title: Plasmid DNA fermentation strain and process-specific effects on vector yield, quality, and transgene expression.
abstract: Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase-directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm- versions of CMV and CMV-HTLV-I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm- plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm- strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial.
authors.affiliation: Nature Technology Corporation, Lincoln, Nebraska 68521, 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: Angela
authors.initials: A
authors.lastname: Schukar
authors.forename: Sheryl
authors.initials: S
authors.lastname: Anderson
authors.forename: Clague P
authors.initials: CP
authors.lastname: Hodgson
authors.forename: James A
authors.initials: JA
authors.lastname: Williams
mesh_terms: D000085:Acetates / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000096:biosynthesis*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors; D015368:Human T-lymphotropic virus 1 / Q000235:genetics; D006801:Humans; D010957:Plasmids; D019076:Transgenes; D019444:Vaccines, DNA / Q000276:immunology
chemical_list: D000085:Acetates; D003470:Culture Media; D019444:Vaccines, DNA; D004247:DNA; D015257:DNA-Cytosine Methylases