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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I7 - Rationally designed tau-specific receptors for spatiotemporally regulated gene and cell therapies against neurodegeneration

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Dylan J. Conger (he/him/his)

Research Assistant
Brunger Lab, Department of Biomedical Engineering, Vanderbilt University
Scottsdale, United States

Co-Author(s)

Madeline R. Spetz (she/her/hers)

Graduate Student
Brunger lab, Department of Biomedical Engineering, Vanderbilt University
Nashville, Tennessee, United States
LD

Lauren E. Drake

Graduate Student
Lippmann lab, Department of Chemical and Biomolecular Engineering, Vanderbilt Universtity, United States
EL

Ethan S. Lippmann, PhD

Associate Professor, Department of Chemical and Biomolecular Engineering
Vanderbilt University
Nashville, Tennessee, United States

Primary Investigator(s)

JB

Jonathan Brunger, PhD

Assistant Professor, Department of Biomedical Engineering
Vanderbilt University
Franklin, Tennessee, United States

Introduction: Tauopathies are a broad continuum of over 26 neurodegenerative diseases, including Alzheimer’s disease (AD), frontotemporal dementia, and Huntington’s disease, which converge on the misfolding and aggregation of tau as an essential mechanism leading to neuron death and cognitive decline. Despite accounting for the vast majority of over 55 million dementia cases worldwide, tauopathies lack disease-modifying molecular therapies. Although tau pathology is spatially correlated with neuron death, monoclonal antibodies that neutralize or clear pathological tau have demonstrated lackluster results in clinical trials, motivating the need for more involved approaches to the treatment of neurodegeneration. Gene therapies are a powerful alternative to small molecule and immunotherapies; however, the expression of therapeutic genes is not restricted to the local extent of neurodegeneration, resulting in potential off-target effects. Prospective cell therapies (e.g. mesenchymal stem cells [MSCs]) possess intrinsic abilities pertinent to neurodegenerative disease, but nonetheless lack disease-relevant sense-and-response behaviors required for the amelioration of tauopathy specifically. The purpose of our research is to combine the specificity of anti-tau immunotherapies with the unique power of gene therapies and the innate agency of cell therapies to generate therapeutic cells with user-defined transcriptional responses to extracellular tau. We do this using the synthetic Notch (synNotch) intramembrane proteolysis receptor platform, which enables the customization of cell behaviors by swapping the extracellular and intracellular domains of the endogenous Notch mechanoreceptor with bespoke recognition domains and transcription factors, respectively. Here, we demonstrate that a panel of tau-sensitive synNotch receptors endow cells with flexible, user-defined transcriptional responses to extracellular tau protein.

Materials and

Methods: SynNotch is a juxtacrine mechanoreceptor, pulsing real-time transcriptional change in response to nearby immobilized or aggregated ligands. Cells were programmed using synNotch receptors constructed with single-chain variable fragment (scFv) recognition domains derived from clinical anti-tau monoclonal antibodies semorinemab, gosuranemab, and bepranemab. Plasmids encoding for anti-tau synNotch receptors and receptor-inducible transgene cassettes were assembled and sequence-confirmed before being packaged into lentivirus used to transduce murine MSCs (mMSCs), L929 mouse fibroblasts, and human iPSC-derived microglia (iMicroglia) (Fig 1A). iMicroglia were deemed ideal candidates for tau-gated behaviors because microglia affect neurodegeneration by sequestering protein aggregates, over-pruning synapses, and inducing other neuroinflammatory states. SynNotch expression was enriched via antibiotic selection. Cell surface receptor expression was evaluated using flow cytometry, probing for an N-terminal c-myc tag present on synNotch constructs. Engineered mMSCs and L929s were plated on passively adsorbed full-length recombinant human tau (rhTau) or cultured in medium containing suspended rhTau (200, 100, or 20 nM). Receptor activation by rhTau resulted in expression of either a SEAP+mCherry transgene cassette or a brain-derived neurotrophic factor (BDNF) transgene. BDNF production was assessed via ELISA, SEAP luminescence was assessed via a chemiluminescent plate reader assay, and mCherry expression was assessed via fluorescence microscopy. iMicroglia were differentiated from Alzheimer’s-neutral KOLF2.1J human iPSCs via forced transcription factor expression over eight days. iMicroglia were subsequently transduced with a Sem-Notch-Gal4 receptor and a receptor-inducible SEAP transgene before being plated in medium-suspended rhTau. SEAP was assessed as aforementioned. Statistics (unpaired t-test and one-way ANOVA with Tukey’s post-hoc) were performed in GraphPad Prism 10.

Results, Conclusions, and Discussions: When expressed in mMSCs, synNotch receptors recognized plate-immobilized rhTau, as indicated by rhTau-dependent SEAP and mCherry upregulation. The bepranemab-based synNotch receptor (Bep-Notch) enacted substantially less transgene production compared to its semorinemab- and gosuranemab-based counterparts (Fig 1B). L929 mouse fibroblasts expressing a Sem-Notch receptor detected rhTau suspended in their culture medium and responded with the expression of firefly luciferase and mCherry, even at the lowest nonzero concentration of 20 uM rhTau (Fig 1C). In mMSCs expressing a Sem-Notch receptor wired to a BDNF transgene, immobilized rhTau stimulated considerable production of BDNF (Fig 1D), a candidate therapeutic transgene shown to encourage neuronal viability and proliferation in disease. Importantly, rhTau treatment enhanced BDNF expression dramatically above baseline levels of mMSC BDNF production. SynNotch constructs described in Fig 1A-D enact transcriptional change by releasing a tTA intracellular domain when activated; receptors boasting an alternate Gal4-VP64 intracellular domain (Sem-Notch-Gal4; Fig 1E) enact comparable expression of SEAP and mCherry in mMSCs. iMicroglia engineered to express a Sem-Notch-Gal4 receptor alongside a receptor-inducible SEAP transgene detected 100 nM medium-suspended rhTau (Fig 1F).

Tau-sensitive receptors enable the programming of versatile cellular responses to extracellular tau. Owing to their different recognition domains, these receptors likely possess differential preferences for certain tau isoforms or posttranslational modifications on tau. Importantly, receptor-gated responses can have therapeutic functions, as demonstrated by BDNF expression and secretion by engineered mMSCs, or reporter functions, as indicated by luciferase, SEAP, or mCherry expression in engineered iMicroglia, mMSCs, and L929s. rhTau-dependent SEAP upregulation in iMicroglia is especially consequential for developing disease-modifying sense-and-response circuits in CNS cells, as microglia play an integral role in the pathogenesis of AD and other tauopathies.

We envision artificial tau receptors being used to outfit cell-based therapies with novel abilities to detect and respond to a hallmark of the neurodegenerative microenvironment. In addition to microglia, astrocytes may serve as useful agents of tau-gated therapeutic behaviors, as astrocytes monitor and aid in neuronal (especially synaptic) health. Moreover, when deployed in neurons, synthetic tau receptors may serve as useful tools for reporting the extent of local tau pathology in real-time when wired to fluorescent or luminescent reporters.

Acknowledgements (Optional): Special thanks to everyone in the Brunger lab for their continued support and encouragement. This work was additionally supported by the Vanderbilt Memory and Alzheimer's Center (VMAC) Pilot and Feasibility Funding Program (P20-AG068082), the NSF Graduate Research Fellowship Program (Grant No.1937963), and the SyBBURE Searle Undergraduate Research and Design Program. Images created using Microsoft PowerPoint, Adobe Illustrator, and BioRender.