A Gene-Switch Platform Interfacing with Reactive Oxygen Species Enables Transcription Fine-Tuning by Soluble and Volatile Pharmacologics and Food Additives

Synthetic biology aims to engineer transgene switches for precise therapeutic protein control in cell-based gene therapies. However, off-the-shelf trigger-inducible gene circuits are usually switched on by single or structurally similar molecules. This study presents a mammalian gene-switch platform that controls therapeutic gene expression by a wide range of molecules generating low, non-toxic levels of reactive oxygen species (ROS). In this system, KEAP1 (Kelch-like ECH-associated protein 1) serves as ROS sensor, regulating the translocation of NRF2 (nuclear factor erythroid 2-related factor 2) to the nucleus, where NRF2 binds to antioxidant response elements (ARE) to activate the expression of a gene of interest. It is found that a promoter containing eight-tandem ARE repeats is highly sensitive to the low ROS levels generated by the soluble and volatile molecules, which include food preservatives, food additives, pharmaceuticals, and signal transduction inducers. In a proof-of-concept study, it is shown that many of these compounds can independently trigger microencapsulated engineered cells to produce sufficient insulin to restore normoglycemia in experimental type-1 diabetic mice. It is believed that this system greatly extends the variety of small-molecule inducers available to drive therapeutic gene switches. ROS-enhancing molecules regulate therapeutic protein expression in mammalian cells. Co-expression of KEAP1 and NRF2 forms a complex in low-ROS conditions. Introduction of ROS-enhancing molecules elevates ROS levels, dissociating the KEAP1/NRF2 complex. NRF2 translocates to the nucleus, activating transgene expression via antioxidant response element (ARE). Alginated-encapsulated engineered cells implanted intraperitoneally into mice control the therapeutic protein production in vivo. image