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1.
Nat Metab ; 5(10): 1787-1802, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37679556

RESUMEN

Neuroinflammatory microglia secrete cytokines to induce neurotoxic reactive astrocytes, which are one of the major causes of neuronal death. However, the intrinsic key regulators underlying neurotoxic reactive astrocytes induction are unknown. Here we show that the transmembrane protein 164 (TMEM164) is an early-response intrinsic factor that regulates neurotoxic astrocyte reactivity. TMEM164 overexpression inhibits the induction of neurotoxic reactive astrocytes, maintains normal astrocytic functions and suppresses neurotoxic reactive astrocyte-mediated neuronal death by decreasing the secretion of neurotoxic saturated lipids. Adeno-associated virus-mediated, astrocyte-specific TMEM164 overexpression in male and female mice prevents the induction of neurotoxic reactive astrocytes, dopaminergic neuronal loss and motor deficits in a Parkinson's disease model. Notably, brain-wide astrocyte-specific TMEM164 overexpression prevents the induction of neurotoxic reactive astrocytes, amyloid ß deposition, neurodegeneration and memory decline in the 5XFAD Alzheimer's disease mouse model, suggesting that TMEM164 could serve as a potential therapeutic target for neurodegenerative disorders.


Asunto(s)
Enfermedad de Alzheimer , Astrocitos , Femenino , Ratones , Animales , Masculino , Astrocitos/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Neuronas/metabolismo
3.
Comput Intell Neurosci ; 2022: 4105942, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35463231

RESUMEN

Aiming at the problem of low accuracy of traditional algorithm model, an intelligent recognition model of business English translation based on an improved GLR algorithm is proposed. Through this algorithm, the automatic sentence recognition technology is established, and according to the characteristics of business English, the improved GLR algorithm is used for collection, sorting, and analysis, so as to realize the intelligent recognition of business English. The results show that based on the improved GLR algorithm, the recognition accuracy is high, and the comprehensive score is 92.5 points, which overcomes the disadvantages of the GLR algorithm, and the operation speed and processing are improved. Based on the improved GLR algorithm, the intelligent translation of business English is realized, which is accurate and fast, and greatly promotes the learning and development of business English.


Asunto(s)
Algoritmos , Redes Neurales de la Computación , Inteligencia , Lenguaje , Traducciones
5.
Vaccine ; 40(9): 1208-1212, 2022 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-35094871

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in catastrophic damage worldwide. Accordingly, the development of powerful, safe, easily accessible vaccines with long-term effectiveness is understood as an urgently needed countermeasure against this ongoing pandemic. Guided by this strong promise of using AAVs, we here designed, optimized, and developed an AAV-based vaccines (including AAV-RBD(max), AAV-RBD(wt), AAV-2xRBD, and AAV-3xRBD) that elicit strong immune responses against the RBD domain of the SARS-CoV-2 S protein. These immunogenic responses have proven long-lived, with near peak levels for at least six months in mice. Notably, the sera immunized with AAV-3xRBD vaccine contains powerful neutralizing antibodies against the SARS-CoV-2 pseudovirus. Further evidence proven that potent specific antibodies could also be elicited in canines after vaccination with AAV-3xRBD vaccine.


Asunto(s)
COVID-19 , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Vacunas contra la COVID-19 , Perros , Humanos , Ratones , Ratones Endogámicos BALB C , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Vacunas Virales/genética
6.
Mol Ther ; 30(1): 105-118, 2022 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-34174443

RESUMEN

Myosin VI(MYO6) is an unconventional myosin that is vital for auditory and vestibular function. Pathogenic variants in the human MYO6 gene cause autosomal-dominant or -recessive forms of hearing loss. Effective treatments for Myo6 mutation causing hearing loss are limited. We studied whether adeno-associated virus (AAV)-PHP.eB vector-mediated in vivo delivery of Staphylococcus aureus Cas9 (SaCas9-KKH)-single-guide RNA (sgRNA) complexes could ameliorate hearing loss in a Myo6WT/C442Y mouse model that recapitulated the phenotypes of human patients. The in vivo editing efficiency of the AAV-SaCas9-KKH-Myo6-g2 system on Myo6C442Y is 4.05% on average in Myo6WT/C442Y mice, which was ∼17-fold greater than editing efficiency of Myo6WT alleles. Rescue of auditory function was observed up to 5 months post AAV-SaCas9-KKH-Myo6-g2 injection in Myo6WT/C442Y mice. Meanwhile, shorter latencies of auditory brainstem response (ABR) wave I, lower distortion product otoacoustic emission (DPOAE) thresholds, increased cell survival rates, more regular hair bundle morphology, and recovery of inward calcium levels were also observed in the AAV-SaCas9-KKH-Myo6-g2-treated ears compared to untreated ears. These findings provide further reference for in vivo genome editing as a therapeutic treatment for various semi-dominant forms of hearing loss and other semi-dominant diseases.


Asunto(s)
Edición Génica , Pérdida Auditiva , Animales , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico/genética , Audición , Pérdida Auditiva/genética , Pérdida Auditiva/terapia , Humanos , Ratones , ARN Guía de Kinetoplastida
7.
Nat Biomed Eng ; 5(2): 144-156, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33398131

RESUMEN

Therapeutic genome editing requires effective and targeted delivery methods. The delivery of Cas9 mRNA using adeno-associated viruses has led to potent in vivo therapeutic efficacy, but can cause sustained Cas9 expression, anti-Cas9 immune responses and off-target edits. Lentiviral vectors have been engineered to deliver nucleases that are expressed transiently, but in vivo evidence of their biomedical efficacy is lacking. Here, we show that the lentiviral codelivery of Streptococcus pyogenes Cas9 mRNA and expression cassettes that encode a guide RNA that targets vascular endothelial growth factor A (Vegfa) is efficacious in a mouse model of wet age-related macular degeneration induced by Vegfa. A single subretinal injection of engineered lentiviruses knocked out 44% of Vegfa in retinal pigment epithelium and reduced the area of choroidal neovascularization by 63% without inducing off-target edits or anti-Cas9 immune responses. Engineered lentiviruses for the transient expression of nucleases may form the basis of new treatments for retinal neovascular diseases.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Lentivirus/fisiología , Degeneración Macular/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Modelos Animales de Enfermedad , Vectores Genéticos/fisiología , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética
8.
Nat Cell Biol ; 23(1): 99-108, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33398178

RESUMEN

Detection of endogenous signals and precise control of genetic circuits in the natural context are essential to understand biological processes. However, the tools to process endogenous information are limited. Here we developed a generalizable endogenous transcription-gated switch that releases single-guide RNAs in the presence of an endogenous promoter. When the endogenous transcription-gated switch is coupled with the highly sensitive CRISPR-activator-associated reporter we developed, we can reliably detect the activity of endogenous genes, including genes with very low expression (<0.001 relative to Gapdh; quantitative-PCR analysis). Notably, we could also monitor the transcriptional activity of typically long non-coding RNAs expressed at low levels in living cells using this approach. Together, our method provides a powerful platform to sense the activity of endogenous genetic elements underlying cellular functions.


Asunto(s)
Células Madre Embrionarias de Ratones/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , ARN Largo no Codificante/metabolismo , ARN Mensajero/metabolismo , Animales , Sistemas CRISPR-Cas , Células HEK293 , Humanos , Ratones , Células Madre Embrionarias de Ratones/citología , Neuroblastoma/patología , ARN Guía de Kinetoplastida/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética
9.
Cell ; 181(3): 590-603.e16, 2020 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-32272060

RESUMEN

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 (Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.


Asunto(s)
Neurogénesis/fisiología , Neuroglía/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Sistemas CRISPR-Cas/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Regulación de la Expresión Génica/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Células Ganglionares de la Retina/fisiología
11.
Natl Sci Rev ; 7(1): 92-101, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34691481

RESUMEN

We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn -/-, SMN2 tg/-). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases.

12.
Natl Sci Rev ; 7(5): 835-837, 2020 May.
Artículo en Inglés | MEDLINE | ID: mdl-34692105

RESUMEN

RNA-targeting CRISPR system Cas13 offers an efficient approach for manipulating RNA transcripts in vitro. In this perspective, we provide a proof-of-concept demonstration that Cas13-mediated Vegfa knockdown in vivo could prevent the development of laser-induced CNV in mouse model of Age-related macular degeneration.

14.
Nature ; 571(7764): 275-278, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31181567

RESUMEN

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA12, and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.


Asunto(s)
ADN/genética , Edición Génica/métodos , Mutagénesis , Mutación , Nucleósido Desaminasas/genética , Ingeniería de Proteínas , ARN/genética , Adenina/metabolismo , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Citosina/metabolismo , Citosina Desaminasa/genética , Citosina Desaminasa/metabolismo , Células HEK293 , Humanos , Nucleósido Desaminasas/metabolismo , Especificidad por Sustrato , Transfección
15.
Exp Ther Med ; 17(3): 1930-1938, 2019 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-30867688

RESUMEN

MicroRNAs (miRs), a class of small non-coding RNAs, have been demonstrated to be involved in the development and progression of human malignancies, including cutaneous squamous cell carcinoma (CSCC). miR-186 serves a suppressive role in certain common types of human cancer; however, its exact function in CSCC has not been reported previously. In the present study, the expression of miR-186 was significantly increased in CSCC tissues compared with adjacent non-tumour tissues. Overexpression of miR-186 significantly promoted CSCC cell proliferation while inhibiting cell apoptosis. Reticulophagy regulator 1 (RETREG1), a gene that is significantly downregulated in CSCC tissues and cell lines, was identified as a novel target of miR-186. In addition, the expression of RETREG1 was inversely correlated with miR-186 expression in CSCC tissues. Furthermore, the expression of RETREG1 was negatively regulated by miR-186 in CSCC cells, and restoration of RETREG1 attenuated the effects of miR-186 on CSCC cells. Taken together, the results of the current study suggest that miR-186 serves an oncogenic role in CSCC and may be used as a potential therapeutic target for the treatment of this disease.

16.
Dev Biol ; 441(1): 95-103, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29953879

RESUMEN

Proliferation of neural stem cells and differentiation of newly generated cells are crucial steps during the development of mammalian neocortex, which are able to generate suitable number of neurons and glial cells to ensure normal formation of cortex. Any disturbance in these processes leads to structural and functional abnormalities of cerebral cortex, such as epilepsy or intellectual disability. Numerous molecules involved in the development of disorders of the nervous system have been discovered in the recent years. The PI3K/AKT signaling pathway has been shown to be widely involved in the corticogenesis. Recently we could show that overexpression of regulatory subunit P85 of PI3K disrupts neuronal migration. However, it remains unclear whether the regulatory subunit P85 plays a role in the proliferation of neural stem cells and differentiation of newly generated cells during mouse brain development. Here, by using in utero electroporation and immunohistochemistry, we show that overexpression of P85 inhibited proliferation of neural progenitor cells and neuronal differentiation. By using 5-bromo-2-deoxyuridine (BrdU) labeling, we reveal that overexpression of P85 extended the cell cycle duration, which may result in developmental retardation during mouse corticogenesis.


Asunto(s)
Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Corteza Cerebral/embriología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Corteza Cerebral/citología , Ratones , Células-Madre Neurales/citología , Fosfatidilinositol 3-Quinasas/genética
17.
Dev Cell ; 45(4): 526-536.e5, 2018 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-29787711

RESUMEN

The targeting efficiency of knockin sequences via homologous recombination (HR) is generally low. Here we describe a method we call Tild-CRISPR (targeted integration with linearized dsDNA-CRISPR), a targeting strategy in which a PCR-amplified or precisely enzyme-cut transgene donor with 800-bp homology arms is injected with Cas9 mRNA and single guide RNA into mouse zygotes. Compared with existing targeting strategies, this method achieved much higher knockin efficiency in mouse embryos, as well as brain tissue. Importantly, the Tild-CRISPR method also yielded up to 12-fold higher knockin efficiency than HR-based methods in human embryos, making it suitable for studying gene functions in vivo and developing potential gene therapies.


Asunto(s)
Sistemas CRISPR-Cas , ADN/administración & dosificación , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Técnicas de Sustitución del Gen/métodos , ARN Guía de Kinetoplastida/administración & dosificación , Animales , Células Cultivadas , Electroporación , Embrión de Mamíferos/citología , Células Madre Embrionarias/citología , Femenino , Fertilización In Vitro , Recombinación Homóloga , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo
18.
Nat Neurosci ; 21(3): 440-446, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29335603

RESUMEN

Despite rapid progresses in the genome-editing field, in vivo simultaneous overexpression of multiple genes remains challenging. We generated a transgenic mouse using an improved dCas9 system that enables simultaneous and precise in vivo transcriptional activation of multiple genes and long noncoding RNAs in the nervous system. As proof of concept, we were able to use targeted activation of endogenous neurogenic genes in these transgenic mice to directly and efficiently convert astrocytes into functional neurons in vivo. This system provides a flexible and rapid screening platform for studying complex gene networks and gain-of-function phenotypes in the mammalian brain.


Asunto(s)
Química Encefálica/genética , Sistemas CRISPR-Cas/genética , Activación Transcripcional/genética , Animales , Astrocitos/fisiología , Proteínas de Unión al ADN , Femenino , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Neuronas/fisiología , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , ARN Largo no Codificante/genética
19.
J Mol Neurosci ; 64(1): 102-110, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29209901

RESUMEN

The process of neuronal migration is precisely regulated by different molecules during corticogenesis. The FAK (focal adhesion kinase) plays a critical role in embryogenesis and is involved in cell motility through focal adhesions, but the underlying mechanisms on inordinate expression are unclear. To investigate the effect of FAK overexpression on neuronal migration spatiotemporally, mice FAK was transfected into the neurons in vivo by electroporation. Results showed that exogenous FAK distributed in the cytoplasm (in vivo) and co-localized with vinculin (in vitro) and induced aberrant neuronal migration via phosphorylation of FAK at Tyr925 during cerebral cortex development. Meanwhile, FAK Y925F mutant also induced aberrant neuronal migration like inordinate FAK/GFP phenotype. All these results implied that FAK-induced abnormal phenotype depended on phosphorylation of FAK at Tyr925, and this demonstrated that the overexpression of FAK impaired neuronal migration through its phosphorylation and activity of FAK during corticogenesis.


Asunto(s)
Movimiento Celular , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Células CHO , Corteza Cerebral/citología , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Cricetinae , Cricetulus , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Ratones , Ratones Endogámicos C57BL , Mutación , Neuronas/fisiología , Fosforilación , Vinculina/metabolismo
20.
Cell Tissue Res ; 372(1): 23-31, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29130119

RESUMEN

In mammalian developing embryonic cortex, projection neurons migrate from the ventricular zone to the cortical plate, guided by radial glial cells with a transformation between bipolar and multipolar morphology. Previous studies have demonstrated that the PI3K-Akt-mTOR signal plays a critical role in brain development. However, the function of P85 in cortical development is still unclear. In the present study, we found that overexpression of P85 impaired cortical neuronal migration. Using in utero electroporation, we revealed that the length of the leading process in P85 overexpressed neurons became shorter than that in the control group but with more branches. Using markers for new-born neurons, we further found that overexpression of P85 did not affect the ultimate fate of these cortical neurons. These findings indicated that the P85 subunit plays an essential role in neuronal migration and neuronal morphology during mouse corticogenesis.


Asunto(s)
Movimiento Celular , Forma de la Célula , Corteza Cerebral/citología , Neurogénesis , Neuronas/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Linaje de la Célula , Ratones Endogámicos C57BL , Neuritas/metabolismo
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