Plasmid DNA fermentation strain and process-specific effects on vector yield, quality, and transgene expression.
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.
Carnes
Aaron E
AE
Nature Technology Corporation, Lincoln, Nebraska 68521, USA.
Luke
Jeremy M
JM
Vincent
Justin M
JM
Schukar
Angela
A
Anderson
Sheryl
S
Hodgson
Clague P
CP
Williams
James A
JA
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
D000085:Acetates; D003470:Culture Media; D019444:Vaccines, DNA; D004247:DNA; D015257:DNA-Cytosine Methylases