(642a) Engineering Gene Expression Dynamics of Mammalian Cells in Culture

Recombinant mammalian cells have been used extensively for the production of therapeutic proteins, accounting for tens of billions per annum of product value and benefiting tens of thousands of patients. Their rise to such prominence was largely attributed to the genetic manipulations which enable cells to produce high levels of recombinant proteins. The transformation of these cells' protein secretion capability from none to a level which rivals professional secretors in vivo during cell development process entails profound changes in gene regulation for which little is known. Empirical selection of cells with high productivity and desired growth characteristics has been key to the success. Substantial effort in metabolic engineering has focused on introducing other metabolic, anti-apoptotic and glycosylation-related genes and significant progress has been reported. Much of these works has been performed using strong and constitutive expression systems. However, cellular needs are dynamic, responding to various environmental perturbations as well as cellular processes, rather than being static. Inducible systems enable time dynamics through external manipulation of inductive conditions. Most ideally the transgenes should be synchronous to the cell's own rhythm. Furthermore, depending on the cellular process to be manipulated and the transgene, the expression level should be controlled to deliver the correct modulating level of gene dose. To that end, we surveyed the transcriptome profiles obtained from time-course experiments spanning all stages of cell growth and encompassing different culture conditions. Clustering of gene expression patterns revealed hundreds of genes with varying dynamics across a wide intensity range. The promoters of genes identified in this analysis can potentially provide a more flexible and dynamic control of the transgenes. In this study, we focused on several genes which exhibit low expression levels in the lag phase and significantly higher expression levels in the stationary phase. We demonstrated that, under the control of these dynamic promoters, a luciferase reporter gene can be expressed in concert with cell growth. This approach illustrates a novel concept in metabolic engineering which can potentially be used to achieve dynamic control of cellular behaviors for enhanced process characteristics.