Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways

Abstract

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 enhancer͞ 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 epitheliumspecific 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.

Epithelium-specific ets transcription factor 2 upregulates cytokeratin 18 expression in pulmonary epithelial cells through an interaction with cytokeratin 18 intron 1

“…Monolayers of IB3-1 lung cystic fibrosis cells (ATCC JHU-52) were grown in LHC-8 medium (Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum. Cotransfections of partially confluent cells (50-80%) were carried out with 0.5 µg of secreted alkaline phosphatase (SEAP) reporter gene plasmid DNA (pK18EpiSEAP) [30], 0.5 µg of expression vector DNA (pcDNA3, pcDNA3·Ese-2, pcDNA3·Ese-2-ets, or pcDNA3·Ese-2-pnt), and 6 µl of Lipofectamine TM Reagent (Invitrogen) per 1 ml of media. Each 1 ml of media was incubated with the cells for 24 h, and then replaced with new media.…”

“…K18 is an intermediate filament expressed solely in differentiated epithelial cells. The first intron of K18, which contains multiple putative EBSs, three negative regulatory elements, and a strong enhancer, has been shown to play an important role in regulating expression of the gene [30][31][32][33]. Other Ets factors, such as Ets-2, a member of the ETS subfamily, have been shown to interact with regions of the first intron of K18 cooperatively with cofactors such as AP-1 [32].…”

Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing

“…Although progress is being made in reducing the cDNA size 33 and developing shorter but reasonably efficient promoters, 34 it remains to be determined whether such advances will be sufficient for long-term correction in vivo. If an epithelial cell specific promoter is deemed necessary, eg the cytokeratin 18 promoter, 35 to provide spatially restricted, more persistent expression, it is likely that more space may be required. Also, in contrast with chimeragens or other repair strategies which correct small genetic defects it is feasible that all mutations within the coding sequence of CFTR could be repaired by one of two PTMs.…”

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells: potential roles in airway inflammation

“…PCR products were confirmed by sequencing before subcloning into a SEAP2 or SEAP reporter plasmid (Clontech, Palo Alto, CA). The mutant NF-κB binding site in the ESE-1 promoter (pESE-1-gga; position -80 to -78) were generated by PCR site-directed mutagenesis [54]. The three ESE-1 promoter deletion plasmids were generated by shortening the 5′ promoter region from pESE-1-SEAP using restriction enzymes.…”