Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system

Abstract To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination.