Synthetic promoters for CHO cell engineering.

We describe for the first time the creation of a library of 140 synthetic promoters specifically designed to regulate the expression of recombinant genes in CHO cells. Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks. The most active second generation promoters achieved approximately twice the activity of a control construct harboring the human cytomegalovirus (CMV) promoter. Lastly, we evaluated the function of a subset of synthetic promoters exhibiting a broad range of activity in different CHO cell host cell lines (CHO-S, CHO-K1, and CHO-DG44) and across extended fed-batch transient expression in CHO-S cells. In general, the different synthetic promoters both maintained their relative activity and the most active promoters consistently and significantly exceeded the activity of the CMV control promoter. For advanced cell engineering strategies our synthetic promoter libraries offer precise control of recombinant transcriptional activity in CHO cells spanning over two orders of magnitude.

Block decoys: transcription-factor decoys designed for in vitro gene regulation studies.

Transcription-factor decoys are short synthetic oligodeoxynucleotides that sequester cognate transcription factors and prevent their binding at target promoters. Current methods of decoy formation have primarily been optimized for potential therapeutic applications. However, they are not ideally suited to in vitro investigations into multi-transcription factor-mediated processes that may require multiple regulatory elements to be inhibited in varying combinations. In this study we describe a novel method for chimeric decoy formation in which blocks containing discrete transcription factor binding sites are combined into circular molecules. Unlike currently available methods, block decoys allow rapid construction of chimeric decoys targeting multiple regulatory elements. Further, they enable fine-tuning of binding-site copy ratios within chimeras, allowing sophisticated control of the cellular transcriptional landscape. We show that block decoys are exonuclease-resistant and specifically inhibit expression from target binding sites. The potential of block decoys to inhibit multiple elements simultaneously was demonstrated using a chimeric decoy containing molar optimized ratios of three regulatory elements, NF-κB-RE, CRE, and E-box. The chimeric decoy inhibited expression from all three elements simultaneously at equivalent levels. The primary intended use of block decoys is in vitro gene regulation studies in which bespoke chimeras can be rapidly constructed and utilized to determine a promoter's functional regulation.

NF-κB, CRE and YY1 elements are key functional regulators of CMV promoter-driven transient gene expression in CHO cells.

Transient gene expression (TGE) in CHO cells is utilized to produce material for use in early stage drug development. These systems typically utilize the cytomegalovirus (CMV) promoter to drive recombinant gene transcription. In this study, we have mechanistically dissected CMV-mediated TGE in CHO cells in order to identify the key regulators of this process. An in silico analysis of the promoter composition of transcription factor regulatory elements (TFREs) and the CHO cell repertoire of transcription factors identified eight TFREs as likely effectors of CMV activity. We determined the regulatory function of these elements by preventing their cognate transcription factors from binding at the CMV promoter. This was achieved by both scrambling promoter binding site sequences and using decoy molecules to sequester intracellular transcription factors. We determined that the vast majority of CMV activity is mediated by just two discrete TFREs, showing that simultaneous inhibition of NF-κB and CRE-mediated transactivation reduced CMV-driven transient secreted alkaline phosphatase (SEAP) production by over 75%. Further, we identified a mechanism by which CMV-mediated TGE is negatively regulated in CHO cells, showing that inhibition of YY1-mediated transrepression increased SEAP production 1.5-fold. This work enables optimization and control of CMV-mediated TGE in CHO cells, in order to improve transient protein production yields.