Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference.
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ABSTRACT:
SUBMITTER: Cao HB
PROVIDER: S-EPMC4497818 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF). However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR). Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.Molecular Therapy-Nucleic Acids (2013) 2, e127; doi:10.1038/mtna.2013.55; published online 8 October 2013.
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Project description:Helper-dependent adenoviral vectors (HD Ad) hold extreme promise for gene therapy of human diseases. All viral genes are deleted in HD Ad vectors, and therefore, the presence of a helper virus is required for their production. Current methods to minimize helper contamination in large-scale preparations rely on the use of the Cre/loxP system. The inclusion of loxP sites flanking the packaging signal results in its excision in the presence of Cre recombinase, preventing helper genome encapsidation. It is well established that the level of Cre recombinase activity is important in determining the degree of helper contamination. However, there is little information on other mechanisms that could also play an important role. We have generated several HD Ad vectors containing a rapalog-inducible system to regulate transgene expression, or LacZ under the control of the elongation factor 1 ? promoter. Large-scale production of these vectors resulted in abundant helper contamination. Viral DNA analysis revealed the presence of rearrangements between vector and helper genomes. The rearrangements involved a helper DNA molecule with a fragment of the left arm of the HD Ad vector, including its ITR, packaging signal, and some stuffer sequence. Overall, our data suggest that helper DNA molecules that accumulate after Cre recombinase activity are prone to rearrangements, resulting in helper genomes that have incorporated a packaging signal from the vector. Helper particles with rearranged genomes have a growth advantage. This study identifies a novel mechanism leading to helper contamination during helper-dependent adenoviral vector production.
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Project description:Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue.
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Project description:Adenoviruses (Ads) infect a broad range of tissue types, and derived vectors have been extensively used for gene therapy. Helper-dependent Ad vectors (HDAds), devoid of viral coding sequences, allow for insertion of large or multiple transgenes in a single vector and have been preclinically used for the study of genetic disorders. However, the clinical application of Ad vectors including HDAds for genetic disorders has been hampered by an acute toxic response. This characteristic, while disadvantageous for gene replacement therapy, could be strategically advantageous for the activation of an immune response if HDAds were used as an adjunct treatment in cancer. Cancer treatments including immunotherapy are frequently limited by the inhibitory environment produced by both tumors and their stroma, each of which express numerous inhibitory molecules. Hence, multiple inhibitory mechanisms must be overcome for development of anti-tumor immunity. The large coding capacity of HDAds can accommodate multiple immune modulating transgenes that could produce a combined effect to overcome tumor-derived inhibition and ensure intratumoral effector T-cell proliferation and function. In this review, we discuss the potential advantages of HDAds to cancer immunotherapy based on potent host immune responses to Ads.
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Project description:The epithelium-derived cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) are important mediators that initiate innate type 2 immune responses in asthma. Leukotriene receptor antagonists (LTRAs) are commonly used to prevent asthma exacerbations. However, the effects of LTRAs on epithelium-derived cytokines expression in airway epithelial cells are unclear. This study aimed to investigate the effects of LTRAs on the expression of epithelium-derived cytokines in human airway epithelial cells and to explore possible underlying intracellular processes, including epigenetic regulation. A549 or HBE cells in air-liquid interface conditions were pretreated with different concentrations of LTRAs. The expression of epithelium-derived cytokines and intracellular signaling were investigated by real-time PCR, enzyme-linked immunosorbent assay, and Western blot. In addition, epigenetic regulation was investigated using chromatin immunoprecipitation analysis. The expression of IL-25, IL-33, and TSLP was increased under LTRAs treatment and suppressed by inhaled corticosteroid cotreatment. Montelukast-induced IL-25, IL-33, and TSLP expression were mediated by the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways and regulated by histone H3 acetylation and H3K36 and H3K79 trimethylation. LTRAs alone might increase inflammation and exacerbate asthma by inducing the production of IL-25, IL-33, and TSLP; therefore, LTRA monotherapy may not be an appropriate therapeutic option for asthma.
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Project description:BackgroundIntroduction of the RNA interference (RNAi) machinery has guided the researchers to discover the function of essential vital or virulence factor genes in the microorganisms such as fungi. In the filamentous fungus Aspergillus nidulans, the gene sidB plays an essential role in septation, conidiation and vegetative hyphal growth. In the present study, we benefited from the RNAi strategy for down-regulating a vital gene, sidB, in the fungus A. nidulans.MethodsThe 21-nucleotide small interfering RNA (siRNA) was designed based on the cDNA sequence of the sidB gene in A. nidulans. Transfection was performed through taking up siRNA from medium by 6 hour-germinated spores. To evaluate the morphologic effects of siRNA on the fungus, germ tube elongation was followed. Moreover, total RNA was extracted and quantitative changes in expression of the sidB gene were analyzed by measuring the cognate sidB mRNA level by use of a quantitative real-time RT-PCR assay.ResultsCompared to untreated-siRNA samples, a significant inhibition in germ tube elongation was observed in the presence of 25 nM of siRNA (42 VS 21 µM). In addition, at the concentration of 25 nM, a considerable decrease in sidB gene expression was revealed.ConclusionUsage of RNAi as a kind of post-transcriptional gene silencing methods is a promising approach for designing new antifungal agents and discovering new drug delivery systems.
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