Efficient coexpression and secretion of antiatherogenic human apolipoprotein Al and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors

Plasma apolipoprotein Al (apoAl) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAl or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of antiatherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAl or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAl or LCAT by transduced cultures was two- to five-fold higher using AA V-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AA V-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAl and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAl and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.