In vitro comparison of Aβ-targeting SNIPR, synNotch, and TRUCK for programmable transgene expression

Background Chimeric antigen receptors (CARs) are being used to direct cytokine secretion and phagocytosis by immune and glial cells toward amyloid β (Aβ) pathology, effectively adapting oncology-derived cell therapies to Alzheimer’s disease (AD). New synthetic receptor systems can take this approach a step further by enabling cells to deliver genetically encoded therapeutics. Among these systems, T cells Redirected for Universal Cytokine Killing (TRUCKs), synthetic Notch (synNotch), and Synthetic Intramembrane Proteolysis Receptors (SNIPRs) have shown promise in oncology and, as CARs, could potentially be adapted to AD. Methods Here, we compared these receptor systems in vitro using Jurkat cells and human regulatory T cells (Tregs). All receptors shared an Aβ-targeting binding domain derived from Aducanumab (Aduhelm) and were coupled to inducible expression cassettes driving identical transgenes: secreted Metridia luciferase (MetLuc) and a Lecanemab (Leqembi)-based chimeric human–mouse antibody (chLecanemab). To model AD pathology in vitro, cell clones were exposed to oligomer-enriched Aβ (AβO) preparations. In Jurkat cells, TRUCK, synNotch, and SNIPR systems were assessed for basal transcriptional leakage, ligand-independent activation, and ligand-dependent activation using flow cytometry and MetLuc reporter assays. Aβ-dependent production of chLecanemab by synNotch and SNIPR systems was further evaluated in Jurkat cells and human Tregs by immunofluorescence (IF). Results We found that the inducible expression cassettes of all receptor systems exhibited transcriptional leakage. Despite confirmed CAR function and transcriptional competence of 6xNFAT response elements paired with different minimal promoters, none of the TRUCK variants produced AβO-induced output significantly above basal leakage. In contrast, synNotch and SNIPR expressed in Jurkat cells and human Tregs responded robustly to AβO. Conclusions We demonstrate that Aβ-targeting SNIPR and synNotch systems can support Aβ-dependent inducible transgene expression in vitro. Their modular architectures enable straightforward substitution of chLecanemab with alternative therapeutic payloads while preserving Aβ-dependent delivery. Because these receptors are orthogonal, the same sequences we provide can be used across different model species and cell types in further studies without requiring receptor redesign.