disease progression.
Asunto(s)
Terapia Genética , Insuficiencia Cardíaca Diastólica/tratamiento farmacológico , Corazón/fisiopatología , Terapia Molecular Dirigida , Oligonucleótidos Antisentido/uso terapéutico , Animales , Vías de Administración de Medicamentos , Evaluación Preclínica de Medicamentos , Electrocardiografía , Perfilación de la Expresión Génica , Insuficiencia Cardíaca Diastólica/diagnóstico por imagen , Insuficiencia Cardíaca Diastólica/etiología , Insuficiencia Cardíaca Diastólica/genética , Hipertensión/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/sangre , Cadenas Pesadas de Miosina/biosíntesis , Cadenas Pesadas de Miosina/genética , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Interferencia de ARN , Ratas , Ratas Endogámicas Dahl , Transcripción Genética/efectos de los fármacos , Ultrasonografía , Remodelación Ventricular/efectos de los fármacosRESUMEN
PURPOSE: miRNA-155 is an oncogenic miRNA highly expressed in B-cell malignancies, particularly in the non-germinal center B-cell or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL), where it is considered a potential diagnostic and prognostic biomarker. Thus, miR-155 inhibition represents an important therapeutic strategy for B-cell lymphomas. In this study, we tested the efficacy and pharmacodynamic activity of an oligonucleotide inhibitor of miR-155, cobomarsen, in ABC-DLBCL cell lines and in corresponding xenograft mouse models. In addition, we assessed the therapeutic efficacy and safety of cobomarsen in a patient diagnosed with aggressive ABC-DLBCL. EXPERIMENTAL DESIGN: Preclinical studies included the delivery of cobomarsen to highly miR-155-expressing ABC-DLBCL cell lines to assess any phenotypic changes, as well as intravenous injections of cobomarsen in NSG mice carrying ABC-DLBCL xenografts, to study tumor growth and pharmacodynamics of the compound over time. To begin to test its safety and therapeutic efficacy, a patient was recruited who underwent five cycles of cobomarsen treatment. RESULTS: Cobomarsen decreased cell proliferation and induced apoptosis in ABC-DLBCL cell lines. Intravenous administration of cobomarsen in a xenograft NSG mouse model of ABC-DLBCL reduced tumor volume, triggered apoptosis, and derepressed direct miR-155 target genes. Finally, the compound reduced and stabilized tumor growth without any toxic effects for the patient. CONCLUSIONS: Our findings support the potential therapeutic application of cobomarsen in ABC-DLBCL and other types of lymphoma with elevated miR-155 expression.
Asunto(s)
Linfoma de Células B Grandes Difuso/tratamiento farmacológico , MicroARNs/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , MicroARNs/metabolismo , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.
Asunto(s)
Matriz Extracelular/metabolismo , MicroARNs/genética , Enfermedades de la Piel/patología , Animales , Biopsia con Aguja , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Fibrosis/genética , Fibrosis/patología , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Ratones , MicroARNs/farmacología , Estudios Prospectivos , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/genética , Resultado del TratamientoRESUMEN
AIMS: Recent studies have shown that microRNAs (miRNAs), besides being potent regulators of gene expression, can additionally serve as circulating biomarkers of disease. The aim of this study is to determine if plasma miRNAs can be used as indicators of disease progression or therapeutic efficacy in hypertension-induced heart disease. METHODS AND RESULTS: In order to define circulating miRNAs that change during hypertension-induced heart failure and that respond to therapeutic treatment, we performed miRNA arrays on plasma RNA from hypertensive rats that show signs of heart failure. Array analysis indicated that approximately one-third of the miRNAs on the array are detectable in plasma. Quantitative real-time polymerase chain reaction (PCR) analysis for a selected panel of miRNAs indicated that circulating levels of miR-16, miR-20b, miR-93, miR-106b, miR-223, and miR-423-5p were significantly increased in response to hypertension-induced heart failure, while this effect was blunted in response to treatment with antimiR-208a as well as an ACE inhibitor. Moreover, treatment with antimiR-208a resulted in a dramatic increase in one miRNA, miR-19b. A time course study indicated that several of these miRNA changes track with disease progression. CONCLUSIONS: Circulating levels of miRNAs are responsive to therapeutic interventions and change during the progression of hypertension-induced heart disease.
Asunto(s)
Biomarcadores/sangre , Progresión de la Enfermedad , Insuficiencia Cardíaca/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , MicroARNs/sangre , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Captopril/uso terapéutico , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/diagnóstico , Hipertensión/inducido químicamente , Hipertensión/diagnóstico , Masculino , MicroARNs/genética , Péptido Natriurético Encefálico/sangre , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Endogámicas Dahl , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Cloruro de Sodio/toxicidad , Resultado del TratamientoRESUMEN
Within the last 15 years, microRNAs (miRNAs) have emerged as powerful regulators of gene expression. Highly conserved, these small RNAs appear to regulate nearly every aspect of eukaryotic biology. Perturbation of miRNA expression has been correlated with cancer and, more recently, a variety of diseases; however, the more striking observation is that manipulation of miRNA levels can control disease phenotypes. Hence, the race to bring the first miRNA therapeutic to the market has begun. This review provides an overview of the history and mechanism of miRNAs, the ongoing efforts to develop miRNA therapies, and the nucleic acid technologies that may be key to the success of a miRNA therapeutic.
Asunto(s)
MicroARNs/uso terapéutico , Animales , Técnicas de Transferencia de Gen , Humanos , MicroARNs/antagonistas & inhibidores , MicroARNs/química , MicroARNs/metabolismoAsunto(s)
Insuficiencia Cardíaca Sistólica/genética , MicroARNs/sangre , Femenino , Humanos , MasculinoRESUMEN
Piwi proteins, a subfamily of Argonaute (Ago) proteins, have recently been shown to bind endogenous small RNAs. However, differences between Ago proteins (which bind microRNAs and small interfering RNAs) and Piwi proteins and Piwi-interacting RNAs (piRNAs) suggest novel functions for Piwi proteins. Here, we highlight the recent progress in understanding Piwi function and the implications for germline and stem cell development.
Asunto(s)
Proteínas/metabolismo , Animales , Epigénesis Genética , Femenino , Silenciador del Gen , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Masculino , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Filogenia , Proteínas/clasificación , Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Células Madre/citología , Células Madre/metabolismoRESUMEN
Small noncoding RNAs regulate processes essential for cell growth and development, including mRNA degradation, translational repression, and transcriptional gene silencing (TGS). During a search for candidate mammalian factors for TGS, we purified a complex that contains small RNAs and Riwi, the rat homolog to human Piwi. The RNAs, frequently 29 to 30 nucleotides in length, are called Piwi-interacting RNAs (piRNAs), 94% of which map to 100 defined (< or = 101 kb) genomic regions. Within these regions, the piRNAs generally distribute across only one genomic strand or distribute on two strands but in a divergent, nonoverlapping manner. Preparations of piRNA complex (piRC) contain rRecQ1, which is homologous to qde-3 from Neurospora, a gene implicated in silencing pathways. Piwi has been genetically linked to TGS in flies, and slicer activity cofractionates with the purified complex. These results are consistent with a gene-silencing role for piRC in mammals.
Asunto(s)
Proteínas/metabolismo , Interferencia de ARN , ARN no Traducido/metabolismo , Ribonucleoproteínas/metabolismo , Testículo/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Animales , Cromosomas de los Mamíferos , Secuencia Conservada , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Biblioteca de Genes , Genoma , Masculino , Ratones , Proteínas/aislamiento & purificación , ARN no Traducido/química , ARN no Traducido/genética , ARN no Traducido/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , RecQ Helicasas , Ribonucleoproteínas/química , Ribonucleoproteínas/aislamiento & purificación , Transcripción GenéticaRESUMEN
It is well established that the template for telomeric DNA synthesis is provided by the RNA subunit of telomerase; however, the additional functions provided by most of the rest of the RNA (>1000 nucleotides in budding yeast) are largely unknown. By alignment of telomerase RNAs of Saccharomyces cerevisiae and six Kluyveromyces species followed by mutagenesis of the S. cerevisiae RNA, we found a conserved region that is essential for telomere maintenance. Phylogenetic analysis and computer folding revealed that this region is conserved not only in primary nucleotide sequence but also in secondary structure. A common bulged-stem structure was predicted in all seven yeast species. Mutational analysis showed the structure to be essential for telomerase function. Suppression of bulged-stem mutant phenotypes by overexpression of Est1p and loss of co-immunoprecipitation of the mutant RNAs with Est1p indicated that this bulged stem is necessary for association of Est1p, a telomerase regulatory subunit. Est1p in yeast extract bound specifically to a small RNA containing the bulged stem, suggesting a direct interaction. We propose that this RNA structure links the enzymatic core of telomerase with Est1p, thereby allowing Est1p to recruit or activate telomerase at the telomere.
Asunto(s)
ARN de Hongos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telomerasa/genética , Secuencia Conservada , Mutación , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.