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) recept or (CAR) on the surface of the target cell leading to receptor-mediated end ocytosis of the vector. The present study evaluates the effect of free chol esterol, a lipid component of endocytic vesicles, on Ad uptake into CAR-def icient 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 a t its solubility maximum in aqueous solution. Increase of Ad vector-mediate d gene transfer with cholesterol was dependent on the lack of CAR receptor expression on the surface and was diminished by overexpression of CAR in CA R-deficient cells. Cholesterol-mediated increase of Ad-mediated gene expres sion was dependent on coincubation of both cholesterol and Ad and was not d ependent on the cholesterol content of the cell. Increased Ad vector-mediat ed gene expression in the presence of free cholesterol was also observed in murine skin in vivo. Structural analysis of the Ad-cholesterol mixture sho wed complexation between Ad particles leading to formation of multivirus ag gregates due to hydrophobic interaction. The addition of free cholesterol w ith 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.