DNA internalized via caveolae requires microtubule-dependent, Rab7-independent transport to the late endocytic pathway for delivery to the nucleus.
Using cationic liposomes to mediate gene delivery by transfection has the advantages of improved safety and simplicity of use over viral gene therapy. Understanding the mechanism by which cationic liposome:DNA complexes are internalized and delivered to the nucleus should help identify which transport steps might be manipulated in order to improve transfection efficiencies. We therefore examined the endocytosis and trafficking of two cationic liposomes, DMRIE-C and Lipofectamine LTX, in CHO cells. We found that DMRIE-C-transfected DNA is internalized via caveolae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome or lysosome. Inhibition of microtubule-dependent transport with nocodazole revealed that DMRIE-C:DNA complexes cannot enter the cytosol directly from caveosomes. Lysosomal degradation of transfected DNA has been proposed to be a major reason for poor transfection efficiency. However, in our system dominant negatives of both Rab7 and its effector RILP inhibited late endosome to lysosome transport of DNA complexes and LDL, but did not affect DNA delivery to the nucleus. This suggests that DNA is able to escape from late endosomes without traversing lysosomes and that caveosome to late endosome transport does not require Rab7 function. Lysosomal inhibition with chloroquine likewise had no effect on transfection product titers. These data suggest that DMRIE-C and LTX transfection complexes are endocytosed by separate pathways that converge at the late endosome or lysosome, but that blocking lysosomal traffic does not improve transfection product yields, identifying late endosome/lysosome to nuclear delivery as a step for future study.
Wong
Athena W
AW
Department of Early Stage Cell Culture, Genentech Inc., South San Francisco, California 94080, USA. wong.athena@gene.com
Scales
Suzie J
SJ
Reilly
Dorothea E
DE
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D016466:CHO Cells; D021941:Caveolae / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D002738:Chloroquine / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D000962:Antimalarials; D000970:Antineoplastic Agents; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D002738:Chloroquine; D004247:DNA; D020691:rab GTP-Binding Proteins; D015739:Nocodazole