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/10⁵ cells per ml (moi 100) correlated with increased activity of 92.7 ± 22.6 mU/10⁵ cells per ml relative to negligible LPL levels in Ad-RSV-LacZ (β-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.

Overview summary

Lipoprotein lipase (LPL) is a nonhepatic, lipolytic enzyme crucial to human triglyceride (TG) and cholesterol metabolism. A major question for LPL gene therapy is whether adenovirus-mediated gene delivery can render appropriate synthesis, modification, and secretion of bioactive LPL from an ectopic, hepatocellular host target. In the present study, the HepG2 cell line was chosen for study because these cells express many traits attributed to normal human hepatocytes; also HepG2 cells are a currently accepted model for studying lipoprotein metabolism in human hepatocytes. The results herein demonstrate that adenovirus-mediated gene transfer resulted in highly efficient, expression of functional human LPL in human HepG2 cells in vitro. Our findings also suggest that the liver may serve as a suitable in vivo target for adenovirus-mediated gene therapy for LPL deficiency.