Critical molecular and cellular biological factors impacting design of licensable DNA vaccine vectors that combine high yield and integrity during bacterial production with increased expression in mammalian cells are reviewed. Food and Drug Administration (FDA), World Health Organization (WHO) and European Medical Agencies (EMEA) regulatory guidance's are discussed, as they relate to vector design and plasmid fermentation. While all new vectors will require extensive preclinical testing to validate safety and performance prior to clinical use, regulatory testing burden for followon products can be reduced by combining carefully designed synthetic genes with existing validated vector backbones. A flowchart for creation of new synthetic genes, combining rationale design with bioinformatics, is presented. The biology of plasmid replication is reviewed, and process engineering strategies that reduce metabolic burden discussed. Utilizing recently developed low metabolic burden seed stock and fermentation strategies, optimized vectors can now be manufactured in high yields exceeding 2 g/L, with specific plasmid yields of 5% total dry cell weight.

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.

DNA hybridization probes can be radiolabelled internally with 32P either by nick-translation (1 2) or by oligonucleotide-priming (oligolabelling, 3-4).The latter method has the advantage that two sources of nuclease activity (DNAse I and 5'-3' exonuclease) are eliminated, allowing greater control over the size of the end product.

Retrovirus-derived vectors are overwhelmingly preferred over other methods for ex vivo gene therapy because they provide permanent integration of foreign genes into cellular DNA. In comparison, cationic lipids mediate efficent gene transfer, but expression is transient. When we combined cationic lipids with retrovirus particles we obtained a significant enhancement of transduction efficiency, depending upon the type of lipid formulation and the dose used. The relative effectiveness of these cytofectins was: DOSPA:DOPE > DOTMA:DOPE > DOTAP, resulting in 60-, 37-, and 5-fold increases in transduction efficiency, respectively, at optimum dosage. The effect of polycationic DOSPA:DOPE was dependent upon the viral envelope glycoprotein, was attainable by lipid treatment of either cells or virus particles, was not enhanced by the addition of polybrene, and was inhibited by chloroquine. These results strongly suggested that DOSPA:DOPE act primarily by modulation of charge associated with the viral envelope and cell membrane, enhancing retroviral transduction, rather than by providing an alternative pathway of transfection. DOSPA:DOPE is useful for improving the efficiency of gene transfer as well as the sensitivity with which retroviruses can be detected in biological fluids.

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We report the first evidence of increased levels of the retinoblastoma (Rb) message in a majority of colorectal cancers when compared with normal mucosa. Southern blot analysis showed an increase in Rb gene copy number in at least 28% of colorectal carcinomas relative to normal mucosa. These results plus previous reports of nonrandom chromosome 13 gains in approximately 50% of colorectal cancers suggest that an increase in Rb gene copy number occurs frequently in these tumors. Possible mechanisms pertaining to overexpression of the Rb gene are discussed in relation to its role as a recessive cancer gene.

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