Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors

Abstract

The Notch protein is one of the most mechanistically direct transmembrane receptors – the intracellular domain contains a transcriptional regulator that is released from the membrane when engagement of the cognate extracellular ligand induces intramembrane proteolysis. We find that chimeric forms of Notch, in which both the extracellular sensor module and the intracellular transcriptional module are replaced with heterologous protein domains, can serve as a general platform for generating novel cell-cell contact signaling pathways. Synthetic Notch (synNotch) pathways can drive user-defined functional responses in diverse mammalian cell types. Because individual synNotch pathways do not share common signaling intermediates, the pathways are functionally orthogonal. Thus multiple synNotch receptors can be used in the same cell to achieve combinatorial integration of environmental cues, including Boolean response programs, multi-cellular signaling cascades, and self-organized cellular patterns. SynNotch receptors provide extraordinary flexibility in engineering cells with customized sensing/response behaviors to user-specified extracellular cues.

Development of an optimized synthetic Notch receptor as an in vivo cell–cell contact sensor

“…1C), suggesting that cis-provided synNQ interacts with the GFP ligand, thus reducing availability of the ligand. Similar cis-inhibition between a synNQ and a ligand has also been reported in mammalian tissue culture cells (19).…”

“…S8B). Previously, in a mammalian tissue system, a similar synNQ detected cell interactions in cocultured primary neurons and leukemia cells; however, in that case, a considerably larger contact surface was formed between the cells (19).…”

“…1 A and B). More recently, similar synthetic Notch systems based on different artificial binding partners [i.e., mCD19, antimCD19 scFv (single-chain variable fragment)] were used to manipulate cell-cell communication in cultured mammalian cells, in the mouse immune system, and between fly neuron and glia cells (19)(20)(21).…”

Immune targets and neoantigens for cancer immunotherapy and precision medicine

“…In these split constructs, antigen-binding and intracellular signaling components are separated without a dimerization agent, and then assembled together only in the presence of a heterodimerization-promoting small molecule, thus allowing one to precisely control the timing, location, and dosage of T cell activity for controlling potential toxicity [196]. Furthermore, several new designs of the CAR technology based on a synthetic Notch receptor system have been reported to enable T cell activation through recognition of combinational antigens, and to allow engineering of T cells with customized therapeutic response programs [197][198][199]. These studies illustrate the potential of combining cellular engineering with synthetic biology tools to produce safer therapeutic CAR-T cells for cancer immunotherapy.…”

Self-organizing multicellular structures designed using synthetic biology

“…Toda et al used an engineered cell-communication system called synNotch 3 to mirror this biological set-up. SynNotch is adapted from Delta-Notch signalling -a signalling pathway found in nature, in which cells that have membrane-spanning Notch receptors sense Delta proteins on the surface of neighbouring cells.…”