Engineering of an enhanced synthetic Notch receptor by reducing ligand-independent activation

Abstract

Notch signaling is highly conserved in most animals and plays critical roles during neurogenesis as well as embryonic development. Synthetic Notch-based systems, modeled from Notch receptors, have been developed to sense and respond to a specific extracellular signal. Recent advancement of synNotch has shown promise for future use in cellular engineering to treat cancers. However, synNotch from Morsut et al. (2016) has a high level of ligandindependent activation, which limits its application. Here we show that adding an intracellular hydrophobic sequence (QHGQLWF, named as RAM7) present in native Notch, significantly reduced ligand-independent activation. Our enhanced synthetic Notch receptor (esNotch) demonstrates up to a 14.6-fold reduction in ligand-independent activation, without affecting its antigen-induced activation efficiency. Our work improves a previously reported transmembrane receptor and provides a powerful tool to develop better transmembrane signaling transduction modules for further advancement of eukaryotic synthetic biology.

Size-dependent protein segregation creates a spatial switch for Notch signaling and function

“…Low level NICD production was observed even for the Notch variants with a partial EGF truncation (NΔEGF 1-25 ), and levels gradually increased upon successive truncations to the NECD size. Our model also explains previous observations where synNotch with a relatively small ECD exhibited significant ligand-independent activation (10-50% of ligand-induced activation) (28).…”

“…Additionally, the substantial NICD production from the cells expressing NΔEGF indicates that, when localized together, γ -secretase can process Notch, bypassing S2 cleavage. Ligand-independent activation of Notch receptors with an intact S2 site was observed previously in Notch truncation variants or synNotch constructs (27)(28)(29), but the mechanism of this activation has been unclear so far. Our observations support the notion that colocalization of Notch with ߛ -secretase is sufficient to trigger S3 proteolysis and signaling.…”

Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing

“…Importantly, synNotch receptors also contain a cytosolic domain that is cleaved upon receptor stimulation. This γ-secretase-dependent cleavage can cause ligand-independent activation [3,6,7] of the SynNotch receptor, but enhanced synthetic notch receptors (esNotch) are reported to significantly reduce the ligand-independent activation [8]. This intracellular domain can be substituted with various proteins that interact with compounds of the cytoplasm or the nucleus [3].…”

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…These include hypoxia-inducible expression systems, 168 as well as synthetic signaling circuits that take advantage of Boolean logic. [169][170][171] One of the best examples of Boolean logicgated CARs is the synthetic notch (synNotch) system, [169][170][171] in which an antigen-specific synNotch receptor induces the expression of a second CAR upon activation. For example, EpCAM-or B7H3-specific synNotch receptors have been used to restrict ROR1-CAR expression to tumor sites, reducing on target/off cancer toxicity in preclinical models.…”