To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a chromosomally integrated constitutively expressed counter-selectable marker (sacB), allowing plasmid selection on sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (>1 gm/L plasmid DNA yields), while improving in vivo expression of encoded proteins and increasing immune responses to target antigens. These vectors are safer, more potent, alternatives for DNA therapy or vaccination. Keywords DNA vaccine; plasmid; antibiotic-free 1) IntroductionPlasmid based DNA vaccines and therapeutics are in development for a variety of human, animal, bird and fish applications. Antibiotic resistance markers, typically kanamycin resistance (kanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance to endogenous microbial fauna; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome [Reviewed in 1,2 ]. For example, a regulatory guidance with regard to DNA vaccine plasmids states: "The use of certain selection markers, such as resistance to antibiotics, which may adversely impact on other clinical therapies in the target population, should be avoided" [ 3 ]. Further, the use of antibiotics in fermentation culture requires expensive process validation of antibiotic removal during plasmid purification, to prevent contamination of the final product with residual antibiotics. Ideally, the plasmid would not contain any protein coding regions other than the gene of interest, since these could *Corresponding Author James A Williams, Nature Technology Corporation., 4701 Innovation Drive Lincoln Nebraska, 68521, Telephone: (402) ., jim@natx.com. Conflict of Interest Statement JL, AEC, CPH and JAW have an equity interest in Nature Technology CorporationPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptVaccine. Author manuscript; available in PMC 2010 October 30. potentially be expressed in mammalian cells. Alternative select...

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is 'plugged' into a validated vector, and rapidly produced in a validated, fermentation -purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably 'toxic' or otherwise low yielding in standard fermentation processes. We report cell bank and fermentation process unit operation innovations that reduce plasmid-mediated metabolic burden, enabling successful production of previously known toxic influenza hemagglutinin antigen genes. These processes, combined with vector backbone modifications, doubled fermentation productivity compared to existing high copy vectors, such as pVAX1 and gWIZ, resulting in high plasmid yields (up to 2220 mg/L, 5% of total dry cell weight) even with previously identified toxic or poor producing inserts.

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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.