Tools for next-generation transcriptional control in Chinese hamster ovary cell factories

• Main Author: Brown, Adam
• Other Authors: James, David
• Published: University of Sheffield 2014
• Subjects: 660
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.631433

Recombinant gene transcription in Chinese hamster ovary (CHO) cells, the dominant cell factory utilised for biopharmaceutical production, is still routinely regulated with a limited set of functionally ill-defined and uncontrollable genetic elements. This study presents novel transcription control technologies that facilitate development of next-generation biopharmaceutical manufacturing systems. Firstly, synthetic promoters designed specifically to harness the pre-existing transcriptional activation machinery of CHO cell factories have been constructed. Transcription factor regulatory element (TFRE) function was screened in CHO cells and active elements were utilised to create synthetic promoter libraries exhibiting 140 discrete activites, operating over two orders of magnitude, where the most active promoters significantly exceeded that of the human cytomegalovirus immediate early 1 (hCMV-IE1) promoter. Through precise control of recombinant gene expression in CHO host cells over a broad dynamic range this technology could be utilised to both maximise transcription of easy-to-express proteins and provide optimised protein-specific transcription levels (synchronised with polypeptide-specific folding kinetics) of difficult-to-express proteins. Further, it will enable construction of bespoke, synthetic cell factories that require the expression of several genes to be stoichiometrically balanced. Secondly, a novel method of transcription factor (TF) decoy (synthetic oligodeoxynucleotides that specifically sequester cognate TFs) formation has been developed, where blocks containing discrete TF binding sites are combined into circular molecules. Unlike currently available methods block-decoys allow rapid construction of chimeric decoys targeting multiple TFREs. Moreover, they enable fine tuning of binding site copy ratios within chimeras, facilitating sophisticated control of the cellular transcriptional landscape. It was demonstrated that a bespoke block-decoy chimera was able to inhibit expression from multiple target elements simultaneously in CHO cells. Block-decoys can be utilised to investigate any multi-TF mediated cell function or phenotype and represent a valuable new tool for characterising and controlling CHO cell transcription. Finally, the mechanistic functionality of the promoter most commonly utilised to drive transgene expression in CHO cells, hCMV-IE1, has been analysed. It was found that hCMV-IE1 promoter activity in CHO cells is predominantly mediated via just two TFREs (CRE and NFkB), where physical prevention of TF-TFRE interactions at these sites, either by intracellular TF sequestration or TFRE deletion, reduced activity by >75%. This mechanistic understanding of hCMV-IE1s functional regulation in CHO cells facilitates strategies to predictably control or improve its activity by engineering the promoter's TFRE composition or the cell factory's TF abundances. This will likely be most useful for optimising transient gene expression systems where hCMV-IE1 is the current promoter of choice. Cumulatively, the tools developed in this thesis enable sophisticated, next-generation transcriptional control in CHO cell factories.