998. Evaluation of Promoter Elements for Specific Expression in Human Airway Epithelia Using a Self-Inactivating (SIN) Equine Lentivector

Abstract

Top of pageAbstract One goal for obtaining effective gene therapy with low overall toxicity is to target the gene of interest to a specific subset of cells that need the correction, while having little or no expression outside of these cells. One way of achieving this is through the use of cell-specific promoters. We hypothesized that we could achieve long-term expression in subsets of well-differentiated human airway epithelial cells by utilizing cellular or viral promoters that drive expression specifically in these cell-types. In this study, we tested the FoxJ1 cell promoter, which is activated during ciliated cell differentiation, and the LTR regions from two sheep viruses, Enzootic Nasal Tumor Virus (ENTV) and Jaagsiekte Sheep Retrovirus (JSRV) whose tropism is restricted to non-ciliated cell types, including Clara cells. An equine infectious anemia virus (EIAV) self-inactivating SIN lentiviral vector system was employed to compare these promoters to the CMV immediate early promoter in cell lines and primary human airway epithelial (HAE) cell cultures. We tested all three promoters for specificity, and for duration and timing of expression. Measurements of timing and duration of expression were taken following the addition of d-luciferin to sets of HAE cultures, which had been infected with FoxJ1, JSRV and ENTV constructs expressing luciferase, at day10 and day 23 after mono-layer formation. Light emission was measured with a cooled-coupled device (CCD) camera. After the final measurement, these same cells were lysed and the levels of luciferase expression were quantified with a luminometer or sectioned for histology. The types of cells within the HAE cultures that expressed the marker gene were determined by histology after infection by constructs that had the different promoters driving lacZ, followed by staining, and sectioning. It was observed that while none of these promoters could compare with the CMV promoter in various cell lines, levels of expression within well-differentiated primary HAE cultures was comparable. FoxJ1-driven expression was highly specific to well-differentiated epithelial cells and was induced during the differentiation process. In contrast, the JSRV and ENTV LTR promoters drove expression in both well- and poorly-differentiated human airway cells. Studies are in progress to determine the frequency of co-localization of expression with ciliated and non-ciliated cells in well-differentiated epithelia. No significant down-regulation of expression driven by these promoters was observed over the duration of the cultures.