DEVELOPMENT OF IMPROVED VECTORS FOR DNA-BASED IMMUNIZATION AND OTHER GENE-THERAPY APPLICATIONS

Optimizing gene expression and delivery are necessary steps in the pro duction of vectors for DNA-based immunization as well as for other gen e therapy applications. A mouse muscle/reporter gene assay system was used to systematically improve a plasmid DNA vector. The optimized vec tor VR1255 contained: (1) CMV promoter and enhancer; (2) CMV IE Intron A; (3) kanamycin resistance gene; (4) deleted SV40 origin of replicat ion; (5) optimized lux coding region; and (6) a minimal synthetic term inator from the rabbit beta globin gene, mRBG. The vector VR1255 expre ssed 137 times greater than an earlier prototype RSV-based vector. For plasmid vector delivery into nonmuscle tissues, a recently synthesize d cationic lipid, GAP-DLRIE, was found to greatly enhance the uptake a nd expression of plasmid DNA by 100-fold when instilled into the mouse lung. The time-course of CAT expression with GAP-DLRIE indicated that peak expression occurs 2-5 days after intranasal administration and e xpression diminished to about one-third the peak value by day 21. This cationic lipid may be useful for immunization by pulmonary and perhap s other nonmuscle routes. (C) 1997 Elsevier Science Ltd.