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Recombinant Adeno-associated Virus (recombinant + adeno-associated_virus)
Selected AbstractsChronic ethanol increases adeno-associated viral transgene expression in rat liver via oxidant and NF,B-dependent mechanismsHEPATOLOGY, Issue 5 2000Michael D. Wheeler Recombinant adeno-associated virus (rAAV) transduction is limited in vivo, yet can be enhanced by hydroxyurea, ultraviolet-irradiation, or adenovirus coinfection, possibly via mechanisms involving stress in the host cell. Because chronic ethanol induces oxidative stress, it was hypothesized that chronic ethanol would increase rAAV transduction in vivo. To test this hypothesis, rAAV encoding ,-galactosidase was given to Wistar rats that later received either ethanol diet or high-fat control diet via an enteral-feeding protocol for 3 weeks. Expression and activity of ,-galactosidase in the liver were increased nearly 5-fold by ethanol. The increase in transgene expression was inhibited by antioxidant diphenylene iodonium (DPI), which is consistent with the hypothesis that ethanol causes an increase in rAAV transduction via oxidative stress. Ethanol increased DNA synthesis only slightly; however, it increased the nuclear transcription factor ,B (NF,B) 4-fold, a phenomenon also sensitive to DPI. Moreover, a 6-fold increase in rAAV transgene expression was observed in an acute ischemia-reperfusion model of oxidative stress. Transgene expression was transiently increased 24 hours after ischemia-reperfusion 3 days and 3 weeks after rAAV infection. Further, adenoviral expression of superoxide dismutase or I,B, superrepressor inhibited rAAV transgene expression caused by ischemia-reperfusion. Therefore, it is concluded that ethanol increases rAAV transgene expression via mechanisms dependent on oxidative stress, and NF,B likely through enhancement of cytomegaloviral (CMV) promoter elements. Alcoholic liver disease is an attractive target for gene therapy because consumption of ethanol could theoretically increase expression of therapeutic genes (e.g., superoxide dismutase). Moreover, this study has important implications for rAAV gene therapy and potential enhancement and regulation of transgene expression in liver. [source] Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathologyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2010Ayse Ulusoy Abstract Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (,syn). Among the different modifications that can promote the formation of toxic ,syn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated ,syn aggregates faster and sub-stoichiometric amounts of C-terminal truncated ,syn promote aggregation of the full-length ,syn (,synFL) and induce neuronal toxicity. To address in vivo the putative stimulation of ,syn-induced pathology by the presence of truncated ,syn, we used recombinant adeno-associated virus to express either ,synFL or a C-terminal truncated ,syn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of ,syn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated ,syn (1-110) but only ,synFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of ,synFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated ,syn can interact with and exacerbate the formation of pathological accumulations containing ,synFL in vivo. [source] Effects of antisense interleukin-5 gene transferred by recombinant adeno-associated virus to allergic ratsRESPIROLOGY, Issue 1 2010Daxiong ZENG ABSTRACT Background and objective: The accumulation of eosinophils in airways is an important characteristic of asthma. The process is primarily mediated by interleukin-5 (IL-5) secreted by Th2 lymphocytes. This study explored a new approach to asthma therapy in which allergic rats were transfected with the IL-5 antisense gene delivered by the recombinant adeno-associated virus (rAAV-ASIL-5). Methods: The viral vector rAAV-ASIL-5 was constructed and the IL-5 antisense gene transfected into allergic rats. The levels of IL-5, IgE, eotaxin and eosinophilic cationic protein (ECP) in sera and bronchoalveolar lavage fluid (BALF) were measured by ELISA. The inflammatory responses in lung tissues were evaluated by histological study. Results: The levels of IL-5 protein in serum and BALF were significantly decreased in the allergic rats treated with rAAV-ASIL-5 (P < 0.05). Serum ovalbumin-specific IgE was reduced in treated rats compared with untreated rats (P < 0.05). rAAV-ASIL-5 treatment also reduced eosinophils in the peripheral blood and BALF, as well as the ECP and eotaxin levels in serum and BALF (P < 0.05). There was significantly less inflammation in the lungs of rAAV-ASIL-5-treated rats than in those of untreated rats. No obvious pathological damage to the kidneys and livers of the rats treated with rAAV was observed. Conclusions: Treatment with rAAV-ASIL-5 inhibited the accumulation of eosinophils and airway inflammation in the rat model of allergic asthma by suppressing IL-5 production. These results suggest that rAAV-ASIL-5-based gene therapy may be used for the treatment of allergic asthma. [source] Enhancing rAAV vector expression in the lungTHE JOURNAL OF GENE MEDICINE, Issue 7 2005Isabel Virella-Lowell Abstract Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (C,) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the C, promoter, and the C, promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the C,-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 × 106 relative light units (RLU)/g tissue and 2.7 × 106 RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful. Copyright © 2005 John Wiley & Sons, Ltd. [source] Scalable production of adeno-associated virus type 2 vectors via suspension transfection,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Joon Young Park Abstract Vectors derived from adeno-associated virus type 2 (AAV2) are promising gene delivery vehicles, but it is still challenging to get the large number of recombinant adeno-associated virus (rAAV) particles required for large animal and clinical studies. Current transfection technology requires adherent cultures of HEK 293 cells that can only be expanded by preparing multiple culture plates. A single large-scale suspension culture could replace these multiple culture preparations, but there is currently no effective co-transfection scheme for generating rAAV from cells in suspension culture. Here, we weaned HEK 293 cells to suspension culture using hydrogel-coated six-well culture plates and established an efficient transfection strategy suitable for these cells. Then the cultures were gradually scaled up. We used linear polyethylenimine (PEI) to mediate transfection and obtained high transfection efficiencies ranging from 54% to 99%, thereby allowing efficient generation of rAAV vectors. Up to 1013 rAAV particles and, more importantly, up to 1011 infectious particles were generated from a 2-L bioreactor culture. The suspension-transfection strategy of this study facilitates the homogeneous preparation of rAAV at a large scale, and holds further potential as the basis for establishing a manufacturing process in a larger bioreactor. © 2006 Wiley Periodicals, Inc. [source] | |||||||||||||