Adenoviral-mediated gene transfer of human surfactant protein B to respiratory epithelial cells.
Human surfactant protein B (SP-B) is a 79-amino acid, phospholipid-associated polypeptide expressed by respiratory epithelial cells of the lung. SP-B is essential for lung function, enhancing the spreading and stability of surfactant phospholipids that serve to reduce surface tension at the alveolar air-liquid interface. Congenital absence of SP-B results in neonatal respiratory failure and death. In the present work, we constructed a replication-deficient adenoviral vector, Av1SP-B1, in which the human SP-B cDNA is expressed under control of the Rous sarcoma virus (RSV) promoter in an E1-E3-deleted adenovirus type 5 (Ad5)-based vector system. Av1SP-B1 was produced in 293 kidney cells, directing the synthesis of the SP-B protein and SP-B peptides. Av1SP-B1 directed the synthesis of SP-B mRNA, precursor and active 8-9 kD polypeptide in immortalized mouse lung epithelial cells (MLE-12 cells), demonstrating complete processing to the human SP-B protein by these cells. Synthesis of human SP-B mRNA was detected as early as 12 h after infection and was maximal 48 h after infection in vitro. Northern blot analysis demonstrated that human SP-B mRNA was expressed in the lungs of cotton rats infected with Av1SP-B1 but not in those of uninfected animals or in animals infected with a reporter adenoviral vector, Av1LacZ4. In situ hybridization demonstrated the abundance and localization of the transferred human SP-B mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Yei
S
S
Genetic Therapy, Inc., Gaithersburg, Maryland.
Bachurski
C J
CJ
Weaver
T E
TE
Wert
S E
SE
Trapnell
B C
BC
Whitsett
J A
JA
D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D002461:Cell Line, Transformed; D018076:DNA, Complementary; D004847:Epithelial Cells; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011510:Proteolipids / Q000096:biosynthesis / Q000235:genetics*; D011663:Pulmonary Surfactants / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins; D016205:Sigmodontinae
D018076:DNA, Complementary; D011498:Protein Precursors; D011510:Proteolipids; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; D011994:Recombinant Proteins