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Enhancement of Adenovirus-Mediated Gene Transfer in Lungs and Epithelial Cells by EGTA* FREE TO VIEW

Carpantato Myles, BS; Eric Sorscher, MD; Sadis Matalon, PhD
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*From the Department of Anesthesiology, Medicine and Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL.

Correspondence to: Sadis Matalon, PhD, Associate Dean of Postdoctural Education, THT 940, 1900 University Blvd, Birmingham, AL 35233; e-mail: Sadis.Matalon@ccc.uab.edu

Chest. 2002;121(3_suppl):35S. doi:10.1378/chest.121.3_suppl.35S
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We investigated the enhancement of gene transfer across epithelial cells using adenoviral vectors by the addition of low doses of ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetra-acetic acid (EGTA). In the first series of experiments, we instilled intratracheally in CD-1 mice 108 plaque-forming units of adenoviral vector-containing reporter genes with and without 100 μM EGTA. Significant expression of luciferase was found in lung tissue 7 days later; addition of 100 μM EGTA in the instillate increased adenoviral luciferase expression by 600%.

To identify potential mechanisms by which EGTA increased gene transfer, we infected nonconfluent monolayers of A549, fetal distal lung epithelial cells, and primary human airway epithelial cells with adenoviral vectors containing LacZ in the presence or absence of 100 μM EGTA. Significantly higher levels of LacZ expression, measured 24 h postinfection, were obtained in the presence of 100 μM EGTA.

These data indicate enhancement of transduction by low doses of EGTA both in vitro and in vivo. The mechanism underlying these effects may be independent or in conjunction with the disruption of tight junctions, since strong increases in gene transfer were demonstrated in vitro in nonconfluent airway epithelial cells grown on plastic, ie, under conditions where tight junctional complexes would be unlikely to play a significant role in refractoriness to gene delivery vectors.

Abbreviation: EGTA = ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetra-acetic acid

Supporsted by National Institutes of Health grant P30DK54781.




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