Design of a synthesis-friendly hypoxia-responsive promoter for cell-based therapeutics

Towards the goal of making 'smart' cell therapies, one that recognizes disease conditions (e.g. hypoxia) and then produces mitigating biologics, it is important to develop suitable promoters. Currently, hypoxia responsive promoters are composed of strongly repeated sequences containing hypoxia response elements upstream of a minimal core promoter. Unfortunately, such repeated sequences have inherent genomic instability that may compromise the long-term consistency of cell-based therapeutics. Thus, we designed a synthesis-friendly hypoxia-inducible promoter (named SFHp) that has GC content between 25% and 75% and no repeats greater than 9 base pairs. In HEK293 cells stably integrated with genes regulated by synthetic SFHp, we demonstrated inducible reporter expression with fluorescent proteins, cell morphology rewiring with our previously engineered RhoA protein and intercellular cell signalling with secreted cytokines. These experiments exemplify the potential usage of SFHp in cell-based therapeutics with integrated genetic circuits that inducibly respond to the disease microenvironment.