Plasmid DNA molecules complexed with cationic liposomes are protected from degradation by nucleases and shearing by aerosolisation.
The cationic liposome DOTAP was complexed with plasmid DNA encoding beta-galactosidase in various ratios. As the concentration of DOTAP increased, the DNA became increasingly refractory to staining with ethidium bromide, presumably because the DNA was becoming condensed and being encapsulated by the liposomes. Transfection by DNA-DOTAP complexes at all ratios tested was unaffected by treatment of the complexes with DNase I. This finding has relevance to clinical trials for gene therapy of cystic fibrosis, in which patients are normally removed from treatment with DNase before receiving administration of DNA. We additionally tested the effect of aerosolisation of the liposome-DNA complex and of the DNA alone on the efficiency of in vitro transfection. Aerosolised DNA complexed with fresh DOTAP led to much lower reporter gene expression in Cos 7 cells than non-aerosolised complex, since aerosolisation appeared to destroy almost all of the plasmid. However, complexing the plasmid before passage through the nebuliser did protect most of the DNA from degradation, as reflected in the levels of transfection obtained. These findings contribute towards an overall understanding of both how DNA-cationic liposome complexes are formed and their fate following administration in vivo.
Crook
K
K
MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK.
McLachlan
G
G
Stevenson
B J
BJ
Porteous
D J
DJ
D000336:Aerosols; D000818:Animals; D019556:COS Cells; D002412:Cations; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005229:Fatty Acids, Monounsaturated; D017930:Genes, Reporter / Q000235:genetics; D008081:Liposomes; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
D000336:Aerosols; D002412:Cations; D004274:DNA, Recombinant; D005229:Fatty Acids, Monounsaturated; D008081:Liposomes; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; C085693:DNASE1 protein, human; D003850:Deoxyribonuclease I; D001616:beta-Galactosidase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane