Polarity of secretion of alpha 1-antitrypsin by human respiratory epithelial cells after adenoviral transfer of a human alpha 1-antitrypsin cDNA.
alpha 1-Antitrypsin (alpha AT) deficiency, a hereditary cause of progressive emphysema, can potentially be treated by transfer of a functional human alpha 1AT gene to the respiratory epithelium. For such an approach to be successful, alpha 1AT must be provided to both the interstitium and the epithelial surface--that is, the alpha 1AT directed by the transferred gene must be secreted to both the apical and basolateral surfaces of the epithelial cells. To evaluate this concept, a recombinant, replication-deficient adenoviral vector (Ad-alpha 1AT) containing a human alpha 1AT cDNA driven by an adenovirus major late promoter was used to infect Bet-1A human respiratory epithelial cells. The infected cells expressed Ad-alpha 1AT-directed mRNA transcripts and synthesized and secreted functional human alpha 1AT as shown by [35S]methionine labeling and immunoprecipitation of a 52-kD glycosylated human alpha 1AT molecule capable of interacting with neutrophil elastase, its natural substrate. Bet-1A cells grown on microporous polycarbonate membranes formed tight junctions (resistance > 150 omega x cm2). After infection with Ad-alpha 1AT, [35S]methionine labeling and enzyme-linked immunoassay demonstrated that alpha 1AT was secreted into both the apical and basolateral compartments, with an average apical to basolateral ratio of 1.9 +/- 0.2. Thus, human respiratory epithelial cells infected with a recombinant adenoviral vector containing a human alpha 1AT cDNA secrete alpha 1AT across both the apical and basolateral cell membranes, suggesting that the respiratory epithelium could serve as a target for in vivo gene therapy of alpha 1AT deficiency.
Siegfried
W
W
Pulmonary Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA.
Rosenfeld
M
M
Stier
L
L
Stratford-Perricaudet
L
L
Perricaudet
M
M
Pavirani
A
A
Lecocq
J P
JP
Crystal
R G
RG
D000256:Adenoviridae / Q000235:genetics; D016764:Cell Polarity; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000008:administration & dosage; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000008:administration & dosage / Q000378:metabolism*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin