180. pDNAVACC Vector Family: High Throughput Intracellular Targeting DNA Vaccine Plasmids Created by Gene Self Assembly

Abstract

DNA vaccines have the potential to provide a safe route for protective immunity to neoplasms and infectious agents. However, current DNA vaccine plasmids are not optimal with additional nonessential DNA, nor do they facilitate high throughput projects, or flexible targeting of antigens to various intracellular destinations. A family of DNA vaccine vectors was constructed by simultaneously joining six fragments, using Gene Self-Assembly (GENSAtm) technology. GENSAtm uses class IIS restriction enzymes to generate unique, non-palindromic overhanging termini that can ligate to only one other terminus in a complex mixture, thus assuring that each fragment ligates in the correct orientation to its correct partner, and none other. All plasmid elements have been optimized and minimized to comply with FDA guidelines regarding content and elimination of extraneous materials. The resulting vector is much smaller than existing vectors such as gWiz (5kb, versus 3|[ndash]|3.5 kb for the pDNAVACC vectors) and drives higher levels of target gene expression. Incorporation of an optimized kanR gene-pUC origin orientation, as well as a rationally designed RNAII mutation, results in 2 fold improved plasmid productivity. These vectors are designed to facilitate high throughput cloning applications, and allow simultaneous cloning into multiple vectors that feature various intracellular targeting destinations for the protein product. The ability to control expression and trafficking in a modular format is intended to provide a rapid, rational approach to cancer therapy and emerging infectious diseases.