Increasing epithelial junction permeability enhances gene transfer to airway epithelia In vivo.
Gene transfer to airway epithelia is the most direct approach for treating the progressive lung disease associated with cystic fibrosis. However, the transduction efficiency is poor when viral vectors are applied to the mucosal surface. We reported previously that gene transfer via the apical surface of human airway epithelia in vitro was improved by formulating vectors with ethyleneglycol-bis-(2-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in a hypotonic buffer. First, we investigated the mechanism for this enhancement. When 100-nm fluorescent beads were applied to the apical surface in the presence of EGTA, paracellular deposition of the particles was noted. Transmission electron microscopy verified that the epithelial junction complex was disrupted under these conditions. The Ca(2+) chelators EGTA, 1,2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), and ethylenediaminetetraacetic acid all caused a rapid, reversible drop in transepithelial resistance and facilitated gene transfer with retrovirus or adenovirus in vitro. When Ca(2+) chelators were applied to rabbit tracheal epithelia or human nasal epithelia in vivo, the transepithelial voltage decreased, and amiloride sensitivity was lost, suggesting that epithelial junctions opened. Importantly, this novel formulation enhanced both retroviral- and adenoviral-mediated gene transfer to rabbit tracheal epithelia in vivo. This technique may have applications for vector or drug delivery to airway epithelia and other polarized cells.
Wang
G
G
Departments of Pediatrics and Internal Medicine, Program in Gene Therapy, Central Microscopy Research Facility, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Zabner
J
J
Deering
C
C
Launspach
J
J
Shao
J
J
Bodner
M
M
Jolly
D J
DJ
Davidson
B L
BL
McCray
P B
PB
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001980:Bronchi / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability; D002614:Chelating Agents / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D009297:Nasal Mucosa / Q000187:drug effects; D009994:Osmolar Concentration; D011817:Rabbits; D014132:Trachea / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure
D002614:Chelating Agents; D004533:Egtazic Acid; D000584:Amiloride; D002118:Calcium