title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.
abstract: BACKGROUND
  For safety considerations, regulatory agencies recommend the elimination of antibiotic resistance markers and non-essential sequences from plasmid DNA-based gene medicines. In the present study, we analyzed antibiotic-free (AF) vector design criteria impacting upon bacterial production and mammalian transgene expression.
  
  
  METHODS
  Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed enhanced green fluorescent protein and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction, respectively, after transfection of human HEK293 cells.
  
  
  RESULTS
  Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1000 mg/l plasmid DNA) and high-level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1740 mg/l was developed.
  
  
  CONCLUSIONS
  We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly-identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors.
authors.affiliation: Nature Technology Corporation, Lincoln, NE, USA.
authors.forename: Aaron E
authors.initials: AE
authors.lastname: Carnes
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Luke
authors.forename: Justin M
authors.initials: JM
authors.lastname: Vincent
authors.forename: Sheryl
authors.initials: S
authors.lastname: Anderson
authors.forename: Angela
authors.initials: A
authors.lastname: Schukar
authors.forename: Clague P
authors.initials: CP
authors.lastname: Hodgson
authors.forename: James A
authors.initials: JA
authors.lastname: Williams
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D051379:Mice; D012313:RNA / Q000235:genetics*; D012319:RNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D019076:Transgenes / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012313:RNA; D012319:RNA Polymerase II; D012320:RNA Polymerase III
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