359. Specific Modulation of Tight Junctions to Enhance Airway Gene Transfer

Abstract

Tight junctions (TJs) are directly involved in the gate functions of epithelial and endothelial cells. The transient up-or down-regulation of these gate functions could have a therapeutic advantage in the treatment of lung diseases via delivery of pharmacological agents or gene transfer vectors. Because airway epithelia are resistant to luminal infection by viral vectors, one strategy for enhancing gene transfer is to modify TJ permeability. The feasibility of this approach has been demonstrated with medium-chain fatty acids, calcium chelators and detergents. However, the toxicity of these agents may be limiting for clinical application. The growth in the knowledge of key TJ proteins responsible for regulating TJ permeability has presented opportunities for specific modulation that may limit toxicity. Recently, synthetic peptides corresponding to the extracellular loops of the TJ protein occludin were utilized to modulate TJ permeability in intestinal epithelia (Tavelin et al., 2003). We hypothesized that synthetic occludin peptides (OPs) might modulate TJ permeability in airway epithelia by specifically targeting occludin to limit toxicity while enhancing airway gene transfer. To test this hypothesis, we investigated the effects of luminal delivery of OPs (a gift from Dr. Istvan Toth, Brisbane, Australia) on the TJ permeability of molecular tracers and viral vectors in lung epithelia. To determine if OPs disrupted the transepithelial permeability gate, the transepithelial electrical resistance (RT) of well differentiated human airway epithelia (WD HAE) luminally exposed to OPs was measured. RT measurements at 10 min intervals showed a rapid decrease with exposure to OPs, and TJ permeability to 70-kDa or 2000 kDa FITC-dextrans increased in a concentration-dependent manner. WD HAE epithelia luminally exposed to OPs and infected with a LacZ-encoding adenoviral vector, showed a significant increase in transduction efficiency. The reversibility of this strategy was evaluated by measuring RT recovery after removal of OPs. Only a transient alteration in TJ permeability was observed with a restoration of RT to normal levels within 24 hr following OP-treatment. The toxic effects of OPs were evaluated by measurements of lactate dehydrogenase (LDH) levels in the culture media. Dose dependent cellular cytotoxicity was observed only at a concentration of 1000 |[mu]|M at 6 hr post treatment, and LDH levels were back to normal by 24 hr. The measurement IL-6 and IL-8 to determine inflammatory responses to the OPs is underway. Cellular toxicity to OPs will be evaluated by assessing the fence function in OP-treated cultures using fluorescent lipid labeling and confocal microscopy analysis of lipid diffusion. The specificity of OPs on TJ protein expression and localization will be assessed by western blotting and immunofluorescent labeling. The transduction efficiency of other viral vectors such as AAV following OP-treatment will also be evaluated. The issues addressed in this study will provide insights into TJ permeability to solutes in airway epithelia, and the specific modulation of TJ function could potentially be useful in safely enhancing gene delivery across lung epithelia.