PITA Fiscal Year 2009 Projects - Biomedical and Health Engineering

Enhanced Therapeutic DNA Production Through Cell & Batch Process Design

Principal Investigators: Michael M. Domach, Ignacio E. Grossmann, Mohammad M. Ataai, Saleem A. Khan

This seed proposal aims to pursue a finding that could favorably impact gene therapy and DNA vaccine technologies. Prior work involved, in part, measuring the production of a protein product from cells that maintain high copy number plasmids when different carbon sources and mutational backgrounds were used. Not only did we find that glycerol-grown cells and those lacking the enzyme pyruvate kinase (PYK-) exceed glucose-grown cells in target protein production as was predicted, we found that PYK- cells also produced 3- fold or more plasmid DNA. Subsequent Metabologica simulations concurred with the finding as well as suggested that even higher plasmid yield could be obtained. Additionally, the simulations indicated that PYKresults in coordinated increases in the fluxes from metabolic subsystems that support plasmid DNA synthesis. The finding is significant because plasmid DNA is the agent of gene therapy and new vaccines. For gene therapy, a multi-step process is used where first bacteria are used to make the therapeutic agent (i.e. plasmid DNA), and then an experiment or clinical trial is performed using the agent. Because the delivery of the agent into a cell or the human body is a multi-step process where each step’s efficiency is much less than 1, significant doses of agent are required per experiment or person. Thus, any development that improves the yield of the agent-producing process would be a welcome advance, especially when quantities that are sufficient for treating patient populations become needed. Indeed, one PA company we interact with has a strong interest in improving agent yield. We propose to test and initially optimize a multi-step process that is expected to increase plasmid yield by more than 10-fold. Also, computations enabled with Metabologica are proposed to deduce further other subsequent yield-enhancing steps to take in cell and plasmid design.