Large scale manufacture of plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Although extensive research on plasmid DNA purification methodologies has resulted in efficient purification processes, plasmid DNA production capacity is severely limited by low fermentation yield. The hypothesis of the proposed work is that E. coli plasmid DNA can be more efficiently produced by co-expression of an antisense RNA targeting the ColEI origin represser RNA (RNAI). The strategy will be to make designer antisense RNAI inhibitors, inducing them at the appropriate time to allow runaway replication of target plasmids. In Specific Aim 1, we will construct recombinant plasmids for expression of RNAI inhibitors. In Specific Aim 2, we will screen for antisense RNAI inhibitors useful for the production of plasmids. The overall goal of the proposed feasibility study is to determine whether such a bacterial strain is a significant advantage in the production of plasmid DNA using existing replicons (milestone I). If successful, Phase II studies will include: 1) improved constructs for insertion of the antisense RNA into the E. coli chromosome; 2) creating the strains needed and 3) fermentation process development. In Phase III, NTC will introduce the improved strains into commercial plasmid production. Potential commercial applications Success in constructing one or more useful strains of bacteria expressing RNAI inhibitor in a controllable fashion (milestone II) will help to overcome a major obstacle in the economical production of highly purified plasmid DNA for use in human Pharmaceuticals and DNA vaccines. Such strains will be rapidly introduced into Nature Technology's large scale DNA manufacturing process, and will also be made available for licensing to commercial 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 likelihood of a commercially useful and valuable product resulting from the proposed work is considered high.