Clathrin-mediated Endocytosis and Subsequent Endo-Lysosomal Trafficking of Adeno-associated Virus/Phage |
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Authors: | Charlotte A Stoneham Michael Hollinshead Amin Hajitou |
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Institution: | From the ‡Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, 160 Du Cane Road, London W12 0NN, United Kingdom and ;the §Section of Virology, Department of Medicine, Faculty of Medicine, Imperial College London, St. Mary''s Campus, Norfolk Place, London W2 1PG, United Kingdom |
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Abstract: | Adeno-associated virus/phage (AAVP) is a gene delivery vector constructed as a hybrid between adeno-associated virus and filamentous phage. Tumor targeting following systemic administration has previously been demonstrated in several in vivo cancer models, with tumor specificity achieved through display of an αv integrin-targeting ligand on the capsid. However, high titers of AAVP are required for transduction of large numbers of mammalian cells. This study is the first to investigate the mechanisms involved in entry and intracellular trafficking of AAVP. Using a combination of flow cytometry, confocal, and electron microscopy techniques, together with pharmacological agents, RNAi and dominant negative mutants, we have demonstrated that targeted AAVP endocytosis is both dynamin and clathrin-dependent. Following entry, the majority of AAVP particles are sequestered by the endosomal-lysosomal degradative pathway. Finally, we have demonstrated that disruption of this pathway leads to improved transgene expression by AAVP, thus demonstrating that escape from the late endosomes/lysosomes is a critical step for improving gene delivery by AAVP. These findings have important implications for the rational design of improved AAVP and RGD-targeted vectors. |
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Keywords: | Bacteriophage Cancer Therapy Endocytosis Gene Therapy intracellular Trafficking Lysosomes AAVP Bacteriophage Endocytosis Gene Delivery |
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