Biology subjects

Gao, X. J.

Publications and source records attributed to Gao, X. J..

14 recordsLinked to original sources

bioRxiv2026

Targeted cancer ablation via programmable sensing of amplified oncogenic RNA

Selectively eliminating cancer cells based on their oncogenic drivers holds great promise but remains challenging for targeted cancer therapy. Here, we present ORCA (Oncogenic RNA-triggered Cancer Ablation), a programmable and modular platform that couples RADAR-based detection of oncogenic RNA with conditional expression of therapeutic payloads in cancer cells. We identify RADAR sensors targeting the transcripts of endogenously amplified oncogenes, including MYC, FGFR2 and wild-type KRAS in colorectal and gastric cancer cells. Focusing on colorectal cancer (CRC) driven by extrachromosomal DNA-amplified MYC, we demonstrate targeted ablation of high-MYC cancer cells using two orthogonal strategies: anti-CD19 CAR-T therapy and enzyme-prodrug therapy. In vivo delivery of ORCA using adeno-associated virus (AAV) suppresses tumor growth in a subcutaneous xenograft model and prolongs survival in a CRC liver metastasis model. ORCA establishes oncogenic RNAs as actionable inputs for targeted cancer therapy via programmable RNA sensing, thus offering a strategy to target tumors through the transcripts of otherwise difficult-to-drug oncogenes such as MYC.

synthetic biology↗

bioRxiv2025

Efficient generation of epitope-targeted de novo antibodies with Germinal

Obtaining novel antibodies against specific protein targets is a widely important yet experimentally laborious process. Meanwhile, computational methods for antibody design have been limited by low success rates that currently require resource-intensive screening. Here, we introduce Germinal, a broadly enabling generative pipeline that designs antibodies against specific epitopes with nanomolar binding affinities while requiring only low-n experimental testing. Our method co-optimizes antibody structure and sequence by integrating a structure predictor with an antibody-specific protein language model to perform de novo design of functional complementarity-determining regions (CDRs) onto a user-specified structural framework. When tested against four diverse protein targets, Germinal successfully designed functional antibodies across all targets and binder formats, testing only 43-101 designs for each antigen. Validated designs also exhibited robust expression in mammalian cells and high sequence and structural novelty. We provide open-source code and full computational and experimental protocols to facilitate wide adoption. Germinal represents a milestone in efficient, epitope-targeted de novo antibody design, with notable implications for the development of molecular tools and therapeutics.

synthetic biology↗

bioRxiv2025

Sequence-Independent RNA Sensing in Living Mammalian Cells

Recently, several groups described sensors in living cells that take advantage of adenosine deaminases acting on RNA (ADARs) to link the presence of an RNA (a "target transcript") to the translation of a payload from a second, exogenously introduced mRNA. These sensors share the key mechanism of editing a stop codon opposite a specific sequence motif in the target transcript, where this motif requirement is dictated by ADARs strong sequence preference. This constrains sensor design and precludes the sensing of short sequences that lack such motifs, often essential for key applications such as sensing viral RNAs and differentiating splice isoforms. Here we address this limitation with modular RNA sensors using adenosine deaminases acting on RNA ("modulADAR"). ModulADAR features two key elements that mirror the modularity of ADARs: regions that hybridize with the target transcript to recruit ADARs dsRNA-binding domains, and a stem-loop for stop-codon editing by ADARs catalytic domain. We optimize modulADAR and apply it to detect short subsequences that cannot be sensed by prior-generation sensors. We anticipate that modulADAR will empower broader basic science and therapeutic applications, especially those that will uniquely benefit from programmable RNA detection in living cells.

synthetic biology↗

bioRxiv2025

An in planta single-cell screen to accelerate functional genetics

Genetic screens in whole plants are a powerful tool for functional genetics. However, elucidating gene function in highly redundant genetic programs such as signaling pathways remains challenging in both model and non-model plants. Here, we report a single-cell screening platform, PIVOT (Protoplast Isolation after Virus Overexpression in planTa), to accelerate identification and functional characterization of plant genes. We used Nicotiana benthamiana as a heterologous host to test gene libraries arrayed in a single leaf. Two elements of our system made pooled screens possible in planta: (1) we harnessed viral superinfection exclusion to ensure single multiplicity of infection per cell during pooled library delivery, and (2) we engineered a cell surface protein as a phenotypic marker for isolating cells of interest from a heterogeneous population. Using this system, we recovered known and new regulators of cytokinin signaling from an Arabidopsis open reading frame library. We anticipate PIVOT will be broadly applicable for high-throughput, single-cell functional genetic screening across the plant kingdom.

plant biology↗

bioRxiv2025

ProVADA: Generation of Subcellular Protein Variants via Ensemble-Guided Test-Time Steering

Engineering protein variants to function in exogenous environments remains a significant challenge due to the complexity of sequence and fitness landscapes. Experimental strategies often require extensive labor and domain expertise. While recent advances in protein generative modeling offer a promising in silico alternative, many of these methods rely on differentiable fitness predictors, which limits their applicability. To this end, we introduce Protein Variant ADAptation (ProVADA), an ensemble-guided, test-time steering framework that combines implicit generative priors with fitness oracles via a composite functional objective. ProVADA leverages Mixture-Adaptation Directed Annealing (MADA), a novel sampler integrating population-annealing, adaptive mixture proposals, and directed local mutations. Furthermore, ProVADA requires no gradients or explicit likelihoods, yet efficiently concentrates sampling on high-fitness, low-divergence variants. We demonstrate its effectiveness by in silico redesigning human renin for cytosolic functionality. Our results achieve significant gains in predicted localization fitness while preserving structural integrity.

bioengineering↗

bioRxiv2025

Engineered protein circuits for cancer therapy

A central challenge in cancer therapy is selectively killing cancer cells while minimizing resistance. Here, we engineer modular protease-based protein circuits that sense mutant RAS, the most frequently mutated oncogene in cancer, and conditionally activate cell death. Delivered transiently as mRNA in lipid nanoparticles (LNPs), circuits selectively eliminated RAS-mutant cancer cells in culture and suppressed aggressive, multifocal RAS-driven liver tumors. Compared to RAS inhibitors, circuits killed cancer cells independently of oncogene addiction, achieved potent cytotoxicity rather than cytostasis, and functioned at low RAS occupancy through catalysis rather than stoichiometric inhibition. Critically, circuits acquired minimal to no resistance under prolonged selection, while remaining potent against prevalent drug-resistance mechanisms including RAS amplification and bypass signaling. Together, these results establish design principles for engineering therapeutic protein circuits and highlight their potential to overcome longstanding limitations of existing therapeutic modalities.

synthetic biology↗

bioRxiv2025

Machine-Guided Dual-Objective Protein Engineering for Deimmunization and Therapeutic Functions

Cell and gene therapies often rely on the expression of exogenous proteins derived from nonhuman organisms. An emerging consensus is to reduce the potential immunogenicity of such therapies by instead using human protein domains. However, as we engineer these human-derived proteins, we create nonhuman peptides at the linkers or junctions between domains and at mutated residues within them, which still pose a risk of immunogenicity that has largely been left unaddressed. Here, we present a modular workflow to simultaneously optimize the functions of proteins and minimize their immunogenic risk using existing machine learning models that predict protein function and nonhuman peptide immunogenicity from their sequences. We first applied this workflow to existing transcriptional activation and bio-orthogonal RNA binding domains. Then we generated a set of small molecule-controllable transcription factors with human-derived zinc finger DNA-binding domains for targeting orthogonal non-genomic DNA sequences. Finally, we established a workflow for creating deimmunized zinc finger arrays to target arbitrary genomic DNA sequences and used it to upregulate expression of two therapeutically relevant genes, UTRN and SCN1A. Our future-proof, modular workflow offers a proof of principle for making cell and gene therapies safer and more efficacious through dual-objective protein optimization using state-of-the-art algorithms.

bioengineering↗

bioRxiv2024

Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems

The ability to modulate gene expression through modular and universal genetic tools like CRISPR-Cas has greatly advanced gene therapy for therapeutics and basic science. Yet, the inherent stochasticity of delivery methods cause variation in target gene expression at the single-cell level, limiting their applicability in systems that require more precise expression. Thus, we implement a modular incoherent feedforward loop based on proteolytic cleavage of Cas to reduce gene expression variability against the variability of vector delivery. We target a genome-integrated marker and demonstrate dosage control of gene activation and repression, post-delivery tuning, and RNA-based compatibility of the system. To illustrate therapeutic relevance, we target the gene RAI1, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes. We demonstrate dosage-controlled gene activation for both human and mouse Rai1 via viral delivery to patient-derived cell lines and mouse cortical neurons. Overall, we established a robust dosage control circuit for uniform gene expression, beneficial for basic and translational research.

synthetic biology↗

bioRxiv2024

Post-Transcriptional Modular Synthetic Receptors

Inspired by the power of transcriptional synthetic receptors and hoping to complement them to expand the toolbox for cell engineering, we establish LIDAR (Ligand-Induced Dimerization Activating RNA editing), a modular post-transcriptional synthetic receptor platform that harnesses RNA editing by ADAR. LIDAR is compatible with various receptor architectures in different cellular contexts, and enables the sensing of diverse ligands and the production of functional outputs. Furthermore, LIDAR can sense orthogonal signals in the same cell and produce synthetic spatial patterns, potentially enabling the programming of complex multicellular behaviors. Finally, LIDAR is compatible with compact encoding and can be delivered as synthetic mRNA. Thus, LIDAR expands the family of synthetic receptors, holding the promise to empower basic research and therapeutic applications.

synthetic biology↗

bioRxiv2024

Orthogonalized human protease control of secreted signals

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines (IL-2, IL-6, and IL-10) whose activities can be activated and abrogated by proteolytic cleavage. We utilized species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health.

synthetic biology↗

bioRxiv2023

Compact Programmable Control of Protein Secretion in Mammalian Cells

Synthetic biology has developed powerful tools to program complex behaviors, often using genetic control. Protein circuits offer a compact alternative, yet applications with intercellular signals often lack key regulatory capabilities and tunability. Here, we employ a parts-based engineering strategy to develop a single processing and output module for synthetic protein circuits, enabling complex logic, tunable sensitivity, and control over output magnitude. Using high-throughput assays, we systematically analyze the impact of human transmembrane domains on surface expression and circuit performance. We demonstrate the utility of these optimizations by encoding an open-loop circuit within translational delivery vectors, including viral and mRNA platforms, and validate its performance in vivo. Furthermore, we demonstrate multi-input logic and showcase a novel, protein-level NIMPLY gate to regulate CAR T-cell activation. Our modular design strategy provides new insights into domain-based protein engineering and establishes a versatile and complete protein-level platform to control intercellular signaling for translational cell therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/560774v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@4f96f7org.highwire.dtl.DTLVardef@1404cf5org.highwire.dtl.DTLVardef@7c948corg.highwire.dtl.DTLVardef@fcad06_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Modular and programmable RNA sensing using ADAR editing in living cells

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/stats in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular and programmable design for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validated and then expanded our basic design, characterized its performance, and thoroughly analyzed its compatibility with the human/mouse transcriptomes. We also identified strategies to further boost output levels and improve the dynamic range. We show that RADAR is programmable and modular, and uniquely enables compact AND logic. In addition to being quantitative, RADAR can distinguish disease-relevant point mutations. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

bioengineering↗

bioRxiv2021

Protease-controlled secretion and display of intercellular signals

To program intercellular communication for biomedicine, it is crucial to regulate the secretion and surface display of signaling proteins. If such regulations are at the protein level, there are additional advantages, including compact delivery and direct interactions with endogenous signalling pathways. We created a modular, generalizable design called Retained Endoplasmic Cleavable Secretion (RELEASE), with engineered proteins retained in the endoplasmic reticulum and displayed/secreted in response to specific proteases. The design allows functional regulation of multiple synthetic and natural proteins by synthetic protease circuits to realize diverse signal processing capabilities, including logic operation and threshold tuning. By linking RELEASE to additional novel sensing and processing circuits, we were able to achieve elevated protein secretion in response to "undruggable" oncogene KRAS mutants. RELEASE should enable the local, programmable delivery of intercellular cues for a broad variety of fields such as neurobiology, cancer immunotherapy and cell transplantation.

synthetic biology↗

bioRxiv2020

Engineering multiple levels of specificity in an RNA viral vector

Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal “smart” viral delivery system would enable controlled release of viral vectors from “sender” cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity.Competing Interest StatementX.J.G, L.S.C., M.S.K., and M.B.E. are inventors on a U.S. patent provisional application related to this work.View Full Text

synthetic biology↗