Development of antibiotic-free selection system for safer DNA vaccination.

The use of antibiotic-resistance markers in DNA vaccines is discouraged by regulatory agencies due to various theoretical safety concerns. This chapter presents methodologies for the design and cloning of synthetic antigen genes into RNA-OUT encoding antibiotic-free DNA vaccine vectors that are additionally optimized to improve protein expression, and immunogenicity, compared to alternative kanamycin-resistant vectors. First, antigen targeting considerations are discussed in the context of immune response customization through MHC class I or class II directed antigen presentation; the example NTC868 series RNA-OUT vector system allows simultaneous cloning into multiple vectors that feature various transgene intracellular targeting destinations. Then a detailed flowchart for codon optimization and synthetic transgene design is presented. Finally in-depth methodologies for cloning transgenes into the NTC868 series RNA-OUT vector system are presented. The resultant antibiotic-free DNA vaccine vectors are a more potent, safer alternative to existing kanamycin resistance marker encoding vectors.

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.

Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector. Alternative pVEX vectors encoding Pyrococcus furiosus maltodextrin-binding protein (pfMBP) as a fusion partner improved Influenza hemagglutinin antigen vaccine solubility and thermostability. A pfMBP hemagglutinin HA2 domain fusion protein was a potent immunogen. Manufacturing processes that combined up to 5 g/L defined media fermentation yields with rapid, selective, thermostable pfMBP fusion protein purification were developed. The pVEX pfMBP-based thermostable tag (TST) platform is a generic protein engineering approach to enable high yield manufacture of thermostable recombinant protein vaccine components.

Coexpressed RIG-I agonist enhances humoral immune response to influenza virus DNA vaccine.

Increasing levels of plasmid vector-mediated activation of innate immune signaling pathways is an approach to improve DNA vaccine-induced adaptive immunity for infectious disease and cancer applications. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA (dsRNA) pattern receptor required for innate immune activation in response to viral infection. Activation of RIG-I leads to type I interferon (IFN) and inflammatory cytokine production through interferon promoter stimulator 1 (IPS-1)-mediated activation of interferon regulatory factor 3 (IRF3) and NF-κB signaling. DNA vaccines coexpressing antigen and an expressed RNA (eRNA) RIG-I agonist were made, and the effect of RIG-I activation on antigen-specific immune responses to the encoded antigen was determined. Plasmid vector backbones expressing various RIG-I ligands from RNA polymerase III promoters were screened in a cell culture assay for RIG-I agonist activity, and optimized, potent RIG-I ligands were developed. One of these, eRNA41H, combines (i) eRNA11a, an immunostimulatory dsRNA expressed by convergent transcription, with (ii) adenovirus VA RNAI. eRNA41H was integrated into the backbone of DNA vaccine vectors expressing H5N1 influenza virus hemagglutinin (HA). The resultant eRNA vectors potently induced type 1 IFN production in cell culture through RIG-I activation and combined high-level HA antigen expression with RNA-mediated type I IFN activation in a single plasmid vector. The eRNA vectors induced increased HA-specific serum antibody binding avidity after naked DNA intramuscular prime and boost delivery in mice. This demonstrates that DNA vaccine potency may be augmented by the incorporation of RIG-I-activating immunostimulatory RNA into the vector backbone.

Vector insert-targeted integrative antisense expression system for plasmid stabilization.

Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.

BACKGROUND: For safety considerations, regulatory agencies recommend the elimination of antibiotic resistance markers and non-essential sequences from plasmid DNA-based gene medicines. In the present study, we analyzed antibiotic-free (AF) vector design criteria impacting upon bacterial production and mammalian transgene expression. METHODS: Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied. Plasmid production yield was assessed through inducible fed-batch fermentation. RNA Pol II-directed enhanced green fluorescent protein and RNA Pol III-directed RNA expression were quantified by fluorometry and quantitative real-time polymerase chain reaction, respectively, after transfection of human HEK293 cells. RESULTS: Sucrose-selectable minimalized protein and therapeutic RNA expression vector designs that combined an RNA-based AF selection with highly productive fermentation manufacturing (>1000 mg/l plasmid DNA) and high-level in vivo expression of encoded products were identified. The AF selectable marker was also successfully applied to convert existing kanamycin-resistant DNA vaccine plasmids gWIZ and pVAX1 into AF vectors, demonstrating a general utility for retrofitting existing vectors. A minimum vector size for high yield plasmid fermentation was identified. A strategy for stable fermentation of plasmid dimers with improved vector potency and fermentation yields up to 1740 mg/l was developed. CONCLUSIONS: We report the development of potent high yield AF gene medicine expression vectors for protein or RNA (e.g. short hairpin RNA or microRNA) products. These AF expression vectors were optimized to exceed a newly-identified size threshold for high copy plasmid replication and direct higher transgene expression levels than alternative vectors.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.