Carnes A.E.,Nature Technology Corporation
Journal of Biotechnology | Year: 2014
The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655. δendAδrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (~141. mg/L) than MG1655. δendAδrecA (~48. mg/L) and DH5α (~40. mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655. δendAδrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200. mg/L. © 2014 Elsevier B.V.
Williams J.A.,Nature Technology Corporation
Current Gene Therapy | Year: 2014
DNA vaccines are a rapidly deployed next generation vaccination platform for treatment of human and animal disease. DNA delivery devices, such as electroporation and needle free jet injectors, are used to increase gene transfer. This results in higher antigen expression which correlates with improved humoral and cellular immunity in humans and animals. This review highlights recent vector and transgene design innovations that improve DNA vaccine performance. These new vectors improve antigen expression, increase plasmid manufacturing yield and quality in bioreactors, and eliminate antibiotic selection and other potential safety issues. A flowchart for designing synthetic antigen transgenes, combining antigen targeting, codon-optimization and bioinformatics, is presented. Application of improved vectors, of antibiotic free plasmid production, and cost effective manufacturing technologies will be critical to ensure safety, efficacy, and economically viable manufacturing of DNA vaccines currently under development for infectious disease, cancer, autoimmunity, immunotolerance and allergy indications. © 2014 Bentham Science Publishers.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 247.93K | Year: 2013
DESCRIPTION (provided by applicant): To commercialize non-viral gene medicines, it is critical that both vector potency (i.e. therapeutic transgene expression levels) and the duration of the therapeutic effect be improved. Potent dose-sparing extended duration gene therapies will have a cost and efficacy competitive advantage over alternative technologies. In this Phase I proof of concept study, we will create a novel antibiotic-free MiniPlasmid gene therapy platform for extended duration gene therapy. Thevectors combine transient expression enhancers that improve transgene expression levels with a novel 270 base pair replication origin-antibiotic free selection cassette that we hypothesize will promote long duration gene expression after vector delivery tothe body. The MiniPlasmid platform will be applied to create a wound healing gene therapy product to treat diabetic neuropathic foot ulcers. In Specific Aims 1 and 2 a high yielding MiniPlasmid fermentation manufacturing platform is created. In SpecificAim 3 the MiniPlasmid vector platform is validated in vivo for extended duration expression compared to conventional plasmids. A hypoxia- inducible factor 1 (HIF-1 ) based gene medicine for diabetic foot ulcer treatment is developed utilizing an extendedhalf-life oxygen resistant highly active HIF-1 mutant (CA5-HIF-1 ). Specific Aim 3 is performed in collaboration with wound healing gene therapy expert Dr. John Harmon at Johns Hopkins University. The MiniPlasmid vector platform is designed to improvetransgene expression levels and duration to enable gene medicine development for multiple applications requiring extended duration expression. MiniPlasmid vectors developed in Phase I will be marketed to investigators for a variety of gene therapy applications through publications, trade shows, and the Nature Technology Corporation (NTC) website. In Phase II the HIF-1 MiniPlasmid gene therapeutic will undergo preclinical safety and efficacy evaluations for treatment of diabetic foot ulcers prior to clinical development in Phase III. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: The objective of this proposal is to develop a novel antibiotic-free non-viral MiniPlasmid gene therapy platform for extended duration gene therapy, and as such is responsive to NIGMS SBIR high-priority area of interest in development of improved vectors for gene transfer. The vectors combine transient expression enhancers that improve transgene expression levels with a novel replication origin-antibiotic free selectioncassette that affords long duration gene expression after gene delivery to the body. The platform will be applied to create a wound healing gene therapy product to treat diabetic neuropathic foot ulcers.
Nature Technology Corporation | Date: 2011-05-16
Improvements in plasmid DNA production technology are needed to insure the economic feasibility of future DNA vaccines and DNA therapeutics. General methods are described, by means of which it is possible to dramatically increase plasmid DNA productivity in a fermentor. These processes feature fed-batch fermentation strategies, combined with novel growth and induction phase temperature shifts.
Nature Technology Corporation | Date: 2015-02-03
General methods and strains of bacteria are described that dramatically simplify and streamline plasmid DNA production. In one preferred embodiment, endolysin mediated plasmid extraction combined with flocculation mediated removal of cell debris and host nucleic acids achieves increased yield and purity with simplified downstream purification and reduced waste streams, thus reducing production costs.