Abstract
Background:
Synthetic Notch (synNotch) receptors are a powerful gene regulation platform that activate transcription in response to membrane-bound ligands and extracellular matrix components, with emerging applications in cancer, autoimmunity, and regenerative medicine. Whether synNotch can be adapted to detect and respond to extracellular neurotoxic protein aggregates-such as amyloid beta (Aβ), a hallmark of Alzheimer's disease (AD)-remains unknown.
Methods:
To address this, we engineered an Aβ-responsive synNotch receptor (Adu-synNotch) by fusing the single-chain variable fragment (scFv) derived from Aducanumab (Aduhelm®), an FDA-approved anti-Aβ antibody, to the extracellular domain of synNotch. This construct was expressed in NIH 3T3 cells and paired with downstream reporters CLIP-tag, secreted Metridia luciferase (MetLuc), and synthetic expression cassettes encoding chimeric human-mouse versions of the therapeutic antibodies Lecanemab (Leqembi®) and Aducanumab. Cells were exposed to Aβ(1-42) aggregates, and synNotch activation was assessed via CLIP-tag imaging, MetLuc secretion assays, and immunocytochemistry for Lecanemab and Aducanumab.
Results:
NIH 3T3 cells expressing Adu-synNotch responded to extracellular Aβ aggregates with robust induction of CLIP-tag and MetLuc reporters, along with secretion of chimeric Lecanemab and Aducanumab antibodies. Activation was dose-dependent and the secreted antibodies bound to Aβ aggregates, confirming that extracellular Aβ can serve as a functional synNotch ligand.
Conclusions:
These findings establish that synNotch receptors can be engineered to detect and respond to pathological extracellular protein aggregates such as Aβ. This expands the scope of synNotch-based systems and supports their potential for developing precision cell-based therapies for neurodegenerative diseases like AD.