Biology subjects

Ng, A. H.

Publications and source records attributed to Ng, A. H..

9 recordsLinked to original sources

bioRxiv2026

A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

Cytokines mediate cell-cell communication to coordinate immune responses and hold clinical promise as immunotherapies. While natural cytokines are exquisitely regulated by immune cells, engineered cytokines have not yet matched this sophisticated regulation, limiting clinical efficacy and safety. IL-2 is an important cytokine involved in antitumor response, but its production is disrupted in the immune-suppressive tumor microenvironment. Here we designed a regulated IL-2 module, OUTSMART designed IL-2 (dIL-2), that functions robustly in solid tumors and reduces systemic activity that could trigger toxicity: we computationally redesigned IL-2 to have a novel topology that increases stability, preserves native IL-2R{beta}{gamma} interfaces, ablates IL-2R binding, and delivers IL-2 signaling to CD8+ immune-effector cells. This designed cytokine is genetically regulated by a T cell activation-responsive promoter, allowing dynamic production from chimeric antigen receptor (CAR) T cells. Like wild type IL-2, OUTSMART dIL-2 drives T cell proliferation and enhances effector function. Unlike wild type IL-2, it preferentially stimulates CD8+ T cells and Natural Killer (NK) cells, avoids T regulatory cells, and stimulates CAR-T proliferation while preserving stemness, achieving durable tumor elimination in two solid tumor animal models. These design principles can be applied to create other dynamically-regulated cytokine systems that address mechanisms of complex disease.

synthetic biology↗

bioRxiv2024

Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show "leaky" Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be distinctly problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we established transgenic platforms that minimize Cas9 functionality in the off-state along with maximized and uncompromised on-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (on vs. off-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we created a branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.

molecular 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↗

bioRxiv2020

Accurate prediction of genetic circuit behavior requires multidimensional characterization of parts

Mathematical models can aid the design of genetic circuits, but may yield inaccurate results if individual parts are not modeled at the appropriate resolution. To illustrate the importance of this concept, we study transcriptional cascades consisting of two inducible synthetic transcription factors connected in series. Despite the simplicity of this design, we find that accurate prediction of circuit behavior requires mapping the dose responses of each circuit component along the dimensions of both its expression level and its inducer concentration. With such multidimensional characterizations, we were able to computationally explore the behavior of 16 different circuit designs. We experimentally verified a subset of these predictions and found substantial agreement. This method of biological part characterization enables the use of models to identify (un)desired circuit behaviors prior to experimental implementation, thus shortening the design-build-test cycle for more complex circuits.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsiSynTFinducible synthetic transcription factorYFPyellow fluorescent proteinGEMGal4 DNA binding domain, estradiol ligand binding domain, Msn2 activating domainZ3PMZ3 DNA binding domain, progesterone ligand binding domain, Msn2 activating domainZ4EMZ4 DNA binding domain, estradiol ligand binding domain, Msn2 activating domainView Full Text

synthetic biology↗

bioRxiv2019

MATE-Seq: Microfluidic Antigen-TCR Engagement Sequencing

Adaptive immunity is based on peptide antigen recognition. Our ability to harness the immune system for therapeutic gain relies on the discovery of the T cell receptor (TCR) genes that selectively target antigens from infections, mutated proteins, and foreign agents. Here we present a method that selectively labels peptide antigen-specific CD8+ T-cells in human blood using magnetic nanoparticles functionalized with peptide-MHC tetramers, isolates these specific cells within an integrated microfluidic device, and directly amplifies the TCR genes for sequencing. Critically, the identity of the peptide recognized by the TCR is preserved, providing the link between peptide and gene. The platform requires inputs on the order of just 100,000 CD8+ T cells, can be multiplexed for simultaneous analysis of multiple peptides, and performs sorting and isolation on chip. We demonstrate 1000-fold sensitivity enhancement of antigen-specific T-cell receptor detection and simultaneous capture of two virus antigen-specific T-cell receptors from samples of human blood.

immunology↗

bioRxiv2019

Optogenetic control reveals differential promoter interpretation of transcription factor nuclear translocation dynamics

The dynamic translocation of transcription factors (TFs) in and out of the nucleus is thought to encode information, such as the identity of a stimulus. A corollary is the idea that gene promoters can decode different dynamic TF translocation patterns. Testing this TF encoding/promoter decoding hypothesis requires tools that allow direct control of TF dynamics without the pleiotropic effects associated with general perturbations. In this work, we present CLASP (Controllable Light Activated Shuttling and Plasma membrane sequestration), a tool that enables precise, modular, and reversible control of TF localization using a combination of two optimized LOV2 optogenetic constructs. The first sequesters the cargo in the dark at the plasma membrane and releases it upon exposure to blue light, while light exposure of the second reveals a nuclear localization sequence that shuttles the released cargo to the nucleus. CLASP achieves minute-level resolution, reversible translocation of many TF cargos, large dynamic range, and tunable target gene expression. Using CLASP, we investigate the relationship between Crz1, a naturally pulsatile TF, and its cognate promoters. We establish that some Crz1 target genes respond more efficiently to pulsatile TF inputs than to continuous inputs, while others exhibit the opposite behavior. We show using computational modeling that efficient gene expression in response to short pulsing requires fast promoter activation and slow inactivation and that the opposite phenotype can ensue from a multi-stage promoter activation, where a transition in the first stage is thresholded. These data directly demonstrate differential interpretation of TF pulsing dynamics by different genes, and provide plausible models that can achieve these phenotypes.

systems biology↗

bioRxiv2018

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

synthetic biology↗

bioRxiv2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

synthetic biology↗

bioRxiv2016

The Effect of Compositional Context on Synthetic Gene Net-works

It is well known that synthetic gene expression is highly sensitive to how comprising genetic elements (promoter structure, spacing regions between promoter and coding sequences, ribosome binding sites, etc.) are spatially configured. An important topic that has received far less attention is how the physical layout of entire genes within a synthetic gene network affects their individual expression levels. In this paper we show, both quantitatively and qualitatively, that compositional context can significantly alter expression levels in synthetic gene networks. We also show that these compositional context effects are pervasive both at the transcriptional and translational level. Further, we demonstrate that key characteristics of gene induction, such as ultra-sensitivity and dynamic range, are heavily dependent on compositional context. We postulate that supercoiling can be used to explain these interference effects and validate this hypothesis through modeling and a series of in vitro supercoiling relaxation experiments. On the whole, these results suggest that compositional context introduces feedback in synthetic gene networks. As an illustrative example, we show that a design strategy incorporating compositional context effects can improve threshold detection and memory properties of the toggle switch.

synthetic biology↗