Soonpin Yei

1012-101 Vascular Smooth Muscle-Directed Adenovlral Vectors

Gene transfer to the vascular wall utilizing locally-delivered recombinant adenoviral vectors has shown promise as a novel technique for therapeutic as well as experimental modulation of vascular wall gene expression. Infusion of such vectors using porous balloon catheters (PBC) has previously been demonstrated to result in transduction of extravascular cells at the delivery site, as well as substantial systemic transduction as a consequence of release of vector into the circulation. Introduction of a vascular-directed promoter into the adenoviral vector should thus contribute to targeting the expression of genes to the vascular wall, while reducing peri-vascular and systemic expression. In order to test the feasibility of utilizing the vascular smooth muscle α-actin (SMA) promoter to confer tissue specificity upon a recombinant adenoviral vector, we constructed an adenovirus (AvLacZ5) employing a 1.1 kilobase region of the murine SMA promoter to direct the expression of the nuclear-targeted beta-galactosidase (lacZ) gene and evaluated gene transduction by this vector, in comparison with a vector differing only by the presence of the RSV-LTR promoter. Several cell types were used as targets, including bovine aortic smooth muscle cells (BASMC). human pulmonary epithelial carcinoma cells (A549 cells), and transformed human embryonic kidney epithelial cells which are competent to replicate these adenoviral vectors (293 cells). The vector incorporating the SMA promoter demonstrated substantial selectivity for vascular smooth muscle gene expression, with typical transductions carried out in parallel under identical conditions manifesting 90–95% lacZ-expressing BASMC, 0.3% lacZ-positive A549 cells, and 4% positive 293 cells. Conversely, parallel transductions with the vector employing the RSV promoter typically resulted in 95–99% lac-expressing 293 cells at vector concentrations yielding only 5–10% positive BASMC. These data support cell lineage-specificity of AvLacZ5 at the level of promoter function rather than due to intrinsic cellular differences in capacity for adenovirally-mediated transduction. However, it is notable that a limited subpopulation of 293 cells clearly are able to direct sufficient transcription from the SMA promoter sequences chosen to yield detectable lacZ expression; the molecular basis for this heterogeneity of expression remains to be determined. Adenoviral vectors utilizing these promoter sequences may render vascular-restricted gene transfer feasible when used in conjunction with mechanical devices providing a component of spatial localization.