Biology subjects

Lim, W. A.

Publications and source records attributed to Lim, W. A..

13 recordsLinked to original sources

bioRxiv2026

Building synthetic cell-cell synapses using engineered scaffold protein condensates

Synaptic assemblies that form at cell-cell junctions are often organized by multivalent scaffold proteins with condensate forming properties. An emerging hypothesis is that these scaffolds provide a thresholding mechanism whereby small increases in the density of cell-cell recognition receptors can trigger all-or-none assembly selectively at the correct partner cell interface. We test this hypothesis by engineering synthetic scaffolded synapses. Coarse-grained simulations suggest that properly-tuned scaffold interactions can yield a sharp condensation threshold. Guided by these simulations, we designed modular scaffold proteins consisting of PDZ domains and intrinsically disordered regions (IDRs) of different molecular grammars. Within a specific region of parameter space, controlled by valency, receptor binding affinity and IDR-driven condensation, the synthetic scaffold proteins exhibit switch-like condensation behaviors, where co-condensates of receptor and scaffold selectively form only at interfaces with the proper partner cell. Notably, orthogonal receptor-scaffold pairs enable a single receiver cell to form multiple distinct interfaces, each associated with a different partner cell. We demonstrate that these synthetic synapse assemblies can recruit different client proteins to enhance intercellular signaling, orient cell division, or sculpt complex synaptic morphologies. Our results illustrate how the ultrasensitive condensation properties of modular scaffold proteins can be harnessed to create novel and highly specific cell-cell interfaces.

synthetic biology↗

bioRxiv2026

Programming Targeted Immune Cell Migration Using Orthogonal Chemokine-Receptor Pairs

Chemokines orchestrate the migration of immune cells throughout the body, but harnessing chemokine signaling to engineer targeted cell trafficking is challenging because of their overlapping interactions and broad expression patterns. Here we used structure-guided protein design to engineer orthogonal chemokine-receptor pairs with minimal crosstalk to native systems. T cells engineered with an ortho-receptor selectively infiltrated tissues producing the ortho-chemokine, in vitro and in vivo. By engineering T cells that both sense and secrete the ortho-chemokine, we generated collective swarming behavior in which pioneer cells attract follower cells via an ortho-chemokine gradient self-organized by positive feedback. By providing orthogonal genetic control over both the generation and sensing of chemokine gradients, this system enables the construction of autonomous spatial targeting programs in engineered cells.

synthetic biology↗

bioRxiv2026

Coupling Mechanical Regulation with Biochemical Reaction-Diffusion Circuits Yields Robust Self-Organized Pattern Formation.

Cell-cell signaling circuits that combine local self-activation with long-range inhibition have long been proposed as a theoretical mechanism sufficient to generate cellular patterns, such as spots and stripes. Here we construct synthetic pattern-forming circuits, implementing local self-activation (positive feedback) using juxtacrine synNotch receptor interactions, and implementing long-range inhibition using diffusible competitor molecules. While combining local positive feedback with long-range inhibition leads to more spatially heterogeneous cell states, these synthetic circuits do not robustly lead to well defined patterns. We find, however, that if we couple these reaction-diffusion circuits with induction of genes that regulate cell mechanics - such Cadherin molecules that promote local cell adhesion and sorting - we observed the emergence of much more well-defined cellular patterns. Theoretical analysis indicates while reaction-diffusion circuits can be sufficient to generate patterns under precisely balanced parameter conditions, the close coupling of cell mechanical/sorting significantly increases the robustness of pattern formation (the parameter space yielding patterns). Thus, circuits that closely integrate signaling and mechanical changes may underlie many evolved morphogenic pattern formation systems.

synthetic biology↗

bioRxiv2026

Spike-in probe-enhanced single-cell RNA-seq reveals post-infusion transcriptomic remodeling of "prime-and-kill" synNotch-CAR-T cells

We previously developed synthetic Notch (synNotch)-chimeric antigen receptor (CAR)-T cells to improve the safety and efficacy of CAR-T therapy for glioblastoma. In this system, an anti-EphA2/IL13R2-dual-CAR is expressed only upon recognition of tumor- or brain-specific "priming" antigens, EGFRvIII (termed E-SYNC cells) or brevican (B-SYNC), respectively, with E-SYNC currently under phase I clinical evaluation (NCT06186401). However, tracking and profiling these engineered cells in vivo remain challenging, limiting our understanding of their activity and therapeutic potential. To address this gap, we developed a single-cell RNA-sequencing (scRNA-seq) workflow with custom spike-in probes for synNotch-CAR transcripts, enabling simultaneous detection of engineered cells and transcriptomic profiling. In vitro, integration of multiple probes using machine-learning-assisted classifiers detected 78.2% of E-SYNC cells and 60.0% of B-SYNC cells with 98.0% specificity. In a xenograft model, synNotch-positive cells were detected across the spleen, lung, and brain, with the highest frequency and most robust priming and activation observed in the brain. Single-cell transcriptomic analyses revealed tissue-specific differentiation programs, including cytotoxicity, proliferation, metabolic activity, and acquisition of tissue-resident memory phenotypes, shaped by both environmental cues and synNotch-mediated antigen recognition. In summary, this spike-in probe-enhanced scRNA-seq workflow enables robust detection and high-resolution characterization of synNotch-CAR-T cell dynamics and provides a broadly applicable platform for monitoring engineered immune cells in diverse clinical contexts. One Sentence SummaryOur spike-in probe-enhanced single-cell RNA-sequencing method enables analysis of tissue-dependent activation and transcriptional states of synNotch-CAR-T cells, providing a robust and scalable platform for in vivo tracking and transcriptomic profiling of engineered cell therapies.

cancer biology↗

bioRxiv2025

Coupling differential adhesion to cell signaling avoids kinetic traps to yield robust multicellular self-organization

Differential adhesion, where cells physically reorganize based on their heterogeneous adhesion preferences, is one of the major models for self-organization in development and tissue formation. However, accumulating evidence suggests that differential adhesion is many times insufficient for robust convergence to a target minimal energy multicellular structure. Here we use computational simulations and engineered synthetic cell circuits to systematically explore alternative mechanisms for programming formation of a simple two-cell type core-shell morphology. Starting with two pre-differentiated cell types with constitutively high differential adhesion leads to kinetic trapping in variable, multi-core structures. In contrast, hybrid mechanisms that gradually induce differential adhesion upon cell-cell contact signaling consistently converge to the target single-core structure, in a manner robust to variation in cell numbers, interaction energy, and noise. This work delineates intrinsic limitations of self-organizing systems based solely on differential adhesion, and shows how inducible systems provide a way to invoke the strong adhesion required to maintain a multicellular structure, while avoiding the pitfall of kinetic traps. This study illustrates how joint computational and experimental exploration of synthetic circuits can be used to probe key developmental principles and tradeoffs and inform the design of synthetic development and self-organization.

biophysics↗

bioRxiv2025

Engineering Inducible Cell Fate Transitions by Harnessing Epigenetic Silencing

During development, cell-cell communication induces state transitions that are maintained by epigenetic regulation. Here, we harness endogenous epigenetic silencing machinery to engineer synthetic circuits that induce stable gene expression changes. Using synthetic Notch receptors that control the chromatin regulators KRAB and Dnmt3L, we developed input-sensing switches that induce self-sustained silencing of target loci. We used these modules to construct circuits in which combinatorial inputs direct a choice among multiple alternative states. These epigenetic switches can also be inverted to yield sustained activation of target genes. We demonstrate that these epigenetic switches can be used to drive morphological changes triggered by transient cell-cell interactions, but that remain stable over many cell divisions, as is observed in development. Additionally, these circuits can be used to drive the reprogramming of fibroblasts to a neuron-like state. These synthetic epigenetic circuits represent an important step towards engineering cell populations capable of coordinated multi-cell state decisions.

synthetic biology↗

bioRxiv2025

Programmable microparticles rewire CAR signaling to enable super-physiological expansion of human T cells in vitro

T cell proliferative capacity and persistence critically determine the therapeutic success of chimeric antigen receptor (CAR) T cells. However, it remains unknown if and how human CAR-T cells can be externally programmed to reach maximal proliferative capacity. Here, we use programmable PLGA microparticles functionalized with CAR-antigens and CD28-costimulatory antibodies (CAREp) to repeatedly stimulate human CD8+ CAR-T cells in vitro. CAREp-stimulated CAR-T cells expanded continuously for over 100 days--versus [~]30 days with tumor cell stimulation--and achieved up to 1018-fold cumulative expansion, greatly surpassing CD3/28-Dynabeads. Early-phase transcriptomic responses-- upregulation of DNA repair, cell cycle, telomere maintenance, and mitochondrial pathways--aligned with long-term outcomes: massive proliferation, telomere stability, robust respiration, and preserved progenitor phenotype by single-cell sequencing. Differentiation and exhaustion signals were broadly suppressed. Transient telomerase activity further supported physiologic expansion. These findings demonstrate that nanoscale-controlled extracellular cues can rewire intracellular signaling to drive durable, super-physiological expansion of functional CAR-T cells.

immunology↗

bioRxiv2025

Engineering lung-sensing T cells using synthetic receptors targeting RAGE

Our goal was to leverage synthetic biology approaches to engineer lung-sensing T cells that trigger synthetic transcriptional programs only when in the lung. First, we identified a lung-specific cell surface protein, receptor for advanced glycation endproducts (RAGE), expressed at high levels exclusively in the lung. Then, we engineered chimeric antigen receptors (CAR) and synthetic notch receptors (SynNotch) to bind this protein. We showed that anti-RAGE CAR-T cells traffic to and proliferate in the lung exclusively. Anti-RAGE SynNotch receptors activate transcription of a fluorescent reporter only when co-cultured with RAGE+ cell lines or primary lung cells. Finally, we tested an anti-RAGE SynNotch to anti-CD19 CAR circuit in vivo in mice implanted with lung and flank tumors and found that this approach cleared CD19-expressing lung tumors without affecting CD19-expressing flank tumors. Thus, we demonstrate that RAGE is a lung-specific target and that T cells expressing anti-RAGE receptors can sense and activate lung-specific therapeutic transcriptional programs. This approach could be extended to allow cell-based therapies that target and deliver genetically encoded payloads to treat lung diseases while avoiding systemic toxicity.

synthetic biology↗

bioRxiv2024

Degron-based bioPROTACs for controlling signaling in CAR T cells

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in pre-clinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous, cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit is able to disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are a powerful tool for expanding the cell engineering toolbox for CAR T cells.

synthetic biology↗

bioRxiv2022

Exploring the rules of chimeric antigen receptor phenotypic output using combinatorial signaling motif libraries and machine learning

Chimeric antigen receptor (CAR) costimulatory domains steer the phenotypic output of therapeutic T cells. In most cases these domains are derived from native immune receptors, composed of signaling motif combinations selected by evolution. To explore if non-natural combinations of signaling motifs could drive novel cell fates of interest, we constructed a library of CARs containing [~]2,300 synthetic costimulatory domains, built from combinations of 13 peptide signaling motifs. The library produced CARs driving diverse fate outputs, which were sensitive to motif combinations and configurations. Neural networks trained to decode the combinatorial grammar of CAR signaling motifs allowed extraction of key design rules. For example, the non-native combination of TRAF- and PLC{gamma}1-binding motifs was found to simultaneously enhance cytotoxicity and stemness, a clinically desirable phenotype associated with effective and durable tumor killing. The neural network accurately predicts that addition of PLC{gamma}1-binding motifs improves this phenotype when combined with TRAF-binding motifs, but not when combined with other immune signaling motifs (e.g. PI3K-or Grb2-binding motifs). This work shows how libraries built from the minimal building blocks of signaling, combined with machine learning, can efficiently guide engineering of receptors with desired phenotypes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/474985v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@8dbf97org.highwire.dtl.DTLVardef@1d8300org.highwire.dtl.DTLVardef@1838ba0org.highwire.dtl.DTLVardef@143d907_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Multi-antigen recognition circuits overcome challenges of specificity, heterogeneity, and durability in T cell therapy for glioblastoma

Treatment of solid cancers with chimeric antigen receptor (CAR) T cells is plagued by the lack of target antigens that are both tumor-specific and homogeneously expressed. We show that multiantigen prime-and-kill recognition circuits have the flexibility and precision to overcome these challenges in attacking glioblastoma. A synNotch receptor that recognizes a specific priming antigen - the heterogeneous glioblastoma neoantigen EGFRvIII or a brain tissue-specific antigen - is used to locally induce expression of a CAR, enabling thorough but controlled tumor killing by targeting of homogeneous antigens that are not absolutely tumor specific. Moreover, regulated CAR expression maintains a higher fraction of the T cells in the naive-like state which is associated with higher durability in vivo. In summary, using circuits that integrate recognition of multiple imperfect but complementary antigens, we improve the specificity and persistence of T cells directed against glioblastoma, providing a general recognition strategy applicable to other solid tumors.

immunology↗

bioRxiv2019

DNA-scaffolded biomaterials enable modular and tunable control of cell-based cancer immunotherapies

Advanced biomaterials provide versatile ways to spatially and temporally control immune cell activity, potentially enhancing their therapeutic potency and safety. Precise cell modulation demands multi-modal display of functional proteins with controlled densities on biomaterials. Here, we develop an artificial immune cell engager (AICE) platform - biodegradable particles onto which multiple proteins are densely loaded with ratiometric control via short nucleic acid tethers. We demonstrate the impact of AICE with varying ratios of anti-CD3 and anti-CD28 antibodies on ex vivo expansion of human primary T cells. We also show that AICE can be used to control the activity of engineered T cells in vivo. AICE injected intratumorally can provide a local priming signal for systemically administered AND-gate chimeric antigen receptor T cells, driving local tumor clearance while sparing uninjected tumors that model potentially cross-reactive healthy tissues. This modularly functionalized biomaterial thus provides a flexible platform to achieve sophisticated control over cell-based immunotherapies.

bioengineering↗

bioRxiv2017

The design principles of biochemical timers: circuits that discriminate between transient and sustained stimulation

Many cellular responses for which timing is critical display temporal filtering - the ability to suppress response until stimulated for longer than a given minimal time. Temporal filtering can play a key role in filtering noise, choreographing the timing of events, and mediating the interpretation of dynamically encoded signals. To define the biochemical circuits capable of kinetic filtering, we comprehensively searched the space of three-node networks. We define a metric of \"temporal ultrasensitivity\", a measure of the steepness of activation as a function of stimulus duration. We identified five classes of core network motifs capable of temporal filtering, each with different functional properties such as rejecting high frequency noise, committing to response (bistability), and distinguishing between long stimuli. Combinations of the two most robust motifs, double inhibition (DI) and positive feedback with AND logic (PFAND), underlie several natural timer circuits involved in processes such as cell cycle transitions, T cell activation, and departure from the pluripotent state. The biochemical network motifs described in this study form a basis for understanding the common ways in which cells make dynamic decisions.

systems biology↗