Marker-free plasmids for gene therapeutic applications—Lack of antibiotic resistance gene substantially improves the manufacturing process

Mairhofer, Juergen; Cserjan‐Puschmann, Monika; Striedner, Gerald; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Grabherr, Reingard

doi:10.1016/j.jbiotec.2010.01.025

Cited by 55 publications

(32 citation statements)

References 42 publications

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“…Additionally, marker-free plasmids are highly feasible for generating induced pluripotent stem cells due to their improved delivery efficiency and increased safety, as they contain no antibiotic resistance genes ( Jia et al, 2010;Maucksch et al, 2009). Finally, because the large-scale production of MINI-plasmids is easier and more straightforward (Mairhofer et al, 2010) than other alternative technologies for the production of marker-free plasmids (e.g., minicircles), MINI-plasmids comprise an efficient alternative for cell-based gene therapy and stem cell technology.…”

Section: Resultsmentioning

confidence: 99%

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Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells

Ribeiro

Mairhofer

Madeira

et al. 2012

Cellular Reprogramming

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Genetic modification of stem cells, prior to transplantation, can enhance their survival and can improve their function in cell therapy settings. Mesenchymal stem cells (MSC) are considered one of the most promising tools for cell-based gene therapy, due to their multipotency, ease of isolation, as well as their high ex vivo expansion potential. Neural stem cells (NSC) may also present an ideal route for gene therapy and have been considered for use in cell replacement therapies in various neurodegenerative diseases. Gene therapy-based applications require the transfer of genetic material, either by viral or nonviral gene delivery methods, although the latter has been associated with low efficiencies, especially within hard to transfect cells as stem cells. Herein, we present results on the influence of plasmid size in gene delivery to human MSC and mouse NSC. We used minimized plasmids encoding a fluorescent protein but lacking the antibiotic resistance gene. This work shows that (1) for smaller plasmids the intracellular plasmid copy number can be up to 2.6-fold higher, and (2) the number of cells presenting fluorescence can be twice the number obtained for larger plasmids. Furthermore, by using plasmid constructs containing different polyA signals, we also demonstrated that differences between the plasmids depend largely on the transgene mRNA level. Based on our data we demonstrate that plasmid size severely affects the efficiency of nuclear uptake and we propose that it can also affect the rate of heterochromatin associated gene silencing in stem cells.

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Section: Resultsmentioning

confidence: 99%

“…RNAI is an intrinsic part of the copy number regulation of ColE1-like plasmids (pUC, pET, pMB1, pBR322), and therefore transcribed from common plasmids used for gene therapeutic applications. This system is scalable and the production of marker-free plasmid in the Gram-scale has been reported recently (Mairhofer et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Plasmid DNA Size Does Affect Nonviral Gene Delivery Efficiency in Stem Cells

Ribeiro

Mairhofer

Madeira

et al. 2012

Cellular Reprogramming

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“…A few high yield fed-batch plasmid fermentation processes (500-2,200 mg/L) have been described (Carnes et al, 2006;Listner et al, 2006;Mairhofer et al, 2010;Phue et al, 2008;Singer et al, 2009;Williams et al, 2009c). These processes all couple reduced growth rate (which generally increases copy number) with high copy replication origins (reviewed in Carnes and Williams, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…Plasmid DNA production is typically performed in Escherichia coli K12 strains such as DH5a (Carnes et al, 2006), DH5 (Listner et al, 2006), DH1 (Cooke et al, 2004), JM108 (Mairhofer et al, 2010), or DH10B (Lahijani et al, 1996). E. coli B strain BL21 recA endA is also a high yielding plasmid producer (Phue et al, 2008) (reviewed in Cai et al, 2009;Williams et al, 2009a).…”

Section: Introductionmentioning

confidence: 99%

Plasmid DNA fermentation strain and process‐specific effects on vector yield, quality, and transgene expression

Carnes

Luke

Vincent

et al. 2010

Biotech & Bioengineering

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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.

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“…Safety concerns have included the dissemination of antibiotic resistance selection markers, integration into the human genome, and the development of autoimmunity [8]. The dissemination of antibiotic resistance to pathogens remains a concern, but it may be resolved by the removal of these selection markers during manufacturing [9]. Although early studies found the risk of genomic integration negligible [10], new highly efficient methods of administration such as electroporation have resurrected this old concern [11].…”

Section: Introductionmentioning

confidence: 99%