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 failure 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 P2Y2 receptor (P2Y2-R) is abundantly expressed on the airway lumenal surface and internalizes into coated pits upon agonist activation. We tested whether a small-molecule-agonist (UTP) could direct vectors to P2Y2-R and mediate attachment, internalization, and gene transfer. Fluorescein-UTP studies demonstrated that P2Y2-R agonists internalized with their receptor, and biotinylated UTP (BUTP) mediated P2Y2-R-specific internalization of fluorescently labeled streptavidin (SAF) or SAF conjugated to biotinylated Cy3 adenoviral-vector (BCAV). BUTP conjugated to BCAV mediated P2Y2-R-specific gene transfer in (1) adenoviral-resistant A9 and polarized MDCK cells by means of heterologous P2Y2-R, and (2) well-differentiated human airway epithelial cells by means of endogenous P2Y2-R. Targeting vectors with small-molecule-ligands to apical membrane G-protein-coupled receptors may be a feasible approach for successful CF gene therapy.
Kreda
S M
SM
CF/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA.
Pickles
R J
RJ
Lazarowski
E R
ER
Boucher
R C
RC
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D019921:Biotinylation; D001980:Bronchi / Q000821:virology; D002460:Cell Line; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000628:therapy; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000378:metabolism / Q000821:virology; D019793:Fluorescein / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008854:Microscopy, Electron; D009297:Nasal Mucosa / Q000821:virology; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058473:Receptors, Purinergic P2Y2; D019809:Streptavidin / Q000378:metabolism; D013696:Temperature; D014132:Trachea / Q000821:virology; D014544:Uridine Triphosphate / Q000627:therapeutic use
D008024:Ligands; C546385:P2RY2 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D019809:Streptavidin; D019204:GTP-Binding Proteins; D019793:Fluorescein; D014544:Uridine Triphosphate