Development of an Epithelium-Specific Expression Cassette with Human DNA Regulatory Elements for Transgene Expression in Lung Airways

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′genomic sequences and one of its introns, we have developed a novel expression ca...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1997-12, Vol.94 (26), p.14695-14700
Main Authors: Chow, Yu-Hua, O’Brodovich, Hugh, Plumb, Jonathan, Wen, Yanxia, Sohn, Kyoung-Jin, Lu, Zhan, Zhang, Fangyuan, Lukacs, Gergely L., Tanswell, A. Keith, Hui, Chi-Chung, Buchwald, Manuel, Hu, Jim
Format: Article
Language:English
Subjects:
Animals
Biological Sciences
Biotechnology
Cell lines
Cells, Cultured
COS Cells
Cystic fibrosis
Cystic Fibrosis Transmembrane Conductance Regulator - genetics
Deoxyribonucleic acid
DNA
DNA - genetics
Enhancer Elements, Genetic
Epithelial cells
Epithelium - physiology
Gene Expression Regulation
Gene therapy
Gene Transfer Techniques
Genes, Reporter
Genetic Therapy
Humans
Introns
Keratins - genetics
Lung - physiology
Lungs
Mice
Mice, Transgenic
Promoter Regions, Genetic
Splicing
Transgenes
Transgenic animals
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Summary:The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.94.26.14695