G-protein-coupled receptors as targets for gene transfer vectors using natural small-molecule ligands

Gene therapy for cystic fibrosis (CF) has focused on correcting electrolyte transport in airway epithelia. However, success has been limited by the fa ilure of vectors to attach and enter into airway epithelia, and may require redirecting vectors to targets on the apical membrane of airway cells that mediate these functions, The G-protein-coupled P2Y(2) receptor (P2Y(2)-R) is abundantly expressed on the airway lumenal surface and internalizes into coated pits upon agonist activation. We tested whether a small-molecule-ag onist (UTP) could direct vectors to P2Y(2)-R and mediate attachment, intern alization, and gene transfer, Fluorescein-UTP studies demonstrated that P2Y (2)-R agonists internalized with their receptor, and biotinylated UTP (BUTP ) mediated P2Y(2)-R-specific internalization of fluorescently labeled strep tavidin (SAF) or SAF conjugated to biotinylated Cy3 adenoviral-vector (BCAV ). BUTP conjugated to BCAV mediated P2Y(2)-R-specific gene transfer in (1) adenoviral-resistant A9 and polarized MDCK cells by means of heterologous P 2Y(2)-R, and (2) well-differentiated human airway epithelial cells by means of endogenous P2Y(2)-R. Targeting vectors with small-molecule-ligands to a pical membrane G-protein-coupled receptors may be a feasible approach for s uccessful CF gene therapy.