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.

Lysosomal capturing of cytoplasmic injected nanoparticles by autophagy: An additional barrier to non viral gene delivery

“…Recently, it was shown for the first time that autophagy is a potential barrier in non-viral gene delivery after transfection of cells with nanoparticles. With electroporation, however, an autophagy response was not noted (17). Also previously, we suggested that autophagy could be responsible for the long term endosomal entrapment of nanoparticles in Retinal Pigment Epithelial (RPE) cells (18).…”

Autophagy Induced by Calcium Phosphate Precipitates Targets Damaged Endosomes

“…In addition, evidence for the fusion between autophagosomes and damaged lysosomes that expose glycans had not been found (11), but amphisome formation cannot be excluded in the CPP treatment condition even though CPP-induced double-membraned autophagosomes seem to contain membranous materials, which could be the damaged endosomes (4,6). Similarly, amphisome formation cannot be excluded during the formation of LC3-TVS induced by cationic liposomes (31). Future studies should be directed to address this critical issue in the treatment with CPPs and cationic lipid agents.…”

“…Notably, inhibition of either autophagy initiation or lysosome degradation could significantly increase the expression of DNA transfected with CPPs (38) or with cationic lipids (31). Thus, autophagy can serve as a defense mechanism against foreign DNA entering via the endosome system.…”

“…Another common transfection carrier, cationic liposomes, is also known to cause autophagy (30,31), which was recently shown to be dependent on endocytosis as well (31).…”

Optimization of a cationic liposome-based gene delivery system for the application of miR-145 in anticancer therapeutics

“…However, the molar ratio of DOTAP/chol has an impact on the transfection efficiency. Previous research has indicated that DOTAP/chol liposomes with a molar ratio of 1:1 or 2:1 had a high transfection efficiency (3,5). To achieve a higher transfection efficiency, on the one hand, it is necessary to identify the optimal molar ratio of DOTAP/chol for use in the preparation of liposomes.…”