Biology subjects

Tran, J. C.

Publications and source records attributed to Tran, J. C..

3 recordsLinked to original sources

bioRxiv2026

ProNotch converts extracellular protease activity into programmable transcriptional outputs

Synthetic receptors that convert extracellular protease activity into programmable transcriptional outputs would expand the toolkit of mammalian cell biology and cell engineering, yet modular platforms for directly coupling extracellular proteolysis to gene expression remain limited. Here we introduce ProNotch, a receptor architecture that harnesses protease-gated derepression of a mutant Notch1 negative regulatory region (NRR) to drive user-defined gene expression. ProNotch tethers destabilized NRR mutants to inhibitory anti-NRR single-chain variable fragments (scFvs) via protease-cleavable linkers. NRR engagement by high-affinity scFvs simultaneously rescues surface trafficking of mutant receptors and suppresses basal signaling until linker cleavage releases the inhibitory scFv module, permitting the destabilized NRR to initiate ligand-independent signaling. Linker substitution reprogrammed protease specificity across diverse enzymes, and tandem substrate repeats enhanced sensitivity without increasing basal activity. Single-chain receptor designs enabled OR and AND logic gates, allowing integration of multi-protease inputs into a single transcriptional output. ProNotch detected endogenous MMP-14 activity from cancer cell lines in cis and in trans and drove protease-dependent cell-state transitions in C3H/10T1/2 fibroblasts. The scFv-NRR module also functioned as a protease-activated pro-antibody that conditionally inhibited DLL4-dependent signaling and ligand-independent activation of mutant NOTCH1 in the T cell acute lymphoblastic leukemia cell line HPB-ALL. Together, these results establish ProNotch as a modular platform for engineering protease-responsive cells and demonstrate that its regulatory module can be extended to a soluble, conditionally activated inhibitor of NOTCH1 signaling.

synthetic biology↗

bioRxiv2024

Therapeutically targeting the classical complement pathway with antisense oligonucleotides in Alzheimer's disease

The complement classical pathway (CP) is a key mediator of synapse loss and neurodegeneration in mouse models of Alzheimers (AD) and other neurodegenerative diseases. We analyzed human brain proteomics and found consistent elevations of all CP proteins, but not other complement pathways, in AD patient brains. We performed human genetics analysis that identified a rare variant in the C1S gene within the Finnish population that is associated with AD and we found that a common AD-associated C1S variant correlates with increased C1S protein levels. A targeted assay detected elevated C1S activation in AD patient CSF. Given this specific implication of the CP in AD, we next evaluated the therapeutic approach of targeting the CP in the brain using antisense oligonucleotides (ASOs). To identify promising CP targets for knockdown using ASOs we first tested for rescue of synapse loss in an AD mouse model using heterozygous and homozygous complement knockout mice and examined the relative brain expression levels of different CP genes. Based on these experiments we prioritized C1r, C1s and C4 as promising targets for therapeutic knockdown using ASOs. We then screened for ASOs for each target, evaluating in vitro and in vivo knockdown and toxicity, and identified optimal ASOs targeting C1r, C1s and C4. Experiments with AD model mice demonstrated significant rescue of synapse loss following treatment with C1r, C1s or C4 ASOs. Overall, our findings provide proof of concept for using nucleic acid-based medicine to target the CP in AD and demonstrate the translational potential of this approach.

neuroscience↗

bioRxiv2024

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o-nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

synthetic biology↗