OPTIMIZATION OF PLASMID VECTORS FOR HIGH-LEVEL EXPRESSION IN LUNG EPITHELIAL-CELLS

Nonviral gene therapy approaches use a plasmid vector to express the d esired transgene. We have systematically examined several regulatory e lements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructin g a series of plasmids that differed only in the particular sequence e lement being evaluated. Of the several promoters and polyadenylation s ignal sequences that were tested, the human cytomegalovirus (CMV) imme diate early gene promoter and the addition of polyadenylation signal s equences from the bovine growth hormone (BGH) gene or rabbit beta-glob in gene produced the highest levels of expression in vitro. The inclus ion of a hybrid intron 3' to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also e nhanced expression. On the basis of these results, the CMV promoter, t he hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. Howe ver, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heter ologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the deve lopment of optimized expression plasmids for gene therapy of lung dise ases.