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Background <p>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.</p> Methods <p>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.</p> Results <p>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.</p> Conclusions <p>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.</p> Graphical Abstract <p></p>

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Detection of extracellular amyloid beta aggregates by an Aducanumab-based synNotch receptor: an in vitro proof-of-concept study

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月10日

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  • Nicholas J. Bergo,
  • Suckwon Lee,
  • Cynthia J. Siebrand,
  • Zachary Mayeri,
  • Julie K. Andersen,
  • Chaska C. Walton

摘要

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.

Graphical Abstract