SBIR-STTR Award

DNA vaccines and gene medicines are emerging as an important new class of pharmaceuticals that are derived from bacterial plasmids. For use in humans, plasmid DNA should be essentially free from bacterial genomic DNA and RNA, protein and endotoxin. The hypothesis is that E. coli plasmid DNA can be more efficiently purified by co-expressing RNase and DNase in plasmid producing strains of bacteria. The strategy will be to make hybrid proteins (expressing RNase A and phage T5 DNA exonuclease) and secrete them into the periplasmic space, releasing them at the appropriate time to allow digestion of E. coli RNA and genomic DNA. In Specific Aim I, we will construct recombinant genes for expression of RNase and T5 exonuclease. In Specific Aim II, we will investigate the use of the recombinant enzymes in the purification of plasmids. The overall goal of the proposed Phase I feasibility study is to determine whether such a bacterial strain is a significant advantage in the purification of plasmid DNA. If successful, Phase II studies will include improved constructs for insertion of the genes into the E. coli chromosome; creating the strains needed; development of a third enzyme (protease); and development of an inducible system for introducing the foreign proteins into the cytoplasm during the final hours of fermentation. In Phase Ill, NTC will introduce the improved strains in commercial plasmid production. Success in constructing one or more useful strains of bacteria expressing RNAse, DNAse and/or protease enzymes in a controllable fashion (milestone II) would be a significant development for DNA purification on a commercial scale. Such strains will be rapidly introduced into NTC's large scale DNA manufacturing process, and will also be made available for licensing to other commercial and academic users through the company's technology transfer and licensing program. Currently, experimental gene drugs and DNA vaccines are routinely made on the gram scale, mostly for use in animal safety and efficacy studies. However, the advent of FDA approved commercial products will require scaling up to kilograms, for example, for a widely used vaccine. Judging from the current price of $22,000- $46,000/gram of DNA produced, a less costly, scalable fermentation process is badly needed. The economic benefits and potential market size for the resulting products can be readily appreciated. The ability to use endogenously produced recombinant enzymes would be a significant advantage (in terms of safety and economy) over the use of animal-derived or exogenously added enzymes. The likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.

Thesaurus Terms:
deoxyribonuclease I, endopeptidase, endoribonuclease, fusion gene, hybrid enzyme, nucleic acid chemical synthesis, nucleic acid purification, protein engineering chimeric protein, gene expression, genetic strain, hydrolysis, plasmid Escherichia coli, biotechnology, high throughput technology, polymerase chain reaction

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. DNA molecules made strictly by rational design allows the manufacturer to bypass years of development for the production of efficacious vaccines, and literally create new vaccine entities in days and mass produce vaccines in 2-3 weeks for rapid deployment against new biological agents. However, existing fermentation and purification production technologies are inefficient, difficult to scale, and require sophisticated production equipment and methodology. This limits their utility to meet cost and capacity needs for existing plasmid applications, or to rapidly produce kilograms of plasmid DNA for pandemic vaccination. Phase I addressed these problems directly and successfully by proving feasibility of a cost effective dramatically streamlined and simple purification process that eliminates specialized and costly alkaline or heat lysis steps and the associated toxic waste streams. In Phase II, we will make and evaluate designer strains and associated fermentation and purification methodology. These strains will utilize novel chimeric nucleases and autolytic bacterial strains developed in Phase I. Four prototypes are envisioned. Prototype I is a rapid deployment plasmid production system linking autolytic plasmid purification to Nature Technology Corporation's (NTC) existing fermentation platform to facilitate immediate production of a variety of plasmid DNAs for pandemic applications. The key drivers for this platform are speed of production, safety, simplicity, scalability and transferability to existing manufacturing facilities in developed and emerging countries. Autolytic strains with an integrated chimeric nuclease will be created, and fermentation, autolysis, and plasmid purification methodologies developed. The system will be integrated into NTC's existing rapid deployment DNA vaccine platform (RapidVACC) for response to influenza H5N1 pandemic. Prototypes II-IV are aimed at facilitating the commercialization of gene medicines for a variety of applications. The key drivers for these prototypes are purity, yield, cost (reagents, equipment, volumes, and time), scalability and safety. Prototype IV links high yield defined media fermentation with new simplified lysis and purification methodologies. This technology will be licensed to plasmid DNA manufacturers; one such commercialization channel has already been identified.

Thesaurus Terms:
Fermentation, Fusion Gene, Method Development, Nucleic Acid Purification, Plasmid, Vaccine Development, Vaccine Evaluation, Vector Vaccine Acidity /Alkalinity, Bacterial Genetics, Cell Autolysis, Cost Effectiveness, Disease Outbreak, Genetic Strain, Growth Media, Immunogenetics, Nuclease, Thermostability Bioengineering /Biomedical Engineering, Transfection