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.