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1.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi ; 54(10): 769-775, 2019 Oct 07.
Artigo em Chinês | MEDLINE | ID: mdl-31606991

RESUMO

Objective: To determine the extent of transfection and expression of adeno-associated virus (AAV) serotype 9 (AAV9) in the cochleae of mice at different ages. Methods: AAV9-green fluorescent protein (GFP) was inoculated into the cochlea of mice via the round window membrane (RWM) or through cochleostomy at different ages. Four groups were divided according to ages and injection sites: P1SM group, AAV9-GFP was delivered to the scala media by cochleostomy at postnatal day 1; P1RW group, AAV9-GFP was delivered to the scala tympani via RWM at postnatal day 1; P9RW group: AAV9-GFP was injected through RWM at postnatal day 9; and P30RW group, adult mice (P30) were injected through RWM. GFP expression in cochlear whole mount was analyzed and auditory brainstem response (ABR) tests were conducted one month after virus injection (for each animal, only left cochlea was injected and the right side was used as a control). GraphPad Prism 5 statistical software was used for data analysis. Results: All of inner hair cells (IHCs) and most of outer hair cells (OHCs) were transfected via two approaches at P1 injection. There was no significant difference in ABR threshold between injected ears and untreated ears (P>0.05). All of the IHCs and parts of OHCs (69% in apical turn) were transfected via RWM at P9. The strongest GFP expression was observed near the apical turn. Cochlear inoculation via RWM at P30 led to transgene expression in only IHCs. The ABR threshold of injected ears in P9RW group and P30RW group was significantly higher than that of contralateral ears (P<0.01). Conclusions: AAV9 can be highly expressed in the inner and outer hair cells of the cochlea and hearing sensitivity can be preserved if virus injections are performed in neonatal mice. After AAV9 is transfected into the inner ear of adult mice, it is only expressed in the inner hair cells, which leads to the increase of the ABR response threshold of mice. Transfection efficiency is significant higher in neonate mice than in P9 and adult mice.


Assuntos
Cóclea/virologia , Dependovirus , Células Ciliadas Auditivas/virologia , Transfecção , Fatores Etários , Animais , Cóclea/metabolismo , Cóclea/fisiopatologia , Dependovirus/genética , Dependovirus/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico , Proteínas de Fluorescência Verde/administração & dosagem , Células Ciliadas Auditivas/fisiologia , Camundongos
2.
Zhongguo Gu Shang ; 32(8): 750-755, 2019 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-31533389

RESUMO

OBJECTIVE: To explore infection rate of different adeno-associated virus (AAV) on knee joint cartilage in mice and to find a good gene editing tool for mice chondrocytes of knee joint. METHODS: Forty-five 4-week-old SPF C57BL/6 weighed(14.3±0.2) g were selected. According to different injections(6 µl) for right knee joint, mice were divided into 9 different groups, 5 mice in each group. The groups were such as following:control group (normal saline), Vigene 2 group (AAV2 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 5 group (AAV5 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 6 group (AAV6 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 7 group (AAV7 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 8 group (AAV8 from vigene biosciences, titer for 1×10¹³ vg/ml), Vigene 9 group (AAV9 from vigene biosciences, titer for 1×10¹³ vg/ml), Hanbio DJ group(AAV2-DJ from Hanbio, titer for 1×10¹² vg/ml), Hanbio 5 group (AAV5 from Hanbio, titer for 1×10¹² vg/ml). All AAVs were over-expressed green fluorescent protein(GFP). Knee joint specimens were taken and observed injury of cartilage under stereomicroscope at 30 days after injection, then 10 µm thick frozen sections were prepared. Distribution of green fluorescent protein of meniscus and cartilage of knee joint was observed under fluorescence microscope. RESULTS: Stereomicroscope observation indicated that no obvious lesion was observed in knee joint cartilage of mice after intra-articular injection of AAV. According to frozen sections of knee joints, strong green fluorescence was observed in knee joint cartilage in all AAV experimental groups. Compared with other groups, significantly stronger green fluorescence were observed both in AAV2 and AAV7 groups, whose average fluorescence density was 0.077±0.020 and 0.061±0.022. There were significant differences between two groups and other groups. CONCLUSIONS: AAV could infect chondrocyte of knee joint in vivo by injecting into knee joint cavity. Higher infection efficiency of AAV2 and AAV7 on knee joint cartilage were observed. Local injection of AAV into knee joint cavity could be used as an effective tool for gene editing of knee joint chondrocyte.


Assuntos
Dependovirus , Articulação do Joelho , Animais , Cartilagem , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL
5.
Nat Med ; 25(7): 1123-1130, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270503

RESUMO

Since most dominant human mutations are single nucleotide substitutions1,2, we explored gene editing strategies to disrupt dominant mutations efficiently and selectively without affecting wild-type alleles. However, single nucleotide discrimination can be difficult to achieve3 because commonly used endonucleases, such as Streptococcus pyogenes Cas9 (SpCas9), can tolerate up to seven mismatches between guide RNA (gRNA) and target DNA. Furthermore, the protospacer-adjacent motif (PAM) in some Cas9 enzymes can tolerate mismatches with the target DNA3,4. To circumvent these limitations, we screened 14 Cas9/gRNA combinations for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, we used Beethoven mice5, which harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner-ear hair cells6. We identified a PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. Adeno-associated virus (AAV)-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post injection. Analysis of current ClinVar entries revealed that ~21% of dominant human mutations could be targeted using a similar approach.


Assuntos
Alelos , Edição de Genes , Perda Auditiva Neurossensorial/prevenção & controle , Proteínas de Membrana/genética , Animais , Proteína 9 Associada à CRISPR/fisiologia , Linhagem Celular , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Perda Auditiva Neurossensorial/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL
6.
Neuron ; 103(4): 583-597.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31272828

RESUMO

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.


Assuntos
Técnicas de Introdução de Genes/métodos , Genômica/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoquímica/métodos , Inteínas , Camundongos , Mutagênese Insercional , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteômica , RNA Guia/genética , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico
7.
Nat Neurosci ; 22(8): 1345-1356, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31285614

RESUMO

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.


Assuntos
Dependovirus/genética , Marcação de Genes/métodos , Neuroglia/virologia , Neurônios/virologia , Animais , Técnicas de Transferência de Genes , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Retina/virologia
8.
Drugs ; 79(11): 1255-1262, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31270752

RESUMO

Onasemnogene abeparvovec (onasemnogene abeparvovec-xioi; formerly AVXS-101; ZOLGENSMA®) is an adeno-associated viral vector-based gene therapy designed to deliver a functional copy of the human survival motor neuron (SMN) gene to the motor neuron cells of patients with spinal muscular atrophy (SMA). It has been developed by AveXis, a Novartis company, and was approved in May 2019 in the USA for the treatment of paediatric patients aged < 2 years with SMA and bi-allelic mutations in the SMN1 gene (the primary gene encoding survival motor neuron protein). Onasemnogene abeparvovec is the first gene therapy to be approved for SMA in the USA. The recommended dose is 1.1 × 1014 vector genomes per kg of bodyweight, administered as a single intravenous infusion over 60 min. Regulatory assessments for this formulation of onasemnogene abeparvovec are underway in the EU and Japan; an intrathecal formulation is currently undergoing clinical development in the USA. This article summarizes the milestones in the development of onasemnogene abeparvovec leading to this first approval for the treatment of paediatric patients aged < 2 years with SMA and bi-allelic mutations in SMN1.


Assuntos
Atrofia Muscular Espinal/terapia , Proteínas do Complexo SMN/metabolismo , Dependovirus/genética , Aprovação de Drogas , Terapia Genética , Vetores Genéticos , Humanos , Lactente , Recém-Nascido , Infusões Intravenosas , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Mutação , Proteínas do Complexo SMN/genética , Estados Unidos , United States Food and Drug Administration
9.
Nat Commun ; 10(1): 2958, 2019 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273195

RESUMO

RNAi-based bone anabolic gene therapy has demonstrated initial success, but many practical challenges are still unmet. Here, we demonstrate that a recombinant adeno-associated virus 9 (rAAV9) is highly effective for transducing osteoblast lineage cells in the bone. The adaptor protein Schnurri-3 (SHN3) is a promising therapeutic target for osteoporosis, as deletion of shn3 prevents bone loss in osteoporotic mice and short-term inhibition of shn3 in adult mice increases bone mass. Accordingly, systemic and direct joint administration of an rAAV9 vector carrying an artificial-microRNA that targets shn3 (rAAV9-amiR-shn3) in mice markedly enhanced bone formation via augmented osteoblast activity. Additionally, systemic delivery of rAAV9-amiR-shn3 in osteoporotic mice counteracted bone loss and enhanced bone mechanical properties. Finally, we rationally designed a capsid that exhibits improved specificity to bone by grafting the bone-targeting peptide motif (AspSerSer)6 onto the AAV9-VP2 capsid protein. Collectively, our results identify a bone-targeting rAAV-mediated gene therapy for osteoporosis.


Assuntos
Reabsorção Óssea/complicações , Reabsorção Óssea/prevenção & controle , Osso e Ossos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dependovirus/metabolismo , Inativação Gênica , Osteoporose/complicações , Animais , Reabsorção Óssea/patologia , Osso e Ossos/virologia , Capsídeo/metabolismo , Cartilagem/virologia , Modelos Animais de Doenças , Deleção de Genes , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Sorogrupo
10.
Genome Biol ; 20(1): 113, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159834

RESUMO

BACKGROUND: Genes are comprised of DNA codes and contain promoters and other control elements for reading these codes. The rapid development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has made possible the construction of a novel code-reading system with low dependency on the native control elements. RESULTS: We develop CRISPReader, a technology for controlling promoterless gene expression in a robust fashion. We demonstrate that this tool is highly efficient in controlling transcription and translation initiation of a targeted transgene. A notable feature of CRISPReader is the ability to "read" the open reading frames of a cluster of gene without traditional regulatory elements or other cofactors. In particular, we use this strategy to construct an all-in-one AAV-CRISPR-Cas9 system by removing promoter-like elements from the expression cassette to resolve the existing AAV packaging size problem. The compact AAV-CRISPR-Cas9 is also more efficient in transactivation, DNA cleavage, and gene editing than the dual-AAV vector encoding two separate Cas9 elements, shown by targeting both reporter and endogenous genes in vitro and in vivo. CONCLUSIONS: CRISPReader represents a novel approach for gene regulation that enables minimal gene constructs to be expressed and can be used in potential biomedical applications.


Assuntos
Sistemas CRISPR-Cas , Expressão Gênica , Técnicas Genéticas , Iniciação Traducional da Cadeia Peptídica , Ativação Transcricional , Animais , Dependovirus , Luciferases de Renilla , Camundongos
11.
Neuron ; 103(3): 445-458.e10, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31202541

RESUMO

To make adaptive decisions, organisms must appropriately filter sensory inputs, augmenting relevant signals and suppressing noise. The prefrontal cortex (PFC) partly implements this process by regulating thalamic activity through modality-specific thalamic reticular nucleus (TRN) subnetworks. However, because the PFC does not directly project to sensory TRN subnetworks, the circuitry underlying this process had been unknown. Here, using anatomical tracing, functional manipulations, and optical identification of PFC projection neurons, we find that the PFC regulates sensory thalamic activity through a basal ganglia (BG) pathway. Engagement of this PFC-BG-thalamus pathway enables selection between vision and audition by primarily suppressing the distracting modality. This pathway also enhances sensory discrimination and is used for goal-directed background noise suppression. Overall, our results identify a new pathway for attentional filtering and reveal its multiple roles in sensory processing on the basis of internal goals.


Assuntos
Gânglios da Base/fisiologia , Vias Neurais/fisiologia , Córtex Pré-Frontal/fisiologia , Filtro Sensorial/fisiologia , Tálamo/fisiologia , Estimulação Acústica , Animais , Condicionamento Operante , Sinais (Psicologia) , Dependovirus/genética , Aprendizagem por Discriminação/fisiologia , Eletrodos Implantados , Vetores Genéticos , Camundongos , Ruído , Optogenética , Estimulação Luminosa , Recompensa , Detecção de Sinal Psicológico/fisiologia
12.
J Biotechnol ; 300: 70-77, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31150679

RESUMO

Adeno-associated viral vectors (AAV) for gene therapy applications are gaining momentum, with more therapies moving into later stages of clinical development and towards market approval, namely for cancer therapy. The development of cytotoxic vectors is often hampered by side effects arising when non-target cells are infected, and their production can be hindered by toxic effects of the transgene on the producing cell lines. In this study, we evaluated the potential of rAAV-mediated delivery of short hairpin RNAs (shRNA) to target basal-like breast cancer genetic vulnerabilities. Our results show that by optimizing the stoichiometry of the plasmids upon transfection and time of harvest, it is possible to increase the viral titers and quality. All rAAV-shRNA vectors obtained efficiently transduced the BLBC cell lines MDA-MB-468 and HCC1954. In MDA-MB-468, transduction with rAAV-shRNA vector targeting PSMA2 was associated with significant decrease in cell viability and apoptosis induction. Importantly, rAAV2-PSMA2 also slowed tumor growth in a BLBC mouse xenograft model, thus potentially representing a therapeutic strategy against this type of cancer.


Assuntos
Neoplasias da Mama/genética , Dependovirus/genética , Neoplasia de Células Basais/genética , RNA Interferente Pequeno/genética , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes/normas , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Neoplasia de Células Basais/patologia , Neoplasia de Células Basais/terapia , Plasmídeos , Complexo de Endopeptidases do Proteassoma/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/farmacologia , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Int J Mol Sci ; 20(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064116

RESUMO

Hypercholesterolemia may be causally related to heart failure with preserved ejection fraction (HFpEF). We aimed to establish a HFpEF model associated with hypercholesterolemia and type 2 diabetes mellitus by feeding a high-sucrose/high-fat (HSHF) diet to C57BL/6J low-density lipoprotein receptor (LDLr)-/- mice. Secondly, we evaluated whether cholesterol-lowering adeno-associated viral serotype 8 (AAV8)-mediated LDLr gene transfer prevents HFpEF. AAV8-LDLr gene transfer strongly (p < 0.001) decreased plasma cholesterol in standard chow (SC) mice (66.8 ± 2.5 mg/dl versus 213 ± 12 mg/dl) and in HSHF mice (84.6 ± 4.4 mg/dl versus 464 ± 25 mg/dl). The HSHF diet induced cardiac hypertrophy and pathological remodeling, which were potently counteracted by AAV8-LDLr gene transfer. Wet lung weight was 19.0% (p < 0.001) higher in AAV8-null HSHF mice than in AAV8-null SC mice, whereas lung weight was normal in AAV8-LDLr HSHF mice. Pressure-volume loop analysis was consistent with HFpEF in AAV8-null HSHF mice and showed a completely normal cardiac function in AAV8-LDLr HSHF mice. Treadmill exercise testing demonstrated reduced exercise capacity in AAV8-null HSHF mice but a normal capacity in AAV8-LDLr HSHF mice. Reduced oxidative stress and decreased levels of tumor necrosis factor-α may mediate the beneficial effects of cholesterol lowering. In conclusion, AAV8-LDLr gene therapy prevents HFpEF.


Assuntos
Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/prevenção & controle , Terapia Genética/métodos , Insuficiência Cardíaca/prevenção & controle , Hipercolesterolemia/terapia , Receptores de LDL/genética , Animais , Colesterol/sangue , Dependovirus/genética , Diabetes Mellitus Tipo 2/etiologia , Cardiomiopatias Diabéticas/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/efeitos adversos , Feminino , Insuficiência Cardíaca/fisiopatologia , Hipercolesterolemia/complicações , Hipercolesterolemia/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo , Receptores de LDL/metabolismo , Volume Sistólico , Fator de Necrose Tumoral alfa/sangue
14.
Nihon Yakurigaku Zasshi ; 153(5): 204-209, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31092752

RESUMO

Viral vectors, including lentiviral vectors and adeno-associated viral vectors, have been used as a delivery tool for transduction of neuronal and glial cells with a variety of genetic tools in vitro and in vivo. Although viral vector technologies are essential for application of genetic tools especially in vivo, less attention has been paid to the biological basis of these technologies than to genetic tools delivered. Here we would like to summarize the biological basis of lentiviral vectors and adeno-associated viral vectors and briefly introduce the recent advances from the perspective of the application of these viral vectors to pharmacological research.


Assuntos
Dependovirus , Vetores Genéticos , Lentivirus , Farmacologia , Pesquisa , Humanos , Neuroglia , Neurônios
15.
Nihon Yakurigaku Zasshi ; 153(5): 219-223, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31092754

RESUMO

The development and persistence of drug addiction are associated with the activation and adaptation of the brain reward circuitry, which consists of dopaminergic projection from the ventral tegmental area to the nucleus accumbens (NAc) and the medial prefrontal cortex (mPFC). In cocaine addiction, cocaine-induced activation and neuroplasticity in the brain reward circuitry may contribute to the acquisition and expression of rewarding memory of cocaine, which is critical for the reinstatement of cocaine seeking. However, it remains unclear which neuronal types causally contribute to the retrieval of cocaine-associated rewarding memory. To address this issue, we used DREADD (Designer Receptors Exclusively Activated by Designer Drugs) technology. To selectively suppress mPFC excitatory neurons, we infused an adeno-associated virus (AAV5 or AAV-DJ) vector expressing hM4Di, an inhibitory DREADD, under the control of CaMKII promotor into the mPFC of wildtype mice. To selectively suppress GABAergic neurons, we infused a Cre-dependent AAV (AAV5 or AAV-DJ) vector expressing hM4Di into the mPFC of GAD67-Cre mice or the NAc of vGAT-Cre mice. We found that, in cocaine conditioned place preference paradigm, the activity of mPFC pyramidal and NAc GABAergic neurons is causally related to the retrieval of cocaine-associated memory. The findings suggest that the mPFC-NAc circuit can be a potential therapeutic target for the drug addiction.


Assuntos
Cocaína/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Vetores Genéticos , Memória , Recompensa , Animais , Dependovirus , Camundongos , Núcleo Accumbens/citologia , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos
16.
Nihon Yakurigaku Zasshi ; 153(5): 224-230, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31092755

RESUMO

Decision-making is a key activity process that influences many aspects of daily living and both mental and physical health. In general, healthy participants reveal rational choice, but patients with neuropsychiatric disorders reveal irrational and risky choice in decision-making. Addiction is one of typical diseases revealed risky decision-making, addicts select risky action and options that confer short-term rewards at the cost of long-term disadvantages. Thus, irrational and risky decision-making is recognized as a core problem in patients with neuropsychiatric disorders, and a better understanding of the mechanisms underlying altered decision-making would provide insights into potential therapeutic approaches for these diseases. However, the neural pathway and substrates underlying these deficits are particularly unknown. Recently, we found that insular cortex is one of key regions for risky decision-making in an animal model of methamphetamine addiction, by using the designer receptor exclusively activated by designer drug (DREADD) technology, and that GABAergic dysfunction in insular cortex is involved in evaluating the subjective value of reward and reward prediction error. These brain dysfunctions would be related to risk taking behavior in addiction. In this review, we introduced the possible neural pathway related to risky decision-making and behavioral changes in choice strategy using adeno associated virus (AAV).


Assuntos
Córtex Cerebral/fisiologia , Tomada de Decisões , Metanfetamina/farmacologia , Assunção de Riscos , Animais , Dependovirus , Neurônios GABAérgicos/citologia , Humanos , Recompensa
17.
Methodist Debakey Cardiovasc J ; 15(1): 62-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31049151

RESUMO

Cardiovascular disease is the leading cause of death worldwide, and elevated lipid levels is a major contributor. Gene delivery, which involves controlled transfer of nucleic acids into cells and tissues, has been widely used in research to study lipid metabolism and physiology. Several technologies have been developed to somatically overexpress, silence, or disrupt genes in animal models and have greatly advanced our knowledge of metabolism. This is particularly true with regard to the liver, which plays a central role in lipoprotein metabolism and is amenable to many delivery approaches. In addition to basic science applications, many of these delivery technologies have potential as gene therapies for both common and rare lipid disorders. This review discusses three major gene delivery technologies used in lipid research-including adeno-associated viral vector overexpression, antisense oligonucleotides and small interfering RNAs, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing system-and examines their potential therapeutic applications.


Assuntos
Doenças Cardiovasculares/prevenção & controle , Dislipidemias/terapia , Terapia Genética/métodos , Metabolismo dos Lipídeos/genética , Lipídeos/sangue , Animais , Sistemas CRISPR-Cas , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Dependovirus/genética , Dislipidemias/sangue , Dislipidemias/epidemiologia , Dislipidemias/genética , Edição de Genes/métodos , Terapia Genética/efeitos adversos , Vetores Genéticos , Humanos , Oligonucleotídeos Antissenso/genética , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Resultado do Tratamento
18.
Nat Commun ; 10(1): 2315, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127098

RESUMO

Encoding and retrieval of contextual memories is initially mediated by sparsely activated neurons, so-called engram cells, in the hippocampus. Subsequent memory persistence is thought to depend on network-wide changes involving progressive contribution of cortical regions, a process referred to as systems consolidation. Using a viral-based TRAP (targeted recombination in activated populations) approach, we studied whether consolidation of contextual fear memory by neurons in the medial prefrontal cortex (mPFC) is modulated by memory strength and CREB function. We demonstrate that activity of a small subset of mPFC neurons is sufficient and necessary for remote memory expression, but their involvement depends on the strength of conditioning. Furthermore, selective disruption of CREB function in mPFC engram cells after mild conditioning impairs remote memory expression. Together, our data demonstrate that memory consolidation by mPFC engram cells requires CREB-mediated transcription, with the functionality of this network hub being gated by memory strength.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Medo/fisiologia , Consolidação da Memória/fisiologia , Memória de Longo Prazo/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Comportamento Animal/fisiologia , Condicionamento (Psicologia)/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microinjeções , Modelos Animais , Neurônios/metabolismo , Técnicas de Patch-Clamp , Córtex Pré-Frontal/citologia , Técnicas Estereotáxicas
19.
Clin Exp Med ; 19(3): 289-298, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31054018

RESUMO

Adeno-associated virus (AAV)-derived vectors are currently the most common type of viral vectors used in gene therapy clinical trials. The presence of neutralizing antibodies (NAbs) against wild-type AAVs in the host body is one of the limitations for the successful use of AAV vectors. AAV capsid manipulation, by which recombinant vectors lose their ability to interact with NAbs, can help overcome this obstacle. Various methods can be used for this purpose, including directed evolution as well as conjugation of certain chemical groups to AAV epitopes. The present review concisely explains the use of AAV vectors in the clinic for gene therapy of some diseases, their limitations, and solutions to these limitations.


Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Dependovirus/imunologia , Portadores de Fármacos , Humanos
20.
Nat Commun ; 10(1): 1802, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996254

RESUMO

The primary cause of heart failure is the loss of cardiomyocytes in the diseased adult heart. Previously, we reported that the miR-17-92 cluster plays a key role in cardiomyocyte proliferation. Here, we report that expression of miR-19a/19b, members of the miR-17-92 cluster, is induced in heart failure patients. We show that intra-cardiac injection of miR-19a/19b mimics enhances cardiomyocyte proliferation and stimulates cardiac regeneration in response to myocardial infarction (MI) injury. miR-19a/19b protected the adult heart in two distinctive phases: an early phase immediately after MI and long-term protection. Genome-wide transcriptome analysis demonstrates that genes related to the immune response are repressed by miR-19a/19b. Using an adeno-associated virus approach, we validate that miR-19a/19b reduces MI-induced cardiac damage and protects cardiac function. Finally, we confirm the therapeutic potential of miR-19a/19b in protecting cardiac function by systemically delivering miR-19a/19b into mice post-MI. Our study establishes miR-19a/19b as potential therapeutic targets to treat heart failure.


Assuntos
Terapia Genética/métodos , Insuficiência Cardíaca/patologia , MicroRNAs/administração & dosagem , MicroRNAs/metabolismo , Infarto do Miocárdio/terapia , Animais , Proliferação de Células/genética , Dependovirus/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Insuficiência Cardíaca/terapia , Ventrículos do Coração/patologia , Humanos , Injeções Intralesionais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Regeneração/genética
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