Sustained transgene expression will be required for the vast majority of
genetic diseases being considered for gene therapy. The initially high
levels of expression attained with plasmid DNA (pDNA) vectors containing
viral promoters, such as that from cytomegalovirus (CMV), decline
precipitously to near background levels within 2 to 3 weeks. We have
constructed pDNA vectors containing the human cellular ubiquitin B (Ub)
promoter and evaluated their expression in the mouse lung. Cationic
lipid-pDNA complexes were instilled intranasally (IN) or injected
intravenously (IV) into immunodeficient BALB/c mice. Chloramphenicol
acetyltransferase (CAT) reporter gene expression from the Ub promoter was
initially very low at day 2 post-administration but by day 35 exceeded
the level of expression attained from a CMV promoter vector by 4- to
9-fold. Appending a portion of the CMV enhancer 5' of the Ub promoter
(CMV-Ub) increased CAT expression to nearly that of the CMV promoter and
expression persisted in the lung for at least three months, with 50% of
day 2 levels remaining at day 84. In the liver, expression from the
CMV-Ub hybrid promoter was sustained for 42 days. Since previous studies
have shown that eliminating immunostimulatory CpG motifs in pDNA vectors
reduces their toxicity, we constructed a CpG deficient version of the
CMV-Ub vector expressing alpha-galactosidase A, the enzyme that is
deficient in Fabry disease, a lysosomal storage disorder. After IN or IV
administration, levels of alpha-galactosidase A from this vector were not
only undiminished but increased 500% to 1500% by day 35. These results
suggest that CpG-reduced plasmid vectors containing a CMV-Ub hybrid
promoter may provide the long-term expression and efficacy required for a
practical gene therapeutic.