Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 154
Filter
1.
Neuroscience Bulletin ; (6): 90-102, 2024.
Article in English | WPRIM | ID: wpr-1010647

ABSTRACT

Retrograde adeno-associated viruses (AAVs) are capable of infecting the axons of projection neurons and serve as a powerful tool for the anatomical and functional characterization of neural networks. However, few retrograde AAV capsids have been shown to offer access to cortical projection neurons across different species and enable the manipulation of neural function in non-human primates (NHPs). Here, we report the development of a novel retrograde AAV capsid, AAV-DJ8R, which efficiently labeled cortical projection neurons after local administration into the striatum of mice and macaques. In addition, intrastriatally injected AAV-DJ8R mediated opsin expression in the mouse motor cortex and induced robust behavioral alterations. Moreover, AAV-DJ8R markedly increased motor cortical neuron firing upon optogenetic light stimulation after viral delivery into the macaque putamen. These data demonstrate the usefulness of AAV-DJ8R as an efficient retrograde tracer for cortical projection neurons in rodents and NHPs and indicate its suitability for use in conducting functional interrogations.


Subject(s)
Animals , Haplorhini , Axons , Motor Neurons , Interneurons , Macaca , Dependovirus/genetics , Genetic Vectors
2.
Journal of Integrative Medicine ; (12): 106-115, 2023.
Article in English | WPRIM | ID: wpr-971643

ABSTRACT

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.


Subject(s)
Mice , Animals , Humans , Melitten/genetics , Dependovirus/genetics , Serogroup , HEK293 Cells , Mice, Nude , Mice, Inbred C57BL , Transgenes , Genetic Vectors/genetics
3.
Journal of Southern Medical University ; (12): 944-948, 2022.
Article in Chinese | WPRIM | ID: wpr-941025

ABSTRACT

OBJECTIVE@#To express and purify the antigenic peptide of adeno-associated virus (AAV) capsid conserved regions in prokaryotic cells and prepare its rabbit polyclonal antibody.@*METHODS@#The DNA sequence encoding the conserved regions of AAV capsid protein was synthesized and cloned into the vector pET30a to obtain the plasmid pET30a-AAV-CR for prokaryotic expression and purification of the conserved peptides. Coomassie blue staining and Western blotting were used to identify the AAV conserved peptides. Japanese big ear white rabbits were immunized with AAV conserved region protein to prepare polyclonal antibody, with the rabbits injected with PBS as the control group. The antibody titer was determined with ELISA, and the performance of the antibody for recognizing capsid protein sequences of AAV1-AAV10 was assessed with Western blotting and immunofluorescence assay.@*RESULTS@#The plasmid pET30a-AAV-CR was successfully constructed, and a recombinant protein with a relative molecular mass of 17000 was obtained. The purified protein induced the production of antibodies against the conserved regions of AAV capsid in rabbits, and the titer of the purified antibodies reached 1:320 000. The antibodies were capable of recognizing a wide range of capsid protein sequences of AAV1-AAV10.@*CONCLUSION@#We successfully obtained the polyclonal antibodies against AAV capsid conserved region protein from rabbits, which facilitate future studies of AAV vector development and the biological functions of AAV.


Subject(s)
Animals , Rabbits , Antibodies , Capsid , Capsid Proteins/genetics , Dependovirus/genetics , Prokaryotic Cells , Recombinant Proteins/genetics
4.
Neuroscience Bulletin ; (6): 1271-1288, 2021.
Article in English | WPRIM | ID: wpr-922636

ABSTRACT

Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.


Subject(s)
Animals , Brain , CRISPR-Cas Systems/genetics , Dependovirus/genetics , Haplorhini , Phenotype , Protein Kinases/genetics
5.
Journal of Integrative Medicine ; (12): 515-525, 2021.
Article in English | WPRIM | ID: wpr-922523

ABSTRACT

OBJECTIVE@#Plant-derived cytotoxic transgene expression, such as trichosanthin (tcs), regulated by recombinant adeno-associated virus (rAAV) vector is a promising cancer gene therapy. However, the cytotoxic transgene can hamper the vector production in the rAAV producer cell line, human embryonic kidney (HEK293) cells. Here, we explored microRNA-122 (miR122) and its target sequence to limit the expression of the cytotoxic gene in the rAAV producer cells.@*METHODS@#A miR122 target (122T) sequence was incorporated into the 3' untranslated region of the tcs cDNA sequence. The firefly luciferase (fluc) transgene was used as an appropriate control. Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells. The effects of miR122 overexpression on cell growth, transgene expression, and rAAV production were determined.@*RESULTS@#The presence of 122T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line (in vitro), fresh human hepatocytes (ex vivo), and mouse liver (in vivo). Also, the normal liver physiology was unaffected by delivery of 122T sequence by rAAV vectors. Compared with the parental cells, the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122T, as well as the ability to produce liver-targeting rAAV vectors. Fascinatingly, the yield of rAAV vectors carrying the tcs-122T gene was increased by 77.7-fold in HEK293-mir122 cells. Moreover, the tcs-122T-containing rAAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.@*CONCLUSION@#HEK293-mir122 cells along with the 122T sequence provide a potential tool to attenuate the cytotoxic transgene expression, such as tcs, during rAAV vector production.


Subject(s)
Animals , Humans , Mice , Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , HEK293 Cells , MicroRNAs/genetics , Trichosanthin
6.
Chinese Journal of Hematology ; (12): 34-39, 2020.
Article in Chinese | WPRIM | ID: wpr-1012136

ABSTRACT

Objective: To evaluate the effects of adeno-associated virus (AAV) carrying hFⅧ by serotype 8 (AAV8/hFⅧ) on hemophilia A (HA) mice by gene therapy strategy. Methods: pAAV-CB-EGFP, pH22 (serotype 2) and pfΔ6 (adenovirus helper) were used to package AAV into HEK-293 cells in different conditions (ratios of cells to plasmids). The efficiency of transfection and infection were evaluated using immunofluorescence microscope to seek an optimized package condition. pAAV-TTR-hFⅧ, pH 28 (serotype 8) and pfΔ6 were applied to package AAV8/hFⅧ in HEK-293 cells using the optimized package condition. The purified AAV8/hFⅧ were intravenously injected into HA mice and the effects of gene therapy were estimated. Results: The efficiency of package was evaluated according to the amount and intensity of enhanced green fluorescent protein (EGFP) under immunofluorescence microscope. Four package conditions including 10 cm-dish to transfect 10 μg plasmids, 20 cm-dish to 20 μg, 30 μg and 40 μg plasmids were employed, and the condition of 20 cm-dish to transfect 20 μg plasmids reached the highest transfection efficiency at 24 h, 48 h and 72 h after transfection. The small scale AAV-EGFP was packaged using the optimized condition and an AAV crude extract was harvested by a freeze-thaw method. HEK-293 and 16095 cells were infected by the AAV crude extract, and the preferential infection efficiency was recognized in 16095 cells under immunofluorescence microscope. Then, AAV8/hFⅧ was packaged and purified based on the optimized transfection condition, and the high purity of AAV8/hFⅧ was detected by Western blot. Fractions of AAV8/hFⅧ at the dose of 8×10(12) vg/kg were injected into HA mice through tail vein, an eye-bleeding was performed at every two weeks, and the activity of FⅧ was measured by aPTT assay. Results showed that the activity of FⅧ maintained at the therapeutic level and lasted up to 12 weeks after injection. Conclusion: The purified AAV8/hFⅧ based on the optimized package condition could play a role in HA mice gene therapy, and the long-term therapeutic effects of AAV8/hFⅧ were observed in vivo.


Subject(s)
Animals , Humans , Mice , Dependovirus , Genetic Therapy , Genetic Vectors , HEK293 Cells , Hemophilia A
7.
Journal of Peking University(Health Sciences) ; (6): 845-850, 2020.
Article in Chinese | WPRIM | ID: wpr-942084

ABSTRACT

OBJECTIVE@#To investigate the expression efficiency of exogenous gene mediated by different serotypes of adeno-associated virus (AAV) vectors in retina, and to compare the expression efficiency of AAV vector and two kinds of promoters commonly used in ophthalmology after transfection into mouse retina, so as to provide the basis for selecting appropriate AAV vector and promoter for gene therapy of retinitis pigmentosa.@*METHODS@#AAV2/2, AAV2/5, AAV2/8 and AAV2/9 were prepared. The C57BL/6J mice were injected subretinally with 1 μL purified AAV vectors (1.00×1013 mg/L). Then the mice were killed 2 or 4 weeks after treatment, and the eyes were enucleated for frozen section. The expression of green fluorescent protein (GFP) was observed under the confocal microscope. Two kinds of promoters, CMV and CAG, were selectd, and the expression of AAV2/8-GFP-CMV and AAV2/8-GFP-CAG was observed under confocal microscope.@*RESULTS@#No bacterial infection or immune response were seen in the injected mice. 2 weeks after injection, the GFP green fluorescence of AAV2/8 and AAV2/9 in the mouse retina was obvious, which indicated that the GFP green fluorescence of AAV2/8 and AAV2/9 was high after transfection into the mouse retina. In these two serotypes, GFP green fluorescence of AAV2/8 was mainly concentrated in photoreceptor cells while AAV2/8 was expressed in the whole retina, indicating that AAV2/8 was more specific to photoreceptors. Further experiments on AAV2/8 showed that the GFP green fluorescence of the mouse retina was obvious 4 weeks after injection, indicating that the exogenous gene mediated by AAV2/8 could be stably expressed in vivo. For CMV and CAG promoters, CMV promoter was expressed stronger in retinal pigment epithelium (RPE)cells, while CAG promoter was stronger in photorecepters. In photorecepters, CAG promoter was expressed almost the same as CMV promoter, while CMV promoter was stronger in RPE cells.@*CONCLUSION@#AAV vectors could express transgene robustly in retinal cells; Among several AAV serotypes, AAV2/2 and AAV2/5 showed weaker GFP fluorescence than AAV2/8 and AAV2/9. AAV2/9 showed expression in each layer of the retina including ganglion cells. AAV2/8 was more specific for photoreceptor; CAG promoters had higher specificity for photoreceptors than CMV promoters.


Subject(s)
Animals , Mice , Dependovirus/genetics , Genetic Vectors , Mice, Inbred C57BL , Retina , Serogroup , Transduction, Genetic
8.
Medicina (B.Aires) ; 79(6): 493-501, dic. 2019. ilus, tab
Article in Spanish | LILACS | ID: biblio-1056758

ABSTRACT

En los ó;ºltimos aó;±os la terapia gó;©nica se ha posicionado como una opció;n real y segura en el desarrollo de alternativas terapó;©uticas para la cura y la prevenció;n de diferentes enfermedades. Consiste en la inserció;n de material genó;©tico en un tejido o có;©lula defectuosa, mediante el uso de un vector. Existen varias consideraciones para seleccionar el vector más apropiado, incluyendo el potencial de unió;n y entrada a la có;©lula diana, la capacidad de transferencia del material genó;©tico al nó;ºcleo, la habilidad de expresió;n del inserto y la ausencia de toxicidad. En el panorama actual, los vectores virales más utilizados son los derivados de los virus adenoasociados (AAV). Características como su bioseguridad, baja toxicidad y tropismo selectivo, han posibilitado su evaluació;n como opció;n terapó;©utica en un amplio nó;ºmero de enfermedades monogó;©nicas o complejas. A pesar de sus ventajas, los vectores AAV presentan inconvenientes, siendo el más importante la respuesta inmune del paciente al vector, especialmente la respuesta mediada por anticuerpos neutralizantes (NAb). Los NAb disminuyen la transducció;n del vector e impiden la expresió;n del gen que transporta, limitando su aplicació;n clínica. Por lo tanto, identificar y cuantificar la presencia y actividad de los NAbs, es el primer paso en cualquier protocolo de terapia gó;©nica con vectores AAV. La presencia de NAb depende principalmente de la exposició;n al virus en la naturaleza y varía drásticamente segó;ºn edad, localizació;n geográfica y estado de salud de la persona evaluada.


In recent years, gene therapy has been positioned as a real and safe option in the development of therapeutic alternatives for the cure and prevention of different diseases. It consists in the insertion of genetic material in a defective tissue or cell, through the use of a vector. There are several considerations for selecting the most appropriate vector, including the potential for binding and entry to the target cell, the ability of the genetic material to transfer to the nucleus, the ability to express the insert, and the absence of toxicity. In the current scenario, the most commonly used viral vectors are those derived from adeno-associated viruses (AAV). Characteristics such as biosafety, low toxicity and selective tropism have enabled its evaluation as a therapeutic option in many monogenic or complex diseases. Despite their advantages, AAV vectors have drawbacks, the most important being the patient’s immune response to the vector, especially the response mediated by neutralizing antibodies (NAb). NAbs decrease the transduction of the vector and prevent the expression of the gene it transports, limiting its clinical application. Therefore, identifying and quantifying the presence and activity of NAbs is the first step in any gene therapy protocol with AAV vectors. The presence of NAbs depends mainly on exposure to the virus in nature and varies drastically according to age, geographic location and health status of the person evaluated.


Subject(s)
Humans , Male , Female , Genetic Therapy/methods , Dependovirus/genetics , Dependovirus/immunology , Parvoviridae Infections/genetics , Parvoviridae Infections/immunology , Parvoviridae Infections/virology , Antibodies, Neutralizing/analysis , Serogroup , Genetic Vectors , Antibodies, Viral/analysis
9.
Journal of Central South University(Medical Sciences) ; (12): 1413-1418, 2019.
Article in Chinese | WPRIM | ID: wpr-812999

ABSTRACT

Gene therapy plays an important role in Alzheimer's disease (AD). In recent years, the research on gene delivery vector has gradually transferred from adenovirus vector, adeno-associated virus vector and lentivirus vector to liposomes and nanomaterial carrier systems. Graphene, the newest member of nanomaterial carrier system, has attracted extensive attention for its well permeability and biocompatibility. The methods of gene therapy can be divided into direct and indirect method. The stem cell therapy, which is the most-well studied one, belongs to the indirect method. In the gene therapy of AD, the selection of appropriate carrier and method will determine the therapeutic effect.


Subject(s)
Humans , Alzheimer Disease , Therapeutics , Dependovirus , Gene Transfer Techniques , Genetic Therapy , Genetic Vectors
10.
China Journal of Orthopaedics and Traumatology ; (12): 750-755, 2019.
Article in Chinese | WPRIM | ID: wpr-773841

ABSTRACT

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.


Subject(s)
Animals , Mice , Cartilage , Dependovirus , Green Fluorescent Proteins , Knee Joint , Mice, Inbred C57BL
11.
Biomédica (Bogotá) ; 38(3): 388-397, jul.-set. 2018. graf
Article in English | LILACS | ID: biblio-973992

ABSTRACT

Abstract Introduction: Cerebral ischemia is the third cause of death risk in Colombia and the first cause of physical disability worldwide. Different studies on the silencing of the cyclin-dependent kinase 5 (CDK5) have shown that reducing its activity is beneficial in ischemic contexts. However, its effect on neural cell production after cerebral ischemia has not been well studied yet. Objective: To evaluate CDK5 silencing on the production of neurons and astrocytes after a focal cerebral ischemia in rats. Materials and methods: We used 40 eight-week-old male Wistar rats. Both sham and ischemia groups were transduced at CA1 hippocampal region with an adeno-associated viral vector using a noninterfering (shSCRmiR) and an interfering sequence for CDK5 (shCDK5miR). We injected 50 mg/kg of bromodeoxyuridine intraperitoneally from hour 24 to day 7 post-ischemia. We assessed the neurological abilities during the next 15 days and we measured the immunoreactivity of bromodeoxyuridine (BrdU), doublecortin (DCX), NeuN, and glial fibrillary acid protein (GFAP) from day 15 to day 30 post-ischemia. Results: Our findings showed that CDK5miR-treated ischemic animals improved their neurological score and presented increased BrdU+ cells 15 days after ischemia, which correlated with higher DCX and lower GFAP fluorescence intensities, and, although mature neurons populations did not change, GFAP immunoreactivity was still significantly reduced at 30 days post-ischemia in comparison with untreated ischemic groups. Conclusion: CDK5miR therapy generated the neurological recovery of ischemic rats associated with the induction of immature neurons proliferation and the reduction of GFAP reactivity at short and longterm post-ischemia.


Resumen Introducción. La isquemia cerebral es la tercera causa de riesgo de muerte en Colombia y la primera causa de discapacidad física en el mundo. En diversos estudios en los que se silenció la cinasa 5 dependiente de la ciclina (CDK5) se ha demostrado que la reducción de su actividad es beneficiosa frente a la isquemia. Sin embargo, su efecto sobre la neurogénesis después de la isquemia no se ha dilucidado suficientemente. Objetivo. Evaluar el silenciamiento de la CDK5 en la neurogénesis y la gliogénesis después de la isquemia cerebral focal en ratas. Materiales y métodos. Se usaron 40 machos de rata Wistar de ocho semanas de edad. Los grupos de control y los isquémicos sometidos a transducción en la región del hipocampo CA1, se inyectaron intraperitonealmente por estereotaxia con 50 mg/kg de bromodesoxiuridina (BrdU) a partir de las 24 horas y hasta el día 7 después de la isquemia, con un vector viral asociado a adenovirus usando una secuencia no interferente (SCRmiR) y una interferente (CDK5miR). Se evaluó la capacidad neurológica durante los quince días siguientes y se detectó la capacidad de inmunorreacción para la BrdU, la proteína doblecortina (DCX), los núcleos neuronales (NeuN), y la proteína fibrilar acídica de la glía (Glial Fibrillary Acidic Protein, GFAP) a los 15 y 30 días de la isquemia. Resultados. Los animales isquémicos tratados con CDK5miR mejoraron su puntuación neurológica y presentaron un incremento de la BrdU+ a los 15 días de la isquemia, lo cual se correlacionó con una mayor intensidad de la DCX+ y una menor de la GFAP+. No hubo modificación de los NeuN+, pero sí una reducción significativa de la GFAP+ a los 30 días de la isquemia en los animales tratados comparados con los animales isquémicos no tratados. Conclusión. La terapia con CDK5miR generó la recuperación neurológica de ratas isquémicas asociada con la inducción de la neurogénesis y el control de la capacidad de reacción de la proteína GFAP a corto y largo plazo después de la isquemia.


Subject(s)
Animals , Male , Rats , Genetic Therapy , Brain Ischemia/therapy , Neuroglia/physiology , RNA, Small Interfering/therapeutic use , RNA Interference , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Neurogenesis/genetics , Molecular Targeted Therapy , Genetic Vectors/therapeutic use , Biomarkers , Genetic Therapy/methods , Brain Ischemia/genetics , Brain Ischemia/pathology , Astrocytes/pathology , Carotid Stenosis , Rats, Wistar , Dependovirus/genetics , RNA, Small Interfering/administration & dosage , DNA Replication , Drug Evaluation , Cyclin-Dependent Kinase 5/genetics , Molecular Targeted Therapy/methods , Doublecortin Protein , Ligation , Neurons/pathology
12.
Experimental Neurobiology ; : 350-361, 2017.
Article in English | WPRIM | ID: wpr-146667

ABSTRACT

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.


Subject(s)
Animals , Humans , Mice , Rats , Amygdala , Astrocytes , Brain , Brain Diseases , Dependovirus , Gene Expression , Genetic Therapy , Glial Fibrillary Acidic Protein , Hippocampus , Neurons , Prefrontal Cortex , Recombinases , Thalamus , Ventral Thalamic Nuclei
13.
Acta Physiologica Sinica ; (6): 241-251, 2017.
Article in Chinese | WPRIM | ID: wpr-348278

ABSTRACT

Rac1 belongs to the family of Rho GTPases, and plays important roles in the brain function. It affects the cell migration and axon guidance via regulating the cytoskeleton and cellular morphology. However, the effect of its dynamic activation in regulating physiological function remains unclear. Recently, a photoactivatable analogue of Rac1 (PA-Rac1) has been developed, allowing the activation of Rac1 by the specific wavelength of light in living cells. Thus, we constructed recombinant adeno-associated virus (AAV) of PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A under the control of the mouse glial fibrillary acidic protein (mGFAP) promoter to manipulate Rac1 activity in astrocytes by optical stimulation. Primary culture of hippocampal astrocytes was infected with the recombinant AAV-PA-Rac1 or AAV-PA-Rac1-C450A. Real-time fluorescence imaging showed that the cell membrane of the astrocyte expressing PA-Rac1 protruded near the light spot, while the astrocyte expressing PA-Rac1-C450A did not. We injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A into dorsal hippocampus to investigate the role of the activation of Rac1 in regulating the associative learning. With optical stimulation, the PA-Rac1 group, rather than the PA-Rac1-C450A group, showed slower learning curve during the fear conditioning compared with the control group, indicating that activating astrocytic Rac1 blocks the formation of contextual memory. Our data suggest that the activation of Rac1 in dorsal hippocampal astrocyte plays an important role in the associative learning.


Subject(s)
Animals , Mice , Astrocytes , Physiology , Cell Membrane , Cell Movement , Conditioning, Classical , Cytoskeleton , Dependovirus , Fear , Hippocampus , Physiology , Memory , Mice, Inbred C57BL , Neuropeptides , Genetics , Physiology , Optogenetics , rac1 GTP-Binding Protein , Genetics , Physiology
14.
Electron. j. biotechnol ; 19(4): 75-80, July 2016. ilus
Article in English | LILACS | ID: lil-793956

ABSTRACT

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results:Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 x 10(4),1 x 10(5),and 1x10(6) of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 x 10(4),1 x 10(5) and 1 x 10(6), respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.


Subject(s)
Dependovirus , Glial Cell Line-Derived Neurotrophic Factor , HEK293 Cells , Recombination, Genetic , Transduction, Genetic , Cell Line , Polymerase Chain Reaction , Green Fluorescent Proteins , Genetic Vectors , Microscopy, Fluorescence
15.
Experimental Neurobiology ; : 48-54, 2016.
Article in English | WPRIM | ID: wpr-169709

ABSTRACT

Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.


Subject(s)
Animals, Laboratory , Antibodies , Astrocytes , Brain , Dependovirus , Fluorescence , Haplorhini , Hypertrophy , Lentivirus , Macaca fascicularis , Neuroglia , Neurons , Putamen , Tropism
16.
Experimental Neurobiology ; : 120-129, 2016.
Article in English | WPRIM | ID: wpr-213645

ABSTRACT

Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population.


Subject(s)
Animals , Mice , Dependovirus , Fluorescence , Genes, Homeobox , Genes, Reporter , Immunohistochemistry , Mice, Transgenic , Neurons , RNA, Small Interfering
17.
Yonsei Medical Journal ; : 790-794, 2016.
Article in English | WPRIM | ID: wpr-205734

ABSTRACT

Recombinant gene expression using adeno-associated viruses (AAVs) has become a valuable tool in animal studies, as they mediate safe expression of transduced genes for several months. The liver is a major organ of metabolism, and liver-specific expression of a gene can be an invaluable tool for metabolic studies. AAV-DJ is a recombinant AAV generated by the gene shuffling of various AAV serotypes and shares characteristics of AAV2 and AAV8. AAV-DJ contains a heparin-binding domain in its capsid, which suggests that a heparin column could be used for the purification of the AAV. Given that AAV-DJ has been only recently available, relatively little is known about the optimal preparation/purification and application of AAV-DJ. Here, we present a simple large-scale preparation method that can generate 3×10(13) viral particles for in vivo experiments and demonstrate liver-specific gene expression via systemic injection in mice.


Subject(s)
Animals , Humans , Mice , Capsid , Capsid Proteins/genetics , Dependovirus/genetics , Gene Expression , Genetic Vectors , Genome, Viral/genetics , Hep G2 Cells , Liver/metabolism , Mice, Inbred C57BL
18.
Annals of Laboratory Medicine ; : 469-474, 2016.
Article in English | WPRIM | ID: wpr-59847

ABSTRACT

BACKGROUND: The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. METHODS: A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. RESULTS: The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). CONCLUSIONS: AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Asian People , Capsid Proteins/genetics , Carcinoma, Hepatocellular/etiology , DNA, Viral/chemistry , DNA-Binding Proteins/genetics , Dependovirus/genetics , Incidence , Inverted Repeat Sequences/genetics , Liver Neoplasms/etiology , Parvoviridae Infections/complications , Polymerase Chain Reaction , Republic of Korea , Sequence Analysis, DNA , Viral Proteins/genetics
19.
Journal of Huazhong University of Science and Technology (Medical Sciences) ; (6): 548-553, 2016.
Article in English | WPRIM | ID: wpr-285231

ABSTRACT

Evidence suggested that glycogen synthase kinase-3β (GSK-3β) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3β-adeno associated virus (GSK-3β-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3β-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.


Subject(s)
Animals , Humans , Rats , Axons , Metabolism , Dependovirus , Genetics , Glycogen Synthase Kinase 3 beta , Genetics , Metabolism , Nerve Regeneration , Genetics , RNA, Small Interfering , Genetics , Sensorimotor Cortex , Pathology , Spinal Cord Injuries , Genetics , Pathology , Therapeutics , Syringomyelia , Genetics , Pathology , Therapeutics
20.
Chinese Journal of Biotechnology ; (12): 1230-1238, 2015.
Article in Chinese | WPRIM | ID: wpr-240561

ABSTRACT

AAV-ITR gene expression mini vector is a double-strand or single-strand DNA that only contains inverted terminal repeats of adeno-associated virus, cis-elements and gene of interest and does not contain any other foreign DNA sequences. We prepared Bac-ITR-EGFP and Bac-inrep. Spodoptera frugiperda cells were infected with Bac-ITR-EGFP (P3) and Bac-inrep (P3). Up to 100 μg of AAV-ITR-EGFP gene expression mini vectors were extracted from 2 x 10(7) cells of Sf9 72 h after infection. The gel electrophoresis analysis shows that most forms of AAV-ITR-EGFP gene expression mini vector were monomer and dimer. The mini vector expression efficacy was examined in vitro with HEK 293T cells. The EGFP expression was observed at 24 h after transfection, and the positive ratio reached 65% at 48 h after transfection.


Subject(s)
Animals , Humans , Baculoviridae , DNA, Single-Stranded , Dependovirus , Gene Expression , Genetic Vectors , HEK293 Cells , Sf9 Cells , Terminal Repeat Sequences , Transfection
SELECTION OF CITATIONS
SEARCH DETAIL