Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

Abstract

Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for followon products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

“…We observed improvements to the transfection efficiency above those achieved with pmGENIE-3-R6K. As a final improvement, we engineered the vectors to feature an antibiotic-free selection (pmhyGENIE-3-RNA-OUT) in bacteria to conform to regulatory recommendations for the elimination of antibiotic resistance markers during in vivo transfection and transgenesis (25). A comparison of colony counts between the original pmGENIE-3 plasmid and the newly developed hyperactive plasmid constructs, as depicted in Fig.…”

Antibiotic-Free Selection for Bio-Production: Moving Towards a New "Gold Standard"

“…There are specific mentions for the nature of the gene encoding resistance to kanamycin, as reviewed by Williams et al, (2009 …”

Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza Virus DNA Vaccine

“…RIG-I activation was obtained using plasmid-driven expression of 10 copies of a native Epstein-Barr virus small RNA (EBER1), another known RIG-I ligand (50). However, plasmids containing 10 copies of a repetitive sequence are unstable in E. coli (65). An alternative approach, generation of RIG-I ligands by cytoplasmic RNA Pol III transcription of polyAT repeats (9), did not result in RIG-I activation when annealed DNA fragments polyAT 40 (40 AT repeats), or AAAAAApolyAT 40 TTTTTT (RNA Pol III terminators flanking polyAT 40 ), were incorporated into pDNAVACCUltra-EGFP and pDNAVACCUltra-VA1-EGFP vector backbones (J. Williams, unpublished observations).…”