MODULATION OF ADENOVIRUS-MEDIATED GENE-TRANSFER BY NITRIC-OXIDE

We assessed the role of . NO in recombinant adenovirus-mediated gene t ransfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfec ted with the human inducible nitric oxide synthase, but lacking tetrah ydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were i nfected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCM V-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 mu M), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decr eased both luciferase and beta-galactosidase protein expression to app roximately 60% of their corresponding control values, 24 h later. NIH3 T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and di d not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N-G-monomethyl-L-arginine (L-NMMA; 1 mM), an inhib itor of iNOS, prevented the sepiapterin mediated induction of . NO and restored gene transfer to baseline values. Incubation bf NIH3T3/iNOS with 8-bromo-cGMP (400 mu M) in the absence of sepiapterin, or exposur e of AdCMV-Luc to large concentrations of . NO, did not alter the effi cacy of gene transfer. . NO produced by NIH3T3/iNOS cells also suppres sed beta-galactosidase expression in NIH3T3 cocultured cells stably tr ansfected with beta-galactosidase gene, suggesting . NO inhibited gene expression at either the transcriptional or posttranscriptional level s. To investigate the effects of inhaled . NO on gene transfer in vivo , CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10 (9) pfu in 80 mu l of saline) and exposed to . NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation wa s detected by histological examination. However, lung luciferase activ ity decreased by 53% as compared with air breathing controls (P < 0.05 ; n greater than or equal to 8). We concluded that overproduction of . NO decreases the efficiency of adenovirus-mediated gene transfer in l ung cells in the absence of cytotoxicity or inflammation.