Biology subjects

Zhang, C.

Publications and source records attributed to Zhang, C..

At least 199 records · Page 11Linked to original sources

bioRxiv2025

High-Fidelity Neural Speech Reconstruction through an Efficient Acoustic-Linguistic Dual-Pathway Framework

Reconstructing speech from neural recordings is crucial for understanding speech coding and developing brain-computer interfaces (BCIs). However, existing methods trade off acoustic richness (pitch, prosody) for linguistic intelligibility (words, phonemes). To overcome this limitation, we propose a dual-path framework to concurrently decode acoustic and linguistic representations. The acoustic pathway uses a long-short term memory (LSTM) decoder and a high-fidelity generative adversarial network (HiFi-GAN) to reconstruct spectrotemporal features. The linguistic pathway employs a transformer adaptor and text-to-speech (TTS) generator for word tokens. These two pathways merge via voice cloning to combine both acoustic and linguistic validity. Using only 20 minutes of electrocorticography (ECoG) data per subject, our approach achieves highly intelligible synthesized speech (mean opinion score = 4.0/5.0, word error rate = 18.9%). Our dual-path framework reconstructs natural and intelligible speech from ECoG, resolving the acoustic-linguistic trade-off.

neuroscience↗

bioRxiv2025

DNA damage hypersensitivity in chicken primordial germ cells limits CRISPR-Cas9 editing and supports an shRNA-based screening approach

Chickens (Gallus gallus) are uniquely suited for germline studies because their primordial germ cells (PGCs) can be propagated long-term in vitro and used for germline transmission. To develop a loss-of-function screening platform in chicken PGCs, we compared three perturbation methods: CRISPR/Cas9 knockout, CRISPR interference (CRISPRi), and shRNA-mediated knockdown. We found that CRISPR/Cas9 editing causes severe toxicity in PGCs, with DNA damage hypersensitivity over 20-fold greater than in somatic cells, and with distinct DNA damage checkpoint responses between male (ZZ) and female (ZW) lines. CRISPRi using dCas9-KRAB was ineffective in chicken--likely because of species-specific epigenetic constraints--whereas shRNA knockdown produced robust, nontoxic gene silencing. These results identify DNA damage hypersensitivity as a major barrier to nuclease-based editing in the germline and establish RNAi as a feasible platform for genome-wide functional screening in chicken PGCs.

cell biology↗

bioRxiv2025

Chemical inhibition of a CBASS anti-bacteriophage defense

Multidrug-resistant (MDR) bacteria pose a significant public health challenge, underscoring the urgent need for innovative antibacterial strategies. Bacteriophages (phages), viruses that specifically target bacteria, offer a promising alternative; however, bacterial immune defenses often limit their effectiveness. Developing small-molecule inhibitors of these defenses can facilitate mechanistic studies and serve as adjuvants to enhance phage therapy. Here, we identify novel inhibitors targeting the bacterial cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector Cap5. Cap5 is an HNH endonuclease activated by a cyclic nucleotide to degrade genomic DNA in virally infected cells, leading to cell death through abortive infection. Guided by the crystal structure of the Cap5 SAVED domain bound to its activating ligand, we performed structure-guided virtual screening to identify candidate inhibitors. Biochemical assays revealed that approximately 16% of the top docking hits exhibited inhibitory activity. Further cellular assays demonstrated that one potent compound could enter E. coli cells and inhibit Cap5 activity. Our integrated approach--combining structure-based virtual screening with biochemical validation--provides a robust framework for discovering small-molecule inhibitors of bacterial immune defenses to advance adjunctive therapies and deepen our understanding of phage-bacteria interactions.

biochemistry↗

bioRxiv2025

Transfer RNA modifications during rhizome development in Oryza longistaminata

The rhizome organ endows many clonal plants with the ability to reproduce under stressful climates. Such rhizome-mediated perenniality also underlies the recent success of perennial rice breeding. Despite the importance of this organ, its developmental regulation is poorly understood. Here, using Oryza longistaminata, an emerging model for rhizome biology, we explored the involvement of tRNA modification in rhizome development. Through comparative profiling of modified nucleotides in rhizome samples at different developmental stages and/or subjected to various treatments, we identified a number of key rhizome-related tRNA modifications and revealed that, akin to the observations in many non-plant organisms, tRNA modifications are associated with hormonal signalling to regulate rhizomes responses to environmental cues. Furthermore, genome-wide analysis of tRNA-modifying genes was performed to facilitate future functional studies. These results and analyses not only deepen our understanding of rhizome development but also underscore the regulatory roles of tRNA modification in plant organ development.

plant biology↗

bioRxiv2025

wspA mutation mediated Pseudomonas aeruginosa rugose small colony variant and its hyper-biofilm

Pseudomonas aeruginosa is a major multidrug-resistant pathogen whose biofilm formation complicates treatment. P. aeruginosa rugose small colony variants (RSCVs), characterized by enhanced biofilm production and resistance, pose significant clinical challenges. However, the formation mechanisms remain unclear in clinical strains. Paired clinical P. aeruginosa isolates AR8023-1 (wild-type) and AR8023-2 (RSCV) were collected from urine samples of a neurosurgery inpatient chronologically. The genome comparison was performed using Breseq, and plasmid complementation was performed to construct complemented strain (AR8023-2RifR::wspAAR8023-1). Then, all the aforementioned strains were examined RSCV phenotype, biofilm formation, and motility, and gene expression differences induced by the wspA mutation were analyzed via transcriptome sequencing, followed by quantification of c-di-GMP. In addition, we assessed antimicrobial susceptibility of the strains under both planktonic and biofilm conditions. The results showed that both AR8023-1 and AR8023-2 were identified as ST3420. AR8023-1 exhibited typical smooth morphology, while AR8023-2 displayed a RSCV phenotype. A single SNP difference was identified between the two strains, characterized by a deletion of glutamine at position 289 (CAG triplet deletion) in the wspA gene of strain AR8023-2. The complement isolates restored wild-type morphology, motility, and biofilm formation. Transcriptomics revealed a significant upregulation of c-di-GMP metabolic genes in RSCVs (P < 0.001), indicating that the WspA{Delta}289Q mutation activates diguanylate cyclase (DGC) activity, thereby elevating c-di-GMP synthesis. Intracellular c-di-GMP levels were significantly higher in RSCVs than those in WT and complement isolates, respectively (P < 0.001). Consequently, biofilm susceptibility testing demonstrated the {beta}-lactams MBIC of RSCVs was [≥]8-512-fold higher than those of WT and complement isolates. This study identifies a clinical WspA{Delta}289Q mutation that elevates c-di-GMP, driving RSCV formation and biofilm-mediated resistance. IMPORTANCEP. aeruginosa is a leading multidrug-resistant pathogen whose biofilm formation capability greatly complicates treatment outcomes. This study identifies a novel clinical mutation in the wspA gene (WspA{Delta}289Q) that drives the emergence of rugose small colony variants (RSCVs), a phenotype linked to heightened biofilm production and antimicrobial resistance. We demonstrate that this mutation upregulates cyclic di-GMP synthesis, leading to increased biofilm formation and markedly reduced susceptibility to {beta}-lactam antibiotics under biofilm conditions. These results provide important insights into the genetic basis of RSCV development in clinical settings and highlight the role of c-di-GMP signaling in biofilm-associated resistance. Our findings underscore the need to target c-di-GMP pathways as a potential strategy for combating persistent P. aeruginosa infections.

microbiology↗

bioRxiv2025

A simplified and highly efficient cell-free protein synthesis system for prokaryotes

Cell-free protein synthesis (CFPS) systems are a powerful platform with immense potential in fundamental research, biotechnology, and synthetic biology. Conventional prokaryotic CFPS systems, particularly those derived from Escherichia coli (E. coli), often rely on complex reaction buffers containing up to thirty-five components, limiting their widespread adoption and systematic optimization. Here, we present an optimized E. coli cell-free protein synthesis (eCFPS) system, which is significantly streamlined for high efficiency. Through systematic screening, we successfully reduced the essential core reaction components from 35 to a core set of 7. The thorough optimization of these seven key components ensured that protein expression levels were not only maintained but even substantially improved. Furthermore, we developed a much simpler procedure for preparing the bacterial cytosolic extracts, a "fast lysate" protocol that eliminates the traditional time-consuming runoff and dialysis steps, thereby enhancing the overall accessibility and robustness of eCFPS. This optimized and user-friendly eCFPS efficiently synthesizes challenging proteins, including functional, self-assembling vimentin, and active restriction endonuclease BsaI despite its strong cytotoxicity, and serves as a powerful tool that will facilitate diverse applications in basic life science research and beyond.

biochemistry↗

bioRxiv2025

S3R: Spatially Smooth and Sparse Regression Reveals High-Dimensional Regulatory Networks in Spatial Transcriptomics

Spatial transcriptomics (ST) data demands models that recover how associations among molecular and cellular features change across tissue while contending with noise, collinearity, cell mixing, and thousands of predictors. We present Spatially Smooth Sparse Regression (S3R), a general statistical framework that estimates location-specific coefficients linking a response feature to high-dimensional spatial predictors. S3R unites structured sparsity with a minimum-spanning-tree-guided smoothness penalty, yielding coefficient fields that are coherent within neighborhoods yet permit sharp boundaries. In synthetic data, S3R accurately recovers spatially varying effects, selects relevant predictors, and preserves known boundaries. Applied to Visium-based ST data, S3R recapitulates layer-specific target-TF associations in human dorsolateral prefrontal cortex with concordant layer-wise correlations in matched single-cell data. In acute Haemophilus ducreyi skin infection, S3R converts spot-level gene expression mixtures into cell type-attributed expression fields, revealing per-cell type spatial gradients, and improving concordance of spatially variable gene calls when tests are applied to these demixed fields. In pancreatic ductal adenocarcinoma, S3R builds cross-cell-type, cross-gene co-variation tensors that quantify cell-cell interaction strength at gene-pair resolution and nominate interacting genes whose pathway enrichments align with established stromal-epithelial and immune crosstalk. An efficient implementation scales to large assays, and on a Xenium-based breast cancer dataset, S3R delineates the contributions at gene-gene, local neighborhood, and global context-level to target gene expression. Because responses and predictors in S3R are user-defined, it could flexibly address diverse biological questions within a single, scalable, and interpretable regression framework.

bioinformatics↗

bioRxiv2025

stk32a links sleep homeostasis to suppression of sensory and motor systems

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood. To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies. We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain. Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

neuroscience↗

bioRxiv2025

A Novel Mechanism for Tauopathy in Progressive Multiple Sclerosis: Excitotoxic Misplacement of a Mitochondrial Anchor into Dendrites Driven by Tau-hyperphosphorylation

On April 2nd, 2025, the FDA approved a Fast Track Designation for Biogen to use Antisense Oligonucleotides (ASO) to treat tauopathy in clinical trials for Alzheimers Disease (AD) to meet an unmet medical need [1]. For Multiple Sclerosis (MS), there is a similar unmet medical need regarding tauopathy when MS transitions into the late, or Progressive MS that is currently incurable. AD and MS share commonality: there is comorbidity between AD and MS [2], and the recent awareness that progressive MS may be considered a secondary tauopathy [3]. This study lays the basic science foundation for a future repurposing of ASO tauopathy therapy from AD to MS. The central hypothesis is that in Progressive MS, tauopathy is not a passive bystander but an active contributor to synaptic degeneration through a novel toxic target known as DSI (Dendritic Syntaphilin Intrusion) discovered in our laboratory. In this hypothesis, the excitotoxic N-methyl-D-aspartate receptor (NMDAR) GluN2B activates Tau hyperphosphorylation (p-Tau), leading to the mislocalization or intrusion of a mitochondrial anchor SNPH into neuronal dendrites (DSI). This causes mitochondrial damage and subsequent synapse/dendrite disintegration. In support of this hypothesis that tauopathy is a key driver of DSI, we demonstrated using primary neuronal cultures that inhibitors of p-Tau kinases and Tau-KO both completely abolish DSI. We propose that a therapy for Progressive MS is repurpose the existing FDA-approved ASO Tau knockdown therapy from AD to treat MS. FundingNIH R01 MSN233781 to SY Chiu

neuroscience↗

bioRxiv2025

STADiffuser: high-fidelity simulation and full-view 3D modeling of spatial transcriptomics

Spatial transcriptomics technologies have provided invaluable insights by profiling gene expression alongside precise spatial information. However, they encounter high costs, data sparsity, and limited resolution, hindering their broader adoption and utility. Moreover, the lack of flexible simulators capable of generating high-fidelity simulated data has impeded the development of computational tools for spatial transcriptomic data analysis. To this end, we introduce STADiffuser, a versatile deep generative model that leverages diffusion modeling for accurate simulation of spatial transcriptomic data. STADiffuser employs a two-stage architecture: an autoencoder with a graph attention mechanism for learning spot embeddings, followed by a latent diffusion model integrated with a spatial denoising network for data generation. STADiffuser is the first simulator designed for spatial transcriptomic data, capable of handling multiple samples and 3D coordinates while supporting user-defined conditions. STADiffuser facilitates various downstream analyses, including accurate imputation, super-resolution, and full-slice generation. Furthermore, its generative scheme enables in silico experiments, thereby enhancing the statistical power in detecting differentially expressed genes and identifying cell-type-specific genes while effectively controlling confounding factors. Notably, STADiffuser scales to millions of spots and supports the full-view 3D modeling of a marmoset cerebellum atlas with over 20 million spots, enabling detailed investigations from arbitrary viewing angles.

bioinformatics↗

bioRxiv2025

JARID2 Inhibition Reprograms Human Hematopoietic Progenitor Cells To Enhance Bone Marrow Transplantation

Hematopoietic stem cell transplantation is a common treatment for many blood disorders and can be a life-saving therapy for patients with leukemias, lymphomas and multiple myeloma. Umbilical cord blood (UCB) serves as a valuable source of hematopoietic stem and progenitor cells (HSPCs) for transplantation, particularly for patients lacking a matched donor. However, the limited number of repopulating cells in UCB units restricts its clinical utility. Our prior studies showed that genetic deletion of the polycomb repressive complex 2 (PRC2) co-factor Jarid2 in mouse multipotent progenitors (MPPs) conveyed ectopic self-renewal capacity. Here, we hypothesized that the function of human HSPCs could be enhanced through JARID2 inhibition. In this study, we demonstrate that both constitutive and transient knockdown of JARID2 increases the number and enhances the functionality of human HSPCs both in vitro and in vivo. This phenotype was distinct from inhibition of EZH2 in UCB cells, suggesting the mechanism was independent of PRC2 co-factor activity of JARID2. Mechanistically, JARID2 knockdown promotes a quiescent, long-term self-renewal gene expression program governed by upregulating STAT1 and characterized by an MHC class II immunophenotype. Analogous to mice, these mechanisms conferred HSC-like potential to human MPPs in vivo. Taken together, these findings highlight JARID2 inhibition as a novel and reversible approach to expand functional UCB-derived HSPCs ex vivo, potentially improving access to stem cell transplantation for a wider patient population. One Sentence SummaryGenetic inhibition of JARID2 enhances repopulating activity of human hematopoietic stem and progenitor cells in vivo via STAT1 upregulation.

developmental biology↗

bioRxiv2025

Structural insights and engineering of deep-sea halophilic PET hydrolytic enzymes

Pervasive use of polyethylene terephthalate (PET) poses tremendous challenges for global waste management and environmental sustainability, fueling growing interests in enzymatic degradation as an eco-friendly solution. While PET hydrolases hold significant promise, their industrial deployment is hindered by insufficient performance, particularly under high-salinity conditions raised from high substrate loads. Building on our previous discovery of three deep-sea PET hydrolases (dsPETase01, dsPETase05 and dsPETase06) with exceptional halophilicity and PET-degrading activity, we here present their three-dimensional structures and mechanistic characterization. Structural comparison, site-directed mutagenesis, and domain swapping reveal key structural features and the essential role of C-terminal acidic residues in salt tolerance. Integrating semi-rational protein design, Transformer-based modeling, and disulfide bond engineering, we synergistically enhance the thermostability and catalytic efficiency of dsPETase05. These findings elucidate the unique structural features and salt adaptation mechanisms of deep-sea halophilic PET hydrolases, and inform the future engineering of biocatalysts for application in harsh industrial environments.

molecular biology↗

bioRxiv2025

A molecularly defined basalo-prefrontal-thalamic circuit regulates sensory and affective dimensions of pain

Both the medial prefrontal cortex (mPFC) and thalamus have been implicated in pain regulation. However, the roles of the mPFC-thalamus connection in pain and how the mPFC modulates nociceptive processing remain unclear. Here, we show that the mPFC neurons projecting to thalamus, marked by Foxp2 expression, are deactivated in both acute and chronic pain in male mice. Persistent inactivation of the mPFC Foxp2+ neurons enhances nociceptive sensitivity, while their activation alleviates multiple aspects of pain. Circuit-specific manipulations revealed that the projections to parataenial nucleus, mediodorsal and ventromedial thalamus differentially modulate sensory and affective pain. Additionally, the mPFC Foxp2+ neurons receive cholinergic input from the basal forebrain, particularly the horizonal diagonal band (HDB). Notably, activation of the 4{beta}2-containing nicotinic acetylcholine receptor in mPFC exerts antinociceptive effects in Foxp2+ neuron-dependent manner. Together, our study defines an HDB[->]mPFCFoxp2[->]thalamus circuit essential for sensory and affective pain modulation and underscores the therapeutic potential of targeting mPFC cholinergic signaling in chronic pain management.

neuroscience↗

bioRxiv2025

Ubiquitin-like SUMO protease expansion in rice (Oryza sativa)

O_LISUMOylation is a protein post-translational modification that is essential for plant growth and response to changing environments. However, past work in this area has mainly focused on simple sequence similarity methods for discovering SUMOylation genes, often using orthologue mapping from yeast or Arabidopsis. C_LIO_LIIn this work, we employed a range of computational techniques and approaches to describe and characterise the SUMOylation machinery in Asian rice (Oryza sativa), as a globally important stable crop, and where the SUMOylation system has been shown to play key roles in responses to biotic and abiotic stresses. We describe and analyse the ULP system at the phylogenetic, transcriptional, and protein structural levels, with a focus on the rice reference genome, a well-annotated Rice Population Reference Panel (RPRP), and wild rice genomes. C_LIO_LIOur analysis revealed the expansion of ULPs in the reference genome and RPRP set (32 - 45 ULPs) compared to wild rice (9-36 ULPs), raising an intriguing hypothesis about the expansion of the ULP family being driven by selective breeding pressure. C_LIO_LIWe provide evidence of potential functional ULPs and their possible roles in biotic and abiotic stress responses in cultivated rice. These insights offer valuable resources for future rice breeding and crop improvement. C_LI

plant biology↗

bioRxiv2025

Structural insights on the ligand selectivity of the mouse trace amine-associated receptor TAAR7e

Trace amine-associated receptors (TAARs), a subclass of G protein-coupled receptors involved in detecting volatile amines, are co-expressed with odorant receptors in the main olfactory epithelium. Although many behaviorally relevant amines have been identified for olfactory TAARs, the molecular mechanisms underlying their selectivity for amines with varying carbon chain lengths and degrees of amine substitution remain unclear. In this study, we used mTAAR7e as a model to investigate these mechanisms. Homology modeling revealed a conserved ligand-binding pocket, supported by sequence-structure covariance analysis across TAARs. Structure-activity relationship profiling revealed key chemical determinants integral to mTAAR7e-mediated odorant recognition and provided, for the first time, a structural explanation for its selective preference toward longer carbon chains and tertiary amines. Computationally predicted interactions between mTAAR7e and the representative ligand N,N-dimethylcyclohexylamine (DMCHA) were validated through site-directed mutagenesis. Furthermore, conformational dynamics of mTAAR7e during receptor activation were characterized, providing insights into activation-related structural rearrangements. Together, these findings offer novel insights into the molecular logic of TAAR ligand selectivity and may advance our understanding of how TAARs mediate both olfactory and systemic aminergic signaling.

molecular biology↗

bioRxiv2025

Genetic entanglement enables ultra-stable biocontainment in the mammalian gut

Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability in vivo limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (Sequence enTAngLEd Multi lAyered geneTic buffEring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudo-essentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganu-clease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement simultaneously inactivated both biocontainment layers, leading to cell death by the ColE9 nuclease and the elimination of escape mutants. By lengthening the entangled region, refining ColE9 expression, and optimizing the TSM sequence against IS911 insertion, we achieved escape rates below 10-10 as compared to rates of 10-5 with the non-entangled TSM. The STALEMATE system contained plasmids in E. coli Nissle 1917 for over a week in the mouse gastrointestinal tract with nearly undetectable escape rates upon excretion. STALEMATE offers a modular and simple biocontainment approach to buffer against mutational inactivation in the mammalian gut without a requirement for engineered bacteria or exogenous signaling ligands.

synthetic biology↗

bioRxiv2025

PAM adenine methylation and flanking sequence regulate SaCas9 activity in bacteria

Cas9 nucleases are the effectors of the class 2 type II CRISPR system in bacteria and function to restrict invading DNA. They can also be used with single guide RNAs (sgRNAs) as antimicrobials and genome engineering tools in bacteria, yet applications are hindered by an incomplete understanding of Cas9-target interactions. Here, we generate large-scale SaCas9/sgRNA in vivo bacterial activity datasets and train a machine learning model (crisprHAL) to predict SaCas9 activity. The highest predictive performance was found when downstream sequence flanking the canonical NNGRRN PAM motif at positions [+1] and [+2] was included in model training, correlating with high in vivo activity on sites that included T-rich di-nucleotides in the [+1] and [+2] flanking positions. Strikingly, model predictions and experimentally determined activity in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium showed an [~]10-fold reduced SaCas9 activity at sites with 5'-NNGGAT[C]-3' PAM [+1] sequences. Cleavage assays using plasmid DNA isolated from E. coli inactivated for DNA adenine methyltransferase (DAM) and SaCas9/sgRNA combinations targeting sites with NNGGAT[C] PAM sequences confirmed that adenine methylation impacts SaCas9 cleavage. Moreover, ablation of a GATC DAM site in a PAM sequence enhanced SaCas9 in vitro activity, whereas creation of a DAM site reduced activity, providing a mechanistic link between adenine methylation and SaCas9 activity. Our results show that a general purpose machine learning architecture can provide biologically relevant insights into SaCas9-PAM interactions that can better inform activity predictions for bacterial applications. Avoidance of adenine methylated PAM sites by SaCas9 may be a mechanism of self versus non-self discrimination or reflect an evolutionary adaptation to counter methylation as an anti-restriction strategy by phage or plasmids.

microbiology↗

bioRxiv2025

CircRNA-based, non-integrated, in vivo panCAR-mediated B cell immune resetting in mouse models and non-human primates

B cell immune resetting, defined as the comprehensive depletion and subsequent reconstitution of B cells, represents a pivotal therapeutic approach for B cell-associated disorders. Recently, chimeric antigen receptor T cell (CAR-T) therapy has demonstrated substantial clinical efficacy in treating B cell malignancies and autoimmune diseases. However, this approach remains constrained by its reliance on lymphodepleting conditioning regimens, high invasiveness, high costs, and the risk of severe cytokine release syndrome (CRS). Antibody-based therapies, including monoclonal antibodies and T cell-engager (TCE) bispecific antibodies, suffer from suboptimal tissue penetration, resulting in inadequate B cell depletion in lymphoid and peripheral tissues, and subsequent rapid resurgence of pathogenic B cells. Here, we engineered highly stable circular RNAs (circRNAs) encoding anti-CD19 CAR (circRNA-CAR), which demonstrated prolonged and enhanced CAR protein expression compared to conventional linear mRNAs. By employing immune cell-tropic lipid nanoparticles (LNPs) for in vivo delivery, circRNA-CAR facilitated the generation of panCAR immune cells (including CAR-T, CAR-NK, and CAR-macrophages) in both murine models and cynomolgus monkeys. Compared to rituximab or teclistamab, in vivo panCAR induced more extensive B cell depletion across multiple tissue compartments in mice. Moreover, in vivo panCAR mediated robust B cell depletion, eliminated autoreactive antibodies, and restored renal function by alleviating proteinuria in a systemic lupus erythematosus (SLE) murine model. It also effectively reduced IgE antibody levels, decreased eosinophil activity, and improved lung pathology in an asthma murine model. Notably, in vivo panCAR-mediated B cell depletion significantly attenuated aging-associated phenotypes in 17- month-old aging mice, remarkably outperforming both rituximab and teclistamab. In cynomolgus monkeys, in vivo panCAR achieved sustained B cell depletion lasting approximately 40 to 50 days, followed by reconstitution of a naive B cell repertoire. Collectively, circRNA-based in vivo panCAR enables complete B cell immune resetting, offering a versatile and promising therapeutic platform for a broad spectrum of B cell- and antibody-mediated disorders.

immunology↗