RESUMEN
Antisense oligonucleotides (ASOs) have emerged as a new class of drugs to treat a wide range of diseases, including neurological indications. Spinraza, an ASO that modulates splicing of SMN2 RNA, has shown profound disease modifying effects in Spinal Muscular Atrophy (SMA) patients, energizing efforts to develop ASOs for other neurological diseases. While SMA specifically affects spinal motor neurons, other neurological diseases affect different central nervous system (CNS) regions, neuronal and non-neuronal cells. Therefore, it is important to characterize ASO distribution and activity in all major CNS structures and cell types to have a better understanding of which neurological diseases are amenable to ASO therapy. Here we present for the first time the atlas of ASO distribution and activity in the CNS of mice, rats, and non-human primates (NHP), species commonly used in preclinical therapeutic development. Following central administration of an ASO to rodents, we observe widespread distribution and target RNA reduction throughout the CNS in neurons, oligodendrocytes, astrocytes and microglia. This is also the case in NHP, despite a larger CNS volume and more complex neuroarchitecture. Our results demonstrate that ASO drugs are well suited for treating a wide range of neurological diseases for which no effective treatments are available.
Asunto(s)
Sistema Nervioso Central/química , Ratones/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Primates/metabolismo , Ratas/metabolismo , Animales , Sistema Nervioso Central/citología , Femenino , Hibridación in Situ , Inyecciones Intraventriculares , Inyecciones Espinales , Macaca fascicularis , Masculino , Neuroglía/química , Neuronas/química , Oligonucleótidos Antisentido/administración & dosificación , Especificidad de Órganos , ARN Largo no Codificante/análisis , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , Ratas Sprague-Dawley , Ribonucleasa H , Distribución TisularRESUMEN
Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington's disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2'-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.
Asunto(s)
Proteínas de Unión al ADN/genética , Enfermedad de Huntington/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Expansión de Repetición de Trinucleótido/genética , Alelos , Animales , Conducta Animal , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Unión al ADN/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Humanos , Proteína Huntingtina , Enfermedad de Huntington/patología , Ratones , Mutación , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Neuronas/patología , Proteínas Nucleares/biosíntesis , Agregación Patológica de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.
Asunto(s)
Exones , Expresión Génica , Atrofia Muscular Espinal/genética , Oligonucleótidos Antisentido/farmacología , Animales , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Ratones , Atrofia Muscular Espinal/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously, we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here, we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared with several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and nonhuman primates (NHPs). Intracerebroventricular bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared with i.c.v. infusion. For both methods of delivery, the duration of ISIS 396443-mediated SMN2 splicing correction is long lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.
Asunto(s)
Encéfalo/efectos de los fármacos , Atrofia Muscular Espinal/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Oligonucleótidos/farmacología , Empalme del ARN/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Encéfalo/metabolismo , Femenino , Infusiones Intraventriculares , Inyecciones Intraventriculares , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Atrofia Muscular Espinal/tratamiento farmacológico , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/farmacocinética , Oligodesoxirribonucleótidos Antisentido/uso terapéutico , Oligonucleótidos/administración & dosificación , Oligonucleótidos/farmacocinética , Oligonucleótidos/uso terapéuticoRESUMEN
We describe a new technology for recruiting specific proteins to RNA through selective recognition of heteroduplexes formed with chemically modified antisense oligonucleotides (ASOs). Typically, ASOs function by hybridizing to their RNA targets and blocking the binding of single-stranded RNA-binding proteins. Unexpectedly, we found that ASOs with 2'-deoxy-2'-fluoro (2'-F) nucleotides, but not with other 2' chemical modifications, have an additional property: they form heteroduplexes with RNA that are specifically recognized by the interleukin enhancer-binding factor 2 and 3 complex (ILF2/3). 2'-F ASO-directed recruitment of ILF2/3 to RNA can be harnessed to control gene expression by modulating alternative splicing of target transcripts. ILF2/3 recruitment to precursor mRNA near an exon results in omission of the exon from the mature mRNA, both in cell culture and in mice. We discuss the possibility of using chemically engineered ASOs that recruit specific proteins to modulate gene expression for therapeutic intervention.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Proteína del Factor Nuclear 45/metabolismo , Proteínas del Factor Nuclear 90/metabolismo , Oligonucleótidos Antisentido/farmacología , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Empalme Alternativo/genética , Animales , Sitios de Unión , Exones , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Ratones , Ratones Noqueados , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteína del Factor Nuclear 45/genética , Proteínas del Factor Nuclear 90/genética , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Precursores del ARN/genética , Sitios de Empalme de ARN , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , TransfecciónRESUMEN
Pathological evidence for selective four-repeat (4R) tau deposition in certain dementias and exon 10-positioned MAPT mutations together suggest a 4R-specific role in causing disease. However, direct assessments of 4R toxicity have not yet been accomplished in vivo. Increasing 4R-tau expression without change to total tau in human tau-expressing mice induced more severe seizures and nesting behavior abnormality, increased tau phosphorylation, and produced a shift toward oligomeric tau. Exon 10 skipping could also be accomplished in vivo, providing support for a 4R-tau targeted approach to target 4R-tau toxicity and, in cases of primary MAPT mutation, eliminate the disease-causing mutation.
Asunto(s)
Modelos Biológicos , Comportamiento de Nidificación , Convulsiones/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Animales , Encéfalo/metabolismo , Exones/genética , Humanos , Infusiones Intraventriculares , Ratones , Mutación/efectos de los fármacos , Comportamiento de Nidificación/efectos de los fármacos , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacología , Fosforilación/efectos de los fármacos , Isoformas de Proteínas/metabolismo , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Convulsiones/inducido químicamente , Convulsiones/genética , Solubilidad , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Proteínas tau/genética , Proteínas tau/toxicidadRESUMEN
The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to â¼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.
Asunto(s)
Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Proteínas de Unión al ADN/genética , Proteína FUS de Unión a ARN/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Regiones no Traducidas 3'/genética , Animales , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibroblastos , Técnicas de Silenciamiento del Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células Madre Pluripotentes Inducidas , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/metabolismo , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within â¼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT.