Increased duration of transgene expression in the lung with plasmid DNA vectors harboring adenovirus E4 open reading frame 3

For gene therapy to be effective in the treatment of chronic diseases, plas mid DNA (pDNA) vectors that provide persistent expression of therapeutic le vels of the transgene product are desirable. Studies in the lung with adeno virus vectors showed that products of the adenovirus E4 region can act both irt cis and in traits to increase the duration of expression when transcri ption of the transgene was under the control of the human cytomegalovirus ( CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex compose d of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-con taining adenovirus vector (Ad2/beta gal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expre ssion were observed for up to 10 weeks postinstillation compared with expre ssion from mice instilled with control complexes containing an adenovirus v ector deleted of most of E4 (Ad2/beta gal-2), This effect could also be obs erved in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of ex pression. In addition to the CMV promoter, a CMV enhancer-human mucin I(MUC -I) hybrid promoter also responded to these E4-encoded proteins with increa sed persistence of transgene expression, but a human interleukin 8 (IL-8) p romoter did not, Ad2/beta gal-4 could be replaced by a pDNA vector expressi ng only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus ge nome. Further analysis indicated that the protein encoded by open reading f rame 3 (ORF3) alone was sufficient for conferring the increase in persisten ce of expression. These data indicate that expression of a single protein f rom the adenovirus genome can significantly improve the duration of transge ne expression from pDNA vectors, and increases the feasibility of using non viral vectors for the treatment of chronic diseases.