Top of pageAbstract Successful gene transfer to vascular tissue requires gene expression cassettes that are capable of achieving both high levels and sustained transgene expression. Regulation of gene expression is achieved at multiple levels: transcription, mRNA processing, nuclear export, translation and post-translational processing. To achieve maximal expression of transgenes, expression cassettes need to be optimised to ensure all of these processes are operating efficiently. The overall design of this project has two steps: (I) in vitro assessment of individual non-viral transcription elements with the aim of maximising expression; (II) construction of adenoviral vectors containing optimised constructs to assess tropism, magnitude and longevity of expression in vitro and in vivo. To this end a number of expression cassettes have been constructed to individually assess selected promoters (ICAM-2, ICAM-1, Lox-1250/1800, Flt-1274/1031, Tie-2 and CMV), enhancers (Tie-2 and eNOS), introns, exonic splicing enhancers and matrix attachment regions to initiate and augment gene expression in primary endothelial cells. Novel findings of this study have identified exonic splicing enhancers (ESEs) as critical elements for achieving high-level transgene expression in primary human umbilical vein endothelial cells (HUVECs). ESEs are multifunctional regulators of mRNA metabolism with diverse roles that couple the process of splicing, mRNA export and translation. The Serine-Arginine rich (SR) proteins that bind to these splicing enhancers are important in regulating splice site usage and nuclear export, regulating alternative splicing in a cell-type specific manner and in response to both inter and intracellular signalling. In addition, at least one member of this family (SF2/ASF) stays associated with the mature mRNA and increases translation efficiency. To assess the ability of these splicing enhancers to increase expression of transgenes, naturally occurring and consensus binding sites for a number of SR proteins (SF2/ASF, SC35, SRp40 and SRp55) were introduced after the splice acceptor site of a short intron. Expression cassettes containing the modified introns showed only modest variations in transgene expression compared to the original intron in HeLa cells. In HUVECs however, all but one of the modified introns demonstrated an increase in expression, by between 20 and 40 fold. In addition, short novel matrix attachment regions (MARs) isolated from the Tie-2 intergenic region, have shown the ability to augment transcription by up to 3-fold in HeLa. 12 different adenoviral vectors are currently in production to assess these constructs in a range of other cell-types as well as in vivo. This study underlines the need to include elements within gene expression cassettes capable of positively influencing all steps that control expression.