Free cholesterol enhances adenoviral vector gene transfer and expression in CAR-deficient cells

Efficient adenovirus vector-mediated gene transfer depends on the presence of sufficient amounts of the high-affinity coxsackie-adenovirus (Ad) receptor (CAR) on the surface of the target cell leading to receptor-mediated endocytosis of the vector. The present study evaluates the effect of free cholesterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-deficient cells. Infection in the presence of free cholesterol at its maximum solubility in water led to increased binding, uptake, and expression of Ad in human skin fibroblasts and alveolar macrophages, two primary human cells known to be deficient in CAR. The effect of free cholesterol was maximal at its solubility maximum in aqueous solution. Increase of Ad vector-mediated gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CAR-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expression was dependent on coincubation of both cholesterol and Ad and was not dependent on the cholesterol content of the cell. Increased Ad vector-mediated gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad-cholesterol mixture showed complexation between Ad particles leading to formation of multivirus aggregates due to hydrophobic interaction. The addition of free cholesterol with Ad vectors may be a simple way to increase Ad-mediated gene transfer to cells that are poor targets due to their lack of a sufficient number of Ad receptors.