Production of a Nanoplasmid™ with a large gene insert using the HyperGRO™ fermentation process

Plasmid based DNA vaccines are emerging as a promising alternative to traditional vaccines due to several advantages, including faster production of DNA plasmids using E. coli. However, further increases in transgene expression are needed to meet efficacy requirements for various non-viral gene therapy and DNA vaccination applications. While existing minicircle DNA technology has been shown to offer improved levels and durations of transgene expression by removal of the bacterial region from the plasmid, low manufacturing yields may be a barrier to widespread use of minicircle DNA for vaccination. NTC’s minimalized Nanoplasmid™ vectors utilize RNA-OUT (ROUT) antibiotic-free selection and replace the large 1000 bp pUC replication origin with a novel, 300 bp, R6K-derived mini-origin (Figure 1). Reduction of the spacer region linking the 5’ and 3’ ends of the transgene expression cassette to \u3c500 bp remarkably increases plasmid-mediated transgene expression. Host strains expressing heat-inducible, high copy R6K replication (Rep) proteins have been developed for selection and propagation of Nanoplasmid. This is an additional Nanoplasmid safety factor since mini-origin vectors can only replicate within the engineered Rep protein-expressing E. coli host strain. Please click Additional Files below to see the full abstract