Enhanced transgene expression in human lung epithelial cells from a recombinant adeno-associated virus vector

Although long-term gene expression with few side effects from rAAV2-mediated gene delivery appears possible and quite promising, gene delivery to and expression in polarized lung epithelial cells from rAAV2 vectors has been met with limited success. We demonstrate that tyrphostin-1, a specific inhibitor of the epidermal growth factor receptor (EGFR), can markedly enhance rAAV2-mediated transgene expression in undifferentiated human lung epithelial cells (1133 cells). Our data suggest that tyrphostin-1 enhances transcription from rAAV2 vector genomes under specific conditions that include the presence of a linear DNA template. Additionally, we did not observe enhanced double-stranded DNA synthesis of the rAAV2 genome or a change in the phosphorylation status of the ssD-BP in 1133 cells treated with tyrphostin-1.; It is not fully understood how tyrphostin-1 treatment alters cellular signal transduction pathways and thereby enhances rAAV2-mediated transgene expression. The EGFR is known to regulate the activities of the ERK1/2 MAPK pathways. We demonstrate that ERK1/2 phosphorylation is decreased in tyrphostin-1 treated cells, but decreased ERK1/2 phosphorylation does not lead to enhanced rAAV2-mediated transgene expression. Tyrphostin-1 appears to be a cellular stressor, and cellular stressors have been shown to activate the JNK and p38 MAPK pathways. We demonstrate that the level of JNK1/2 and p38 phosphorylation is increased in tyrphostin-1 treated cells, however, only an inhibitor of the p38 MAPK was able to decrease rAAV2-mediated transgene expression by nearly 50% in both untreated and tryphostin-1 treated cells. This suggests a role for the p38 MAPK pathway in the enhancement of rAAV2-mediated transgene expression by tryphostin-1 treatment of cells. 248; An accurate means of quantitating reporter gene expression in lung lysates is crucial for progress in animal models for vector gene delivery to lungs. The most quantitative methods for assaying reporter gene activity are luminometric methods. We demonstrate that hemoglobin interferes with P. pyralis firefly luciferase and E. coli β-galactosidase luminometric assays. We present improved methods for luminometrically quantitating P. pyralis firefly luciferase or E. coli β-galactosidase enzymatic activity in blood-contaminated lung lysates that will prove useful in animal models for vector gene delivery to lungs.