RESUMEN
Mutations in PLP1, the gene that encodes proteolipid protein (PLP), result in failure of myelination and neurological dysfunction in the X-chromosome-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD)1,2. Most PLP1 mutations, including point mutations and supernumerary copy variants, lead to severe and fatal disease. Patients who lack PLP1 expression, and Plp1-null mice, can display comparatively mild phenotypes, suggesting that PLP1 suppression might provide a general therapeutic strategy for PMD1,3-5. Here we show, using CRISPR-Cas9 to suppress Plp1 expression in the jimpy (Plp1jp) point-mutation mouse model of severe PMD, increased myelination and restored nerve conduction velocity, motor function and lifespan of the mice to wild-type levels. To evaluate the translational potential of this strategy, we identified antisense oligonucleotides that stably decrease the levels of Plp1 mRNA and PLP protein throughout the neuraxis in vivo. Administration of a single dose of Plp1-targeting antisense oligonucleotides in postnatal jimpy mice fully restored oligodendrocyte numbers, increased myelination, improved motor performance, normalized respiratory function and extended lifespan up to an eight-month end point. These results suggest that PLP1 suppression could be developed as a treatment for PMD in humans. More broadly, we demonstrate that oligonucleotide-based therapeutic agents can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, establishing a new pharmaceutical modality for myelin disorders.
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Modelos Animales de Enfermedad , Proteína Proteolipídica de la Mielina/deficiencia , Enfermedad de Pelizaeus-Merzbacher/genética , Enfermedad de Pelizaeus-Merzbacher/terapia , Animales , Sistemas CRISPR-Cas , Femenino , Edición Génica , Hipoxia/metabolismo , Masculino , Ratones , Ratones Mutantes , Actividad Motora/genética , Proteína Proteolipídica de la Mielina/genética , Proteína Proteolipídica de la Mielina/metabolismo , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/genética , Enfermedad de Pelizaeus-Merzbacher/metabolismo , Mutación Puntual , Pruebas de Función Respiratoria , Análisis de SupervivenciaRESUMEN
Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the central nervous system (CNS). By modulating RNA, they hold the promise of targeting root molecular causes of disease and hold potential to treat myriad CNS disorders. Realization of this potential requires that ASOs must be active in the disease-relevant cells, and ideally, that monitorable biomarkers also reflect ASO activity in these cells. The biodistribution and activity of such centrally delivered ASOs have been deeply characterized in rodent and non-human primate (NHP) models, but usually only in bulk tissue, limiting our understanding of the distribution of ASO activity across individual cells and across diverse CNS cell types. Moreover, in human clinical trials, target engagement is usually monitorable only in a single compartment, CSF. We sought a deeper understanding of how individual cells and cell types contribute to bulk tissue signal in the CNS, and how these are linked to CSF biomarker outcomes. We employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and NHPs treated with an ASO against PRNP. Pharmacologic activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target RNA suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect ASO pharmacodynamic effect in disease-relevant cells in a neuronal disorder. Our results provide a reference dataset for ASO activity distribution in the CNS and establish single nucleus sequencing as a method for evaluating cell type specificity of oligonucleotide therapeutics and other modalities.
Antisense oligonucleotide (ASO) drugs are a type of chemically modified DNA that can be injected into cerebrospinal fluid in order to enter brain cells and reduce the amount of RNA from a specific gene. The brain is a complex mixture of hundreds of billions of cells. When an ASO lowers a target gene's RNA by 50%, is that a 50% reduction in 100% of cells, or a 100% reduction in 50% of cells? Are the many different cell types of the brain affected equally? This new study uses single cell RNA sequencing to answer these questions, finding that ASOs are broadly active across cell types and individual cells, and linking reduction of target protein in cerebrospinal fluid to disease-relevant cells.
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Encéfalo , Oligonucleótidos Antisentido , Animales , Ratones , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/análisis , ARN/metabolismo , Distribución Tisular , Factores de Transcripción/metabolismo , Líquido Cefalorraquídeo/química , Enfermedades del Sistema Nervioso Central/terapiaRESUMEN
Genetic variation in LRRK2 associates with the susceptibility to Parkinson's disease, Crohn's disease, and mycobacteria infection. High expression of LRRK2 and its substrate Rab10 occurs in phagocytic cells in the immune system. In mouse and human primary macrophages, dendritic cells, and microglia-like cells, we find that Rab10 specifically regulates a specialized form of endocytosis known as macropinocytosis, without affecting phagocytosis or clathrin-mediated endocytosis. LRRK2 phosphorylates cytoplasmic PI(3,4,5)P3-positive GTP-Rab10, before EEA1 and Rab5 recruitment to early macropinosomes occurs. Macropinosome cargo in macrophages includes CCR5, CD11b, and MHCII, and LRRK2-phosphorylation of Rab10 potently blocks EHBP1L1-mediated recycling tubules and cargo turnover. EHBP1L1 overexpression competitively inhibits LRRK2-phosphorylation of Rab10, mimicking the effects of LRRK2 kinase inhibition in promoting cargo recycling. Both Rab10 knockdown and LRRK2 kinase inhibition potently suppress the maturation of macropinosome-derived CCR5-loaded signaling endosomes that are critical for CCL5-induced immunological responses that include Akt activation and chemotaxis. These data support a novel signaling axis in the endolysosomal system whereby LRRK2-mediated Rab10 phosphorylation stalls vesicle fast recycling to promote PI3K-Akt immunological responses.
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Proteínas Portadoras/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Macrófagos/metabolismo , Fagocitos/inmunología , Pinocitosis/genética , Proteínas de Unión al GTP rab/metabolismo , Animales , Membrana Celular/metabolismo , Quimiocina CCL5/farmacología , Quimiotaxis/genética , Células Dendríticas/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Macrófagos/efectos de los fármacos , Masculino , Espectrometría de Masas , Ratones , Ratones Transgénicos , Microglía/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Mutación , Fagocitos/efectos de los fármacos , Fagocitos/metabolismo , Fosforilación , Pinocitosis/efectos de los fármacos , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
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.
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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
Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.
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Oligonucleótidos Antisentido/uso terapéutico , Enfermedades por Prión/terapia , Proteínas Priónicas/genética , Tratamiento con ARN de Interferencia/métodos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos Antisentido/química , Proteínas Priónicas/metabolismoRESUMEN
OBJECTIVE: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is the most common dominantly inherited ataxia. Despite advances in understanding this CAG repeat/polyglutamine expansion disease, there are still no therapies to alter its progressive fatal course. Here, we investigate whether an antisense oligonucleotide (ASO) targeting the SCA3 disease gene, ATXN3, can prevent molecular, neuropathological, electrophysiological, and behavioral features of the disease in a mouse model of SCA3. METHODS: The top ATXN3-targeting ASO from an in vivo screen was injected intracerebroventricularly into early symptomatic transgenic SCA3 mice that express the full human disease gene and recapitulate key disease features. Following a single ASO treatment at 8 weeks of age, mice were evaluated longitudinally for ATXN3 suppression and rescue of disease-associated pathological changes. Mice receiving an additional repeat injection at 21 weeks were evaluated longitudinally up to 29 weeks for motor performance. RESULTS: The ATXN3-targeting ASO achieved sustained reduction of polyglutamine-expanded ATXN3 up to 8 weeks after treatment and prevented oligomeric and nuclear accumulation of ATXN3 up to at least 14 weeks after treatment. Longitudinal ASO therapy rescued motor impairment in SCA3 mice, and this rescue was associated with a recovery of defects in Purkinje neuron firing frequency and afterhyperpolarization. INTERPRETATION: This preclinical study established efficacy of ATXN3-targeted ASOs as a disease-modifying therapeutic strategy for SCA3. These results support further efforts to develop ASOs for human clinical trials in this polyglutamine disease as well as in other dominantly inherited disorders caused by toxic gain of function. Ann Neurol 2018;83:64-77.
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Ataxina-3/química , Regulación de la Expresión Génica/efectos de los fármacos , Enfermedad de Machado-Joseph/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Factores de Edad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Ataxina-3/genética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Unión al Calcio/metabolismo , Modelos Animales de Enfermedad , Conducta Exploratoria/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/genética , Gliosis/tratamiento farmacológico , Gliosis/etiología , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/patología , Enfermedad de Machado-Joseph/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación/genética , Células de Purkinje/efectos de los fármacos , Células de Purkinje/patología , Proteínas de Unión al ARN/metabolismoRESUMEN
UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.
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Cuerpos de Inclusión/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , Neuronas/metabolismo , Transcitosis/fisiología , alfa-Sinucleína/metabolismo , Animales , Regulación de la Expresión Génica/genética , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligorribonucleótidos Antisentido/farmacología , Fotoblanqueo , Ratas , Sinucleínas/metabolismo , Transcitosis/genética , Tubulina (Proteína)/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.
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The blood barriers of the nervous system protect neural environments but can hinder therapeutic accessibility. The blood-brain barrier (BBB) is well characterized, consisting of endothelial cells with specialized tight junctions and low levels of transcytosis, properties conferred by contacting pericytes and astrocytes. In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system is poorly defined. Here, we characterize the structure of the mammalian BNB, identify the processes that confer barrier function, and demonstrate how the barrier can be opened in response to injury. The homeostatic BNB is leakier than the BBB, which we show is due to higher levels of transcytosis. However, the barrier is reinforced by macrophages that specifically engulf leaked materials, identifying a role for resident macrophages as an important component of the BNB. Finally, we demonstrate the exploitation of these processes to effectively deliver RNA-targeting therapeutics to peripheral nerves, indicating new treatment approaches for nervous system pathologies.
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Barrera Hematonerviosa , Células Endoteliales , Animales , Barrera Hematonerviosa/fisiología , Células Endoteliales/fisiología , Barrera Hematoencefálica/fisiología , Macrófagos , Pericitos/fisiología , MamíferosRESUMEN
Antisense oligonucleotides (ASOs) dosed into cerebrospinal fluid (CSF) distribute broadly throughout the brain and hold the promise of treating myriad brain diseases by modulating RNA. CNS tissue is not routinely biopsied in living individuals, leading to reliance on CSF biomarkers to inform on drug target engagement. Animal models can link CSF biomarkers to brain parenchyma, but our understanding of how individual cells contribute to bulk tissue signal is limited. Here we employed single nucleus transcriptomics on tissue from mice treated with RNase H1 ASOs against Prnp and Malat1 and macaques treated with an ASO against PRNP . Activity was observed in every cell type, though sometimes with substantial differences in magnitude. Single cell RNA count distributions implied target suppression in every single sequenced cell, rather than intense knockdown in only some cells. Duration of action up to 12 weeks post-dose differed across cell types, being shorter in microglia than in neurons. Suppression in neurons was generally similar to, or more robust than, the bulk tissue. In macaques, PrP in CSF was lowered 40% in conjunction with PRNP knockdown across all cell types including neurons, arguing that a CSF biomarker readout is likely to reflect disease-relevant cells in a neuronal disorder.
RESUMEN
Autosomal dominant pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson's disease (PD). The most common mutation, G2019S-LRRK2, increases the kinase activity of LRRK2 causing hyper-phosphorylation of its substrates. One of these substrates, Rab10, is phosphorylated at a conserved Thr73 residue (pRab10), and is one of the most abundant LRRK2 Rab GTPases expressed in various tissues. The involvement of Rab10 in neurodegenerative disease, including both PD and Alzheimer's disease makes pinpointing the cellular and subcellular localization of Rab10 and pRab10 in the brain an important step in understanding its functional role, and how post-translational modifications could impact function. To establish the specificity of antibodies to the phosphorylated form of Rab10 (pRab10), Rab10 specific antisense oligonucleotides were intraventricularly injected into the brains of mice. Further, Rab10 knock out induced neurons, differentiated from human induced pluripotent stem cells were used to test the pRab10 antibody specificity. To amplify the weak immunofluorescence signal of pRab10, tyramide signal amplification was utilized. Rab10 and pRab10 were expressed in the cortex, striatum and the substantia nigra pars compacta. Immunofluorescence for pRab10 was increased in G2019S-LRRK2 knockin mice. Neurons, astrocytes, microglia and oligodendrocytes all showed Rab10 and pRab10 expression. While Rab10 colocalized with endoplasmic reticulum, lysosome and trans-Golgi network markers, pRab10 did not localize to these organelles. However, pRab10, did overlap with markers of the presynaptic terminal in both mouse and human cortex, including α-synuclein. Results from this study suggest Rab10 and pRab10 are expressed in all brain areas and cell types tested in this study, but pRab10 is enriched at the presynaptic terminal. As Rab10 is a LRRK2 kinase substrate, increased kinase activity of G2019S-LRRK2 in PD may affect Rab10 mediated membrane trafficking at the presynaptic terminal in neurons in disease.
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Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Ratones , Animales , Células Madre Pluripotentes Inducidas/metabolismo , Fosforilación , Enfermedad de Parkinson/genética , Mutación , Encéfalo/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across 6 species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varied dramatically across different brain regions in mice, cynomolgus macaques, and humans. PrP expression did not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP was lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55%, and D178N just 31%, of the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA was reflected in brain parenchyma PrP and, in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.
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Enfermedades por Prión , Proteínas Priónicas , Priones , Animales , Biomarcadores/líquido cefalorraquídeo , Genotipo , Humanos , Ratones , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/tratamiento farmacológico , Proteínas Priónicas/líquido cefalorraquídeo , Proteínas Priónicas/genética , Proteínas Priónicas/farmacología , Priones/genética , Priones/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.omtn.2017.08.002.].
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[This corrects the article DOI: 10.1016/j.omtn.2017.08.002.].
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Parkinson's disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.
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Encéfalo/efectos de los fármacos , Oligonucleótidos Antisentido/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Técnicas de Cultivo de Célula , Líquido Cefalorraquídeo/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Femenino , Humanos , Macaca fascicularis , Masculino , Ratones , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , alfa-Sinucleína/genéticaRESUMEN
Knockout of the memory suppressor gene histone deacetylase 2 (Hdac2) in mice elicits cognitive enhancement, and drugs that block HDAC2 have potential as therapeutics for disorders affecting memory. Currently available HDAC2 catalytic activity inhibitors are not fully isoform specific and have short half-lives. Antisense oligonucleotides (ASOs) are drugs that elicit extremely long-lasting, specific inhibition through base pairing with RNA targets. We utilized an ASO to reduce Hdac2 messenger RNA (mRNA) in mice and determined its longevity, specificity, and mechanism of repression. A single injection of the Hdac2-targeted ASO in the central nervous system produced persistent reduction in HDAC2 protein and Hdac2 mRNA levels for 16 weeks. It enhanced object location memory for 8 weeks. RNA sequencing (RNA-seq) analysis of brain tissues revealed that the repression was specific to Hdac2 relative to related Hdac isoforms, and Hdac2 reduction caused alterations in the expression of genes involved in extracellular signal-regulated kinase (ERK) and memory-associated immune signaling pathways. Hdac2-targeted ASOs also suppress a nonpolyadenylated Hdac2 regulatory RNA and elicit direct transcriptional suppression of the Hdac2 gene through stalling RNA polymerase II. These findings identify transcriptional suppression of the target gene as a novel mechanism of action of ASOs.
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Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP-ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO-PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.
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Oligonucleótidos Antisentido/metabolismo , Proteínas Priónicas/metabolismo , Células HeLa , Humanos , Cinética , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Proteínas Priónicas/química , Proteínas Priónicas/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrP; PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering one target mRNA in the brain, but their development for prion disease has been hindered by three unresolved questions from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here we test antisense oligonucleotides (ASOs) delivered by bolus intracerebroventricular injection to intracerebrally prion-infected wild-type mice. Prophylactic treatments given every 2-3 months extended survival times 61-98%, and a single injection at 120 days post-infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a non-targeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent, or even single, bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.
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Oligonucleótidos Antisentido/farmacología , Oligonucleótidos Antisentido/uso terapéutico , Enfermedades por Prión/tratamiento farmacológico , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Femenino , Terapia Genética , Ratones , Ratones Endogámicos C57BL , Enfermedades por Prión/patología , Tasa de SupervivenciaRESUMEN
There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.
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Ganglios Espinales/efectos de los fármacos , Nocicepción/fisiología , Oligonucleótidos Antisentido/uso terapéutico , Dolor/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/fisiopatología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatologíaRESUMEN
Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by duplication of peripheral myelin protein 22 (PMP22) and is the most common hereditary peripheral neuropathy. CMT1A is characterized by demyelination and axonal loss, which underlie slowed motor nerve conduction velocity (MNCV) and reduced compound muscle action potentials (CMAP) in patients. There is currently no known treatment for this disease. Here, we show that antisense oligonucleotides (ASOs) effectively suppress PMP22 mRNA in affected nerves in 2 murine CMT1A models. Notably, initiation of ASO treatment after disease onset restored myelination, MNCV, and CMAP almost to levels seen in WT animals. In addition to disease-associated gene expression networks that were restored with ASO treatment, we also identified potential disease biomarkers through transcriptomic profiling. Furthermore, we demonstrated that reduction of PMP22 mRNA in skin biopsies from ASO-treated rats is a suitable biomarker for evaluating target engagement in response to ASO therapy. These results support the use of ASOs as a potential treatment for CMT1A and elucidate potential disease and target engagement biomarkers for use in future clinical trials.