Efficient adenovirus-mediated ectopic gene expression of human lipoprotein lipase in human hepatic (HepG2) cells.
Gene therapy to deliver and express a corrective lipoprotein lipase (LPL) gene may improve the lipid profile and reduce the morbidity and potential atherogenic risk from hypertriglyceridemia and dyslipoproteinemia in patients with complete or partial LPL deficiency. We have used an E1-/E3- adenoviral vector, with an RSV-driven human LPL cDNA expression cassette (Ad-RSV-LPL), to achieve high ectopic LPL gene expression in the human hepatoma cell line HepG2, an accepted hepatocellular model of lipoprotein metabolism. Ad-RSV-LPL transduction of HepG2 cells with a multiplicity of infection (moi) between 12.5 and 100 yielded dose-dependent increments in LPL mass and activity. Peak levels of LPL protein of 2,032.1 +/- 274.5 ng/10(5) cells per ml (mol 100) correlated with increased activity of 92.7 +/- 22.6 mU/10(5) cells per ml relative to negligible LPL levels in Ad-RSV-LacZ (beta-galactosidase) controls. Exogenous LPL expression over a 5-day period peaked at day 3. Susceptibility to inhibition by 1 M NaCl and an anti-LPL monoclonal antibody confirmed that lipase activity was indeed derived from human LPL. Hydrolysis, by LPL-overexpressing HepG2 cells, of TG carried in very-low-density lipoprotein (VLDL) showed that greater than 50% of the triglycerides (TG) disappeared after 4 hr of incubation. These results were compatible with FPLC evidence of a marked reduction in VLDL-TG. These results provide strong in vitro evidence that adenoviral-mediated ectopic expression of the human LPL gene could render hepatic cells capable of VLDL catabolism and thus support the possibility for in vivo adenoviral vector-mediated liver-targeted LPL gene therapy.
Liu
G
G
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Excoffon
K J
KJ
Benoit
P
P
Ginzinger
D G
DG
Miao
L
L
Ehrenborg
E
E
Duverger
N
N
Denefle
P P
PP
Hayden
M R
MR
Lewis
M E
ME
D000256:Adenoviridae / Q000235:genetics*; D017498:Adenovirus E1 Proteins / Q000235:genetics; D017503:Adenovirus E3 Proteins / Q000235:genetics; D001358:Avian Sarcoma Viruses / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D008071:Lipoprotein Lipase / Q000235:genetics* / Q000378:metabolism*; D008079:Lipoproteins, VLDL / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014280:Triglycerides / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017498:Adenovirus E1 Proteins; D017503:Adenovirus E3 Proteins; D018076:DNA, Complementary; D008079:Lipoproteins, VLDL; D011994:Recombinant Proteins; D014280:Triglycerides; D008071:Lipoprotein Lipase