Increasing Epithelial Junction Permeability Enhances Gene Transfer to Airway Epithelia In Vivo

Abstract

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.

Role of paracellular junction complexes in baculovirus-mediated gene transfer to nondividing rat hepatocytes

“…Several studies have determined that disruption of calciumdependent intercellular junctions by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 23,25,26,28 Previous evidence demonstrated that polarized apical membranes of normal human bronchial epithelial (NHBE) airway cells are resistant to lipofection. NHBE cells form multicellular islands similar to that of long-term primary rat hepatocyte cultures maintained in DMSO.…”

“…However, interaction of the retroviral vector with the basolateral surface of these cells is hindered by paracellular junctions. 23 2,63 Infection of Caco-2 intestinal epithelial cell cultures by CMV was inefficient and limited to peripheral cells suggesting that CMV enters polarized Caco-2 cells preferentially through the basolateral membrane. 25 To overcome this limitation, the investigators pretreated the cultures with 100 mM EGTA in Krebs Ringer solution to deplete extracellular calcium stores, and therefore disrupt paracellular junctions and polarity, exposing the basolateral cell surface.…”

“…[15][16][17][18][19][20] Tight junctions, partially composed of occludin and zona occludens ZO-1 and -2 among other proteins, are the apical-most complexes that form continuous contacts and limit paracellular movement of molecules between adjoining cells. [21][22][23] Adherens junctions are cadherin-based adhesive contacts located below the tight junction in the junctional complex. Desmosomes, located below the adherens junctions, mediate intercellular adhesion and provide anchoring sites for cytoplasmic intermediate filaments.…”

“…Desmosomes, located below the adherens junctions, mediate intercellular adhesion and provide anchoring sites for cytoplasmic intermediate filaments. 21,23 The gap junctions mediate cell-cell communication by diffusion of molecules through channels composed largely of connexins. 24 Several studies have determined that disruption of calcium-dependent paracellular junction complexes by EGTA improves viral infection efficiency in various tissues and polarized cell cultures.…”

“…24 Several studies have determined that disruption of calcium-dependent paracellular junction complexes by EGTA improves viral infection efficiency in various tissues and polarized cell cultures. 14,23,[25][26][27][28] Transient depletion of extracellular calcium may overcome the inefficiency of baculovirus-mediated gene transfer by disassociating paracellular epithelial junction complexes and exposing the basolateral surface. We hypothesized that in normal medium, which contains calcium, the baculovirus binds and delivers its genome only to peripheral cells and baculovirus attachment, and entry to internal cells is prevented because paracellular junctions mask the baculovirus binding motif of these internal hepatocytes.…”