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 tissu es is an important component of efficient and safe gene therapy. Utili zing regulatory elements from the human cytokeratin 18 (K18) gene, inc luding 5' genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter gene s, as well as the human cystic fibrosis transmembrane conductance regu lator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternati ve splicing products, due to the activation of two cryptic splice site s in the CFTR coding region. Modification of the K18 intron and CPTR c DNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein e xpression as well as biological function. Transgenic expression analys is in mice showed that the modified expression cassette can direct eff icient 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 ca ssette which selectively directs lung transgene expression for CFTR ge ne therapy to airway epithelia.