High and sustained transgene expression in vivo from plasmid vectors containing a hybrid ubiquitin promoter

Sustained transgene expression will be required for the successful treatmen t of most genetic diseases being considered for gene therapy. The initially high levels of expression attained with plasmid DNA (pDNA) vectors contain ing viral promoters, such as that from cytomegalovirus (CMV), decline preci pitously to near-background levels within two to three weeks. Here we const ructed pDNA vectors containing the human cellular UBB (encoding ubiquitin B ; Ub) promoter and evaluated their expression in the mouse lung. Cationic l ipid-pDNA complexes were instilled intranasally (IN) or injected intravenou sly (IV) into immunodeficient BALB/c mice. Chloramphenicol acetyltransferas e (CAT) reporter gene expression from the UBB promoter was initially very l ow at day 2 post-administration, but by day 35 exceeded the level of expres sion attained from a CMV promoter vector by four- to ninefold. Appending a portion of the CMV enhancer 5' of the UBB promoter (CMV-Ub) increased CAT e xpression to nearly that of the CMV promoter and expression persisted in th e lung for at least 3 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. As previous studies have shown that eliminating immunostimulatory CpG motifs in pDNA vectors reduces their toxicity, we constructed a CpG-de ficient version of the CMV-Ub vector expressing alpha -galactosidase A, the enzyme deficient in Fabry disease, a lysosomal storage disorder. After 1N or IV administration, levels of a-galactosidase A from this vector were not only undiminished but increased 500% to 1500% by day 35. Our results indic ate that CpG-reduced plasmid vectors containing a CMV-Ub hybrid promoter ma y provide the long-term expression required for a practical gene therapeuti c.