title: Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery.
abstract: Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5 (-/-) cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.
authors: Norwich Medical School, University of East Anglia, Norfolk, UK.
authors: Rebecca
authors: R
authors: Roberts
authors: Wafa' T
authors: WT
authors: Al-Jamal
authors: Matthew
authors: M
authors: Whelband
authors: Paul
authors: P
authors: Thomas
authors: Matthew
authors: M
authors: Jefferson
authors: Jeroen
authors: J
authors: van den Bossche
authors: Penny P
authors: PP
authors: Powell
authors: Kostas
authors: K
authors: Kostarelos
authors: Thomas
authors: T
authors: Wileman
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D000071187:Autophagy-Related Protein 5; D016466:CHO Cells; D002130:Calcium Phosphates / Q000494:pharmacology; D002412:Cations; D006224:Cricetinae; D003412:Cricetulus; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071456:Sequestosome-1 Protein; D025801:Ubiquitin / Q000378:metabolism; D014780:Viruses / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C495773:Atg5 protein, mouse; D000071187:Autophagy-Related Protein 5; D002130:Calcium Phosphates; D002412:Cations; D006360:Heat-Shock Proteins; D008081:Liposomes; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; C020243:calcium phosphate
keywords: autophagy; cationic liposome; gene therapy; nonviral gene delivery; polyplex