Requirements for enhanced transgene expression by untranslated sequences from the human cytomegalovirus immediate-early gene

Background: The cytomegalovirus immediate early (CMV IE) promoter has been widely used far heterologous expression. Further enhancements of gene expre ssion from this potent promoter may allow for the development of improved g ene transfer strategies. We aimed to determine whether inclusion of the fir st exon (5' untranslated) and first intron of the CMV IE gene would increas e heterologous transgene expression in primary target cells and to determin e the sequences required for any observed increases. Materials and Methods: Comparisons of reporter gene expression were made fo llowing transient transfection of vascular smooth muscle cells (VSMCs) with plasmids containing the first exon and intron from the CMV IE gene or dele tional mutations. Comparisons were also made using a heterologous promoter (RSV). Results: Gene expression from the CMV IE promoter was increased 5.7-fold in VSMC with the inclusion of the first exon and intron. Similar increases we re seen with other target cells and from the heterologous RSV promoter. Thi s increase was associated with an increase in steady-state mRNA. Deletion a nalyses demonstrated that the enhancement was dependent on the presence of the 5' portion of the first exon while deletion of large segments within th e intron was associated with similar levels of expression compared with the parental plasmid. Conclusions: Inclusion of the first exon and intron from the CMV IE gene in creases expression from the CMV IE promoter. This enhancement is seen with the heterologous RSV promoter and is associated with an increase in steady- state mRNA. Deletion analyses suggest that this enhancement is associated w ith inclusion of sequences within the 5' portion of the first exon and incl usion of an intron.