Development of antibiotic-free selection system for safer DNA vaccination

The use of antibiotic-resistance markers in DNA vaccines is discouraged by regulatory agencies due to various theoretical safety concerns. This chapter presents methodologies for the design and cloning of synthetic antigen genes into RNA-OUT encoding antibiotic-free DNA vaccine vectors that are additionally optimized to improve protein expression, and immunogenicity, compared to alternative kanamycin-resistant vectors. First, antigen targeting considerations are discussed in the context of immune response customization through MHC class I or class II directed antigen presentation; the example NTC868 series RNA-OUT vector system allows simultaneous cloning into multiple vectors that feature various transgene intracellular targeting destinations. Then a detailed flowchart for codon optimization and synthetic transgene design is presented. Finally in-depth methodologies for cloning transgenes into the NTC868 series RNA-OUT vector system are presented. The resultant antibiotic-free DNA vaccine vectors are a more potent, safer alternative to existing kanamycin resistance marker encoding vectors.