Plasmid DNA fermentation strain and process-specific effects on vector yield, quality, and transgene expression
| Autor: | Sheryl Anderson, Clague P. Hodgson, James A. Williams, Angela Schukar, Jeremy Luke, Aaron E. Carnes, Justin M. Vincent |
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| Rok vydání: | 2010 |
| Předmět: |
DNA-Cytosine Methylases
Transgene Genetic enhancement Genetic Vectors Cytomegalovirus Gene Expression Bioengineering Acetates Biology medicine.disease_cause Applied Microbiology and Biotechnology Plasmid Gene expression Escherichia coli Vaccines DNA medicine Humans Transgenes Gene Human T-lymphotropic virus 1 Immunogenicity DNA Molecular biology Culture Media Fermentation DNA methylation Plasmids Biotechnology |
| Zdroj: | Biotechnology and Bioengineering. 108:354-363 |
| ISSN: | 0006-3592 |
| DOI: | 10.1002/bit.22936 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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