Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 325 records · Page 18Linked to original sources

bioRxiv2025

Developmental expression of risk genes implicates the age of onset for neuropsychiatric disorders

The functional effects of genetic variants associated with complex diseases exhibit pronounced spatiotemporal specificity. Although spatially resolved studies have advanced, their temporal dynamics remain poorly characterized. Here, we present an analytical framework integrating developmental gene expression with genome-wide association studies to decipher age-specific windows during which genetic variants exert their effects and to elucidate underlying mechanisms. Applying this framework to five major neuropsychiatric disorders, we uncover a fundamental principle: the peak incidence of a disease precisely coincides with the developmental window of peak expression of its associated risk genes in the prefrontal cortex. These risk windows are characterized by distinct biological processes; for instance, childhood risk for attention-deficit/hyperactivity disorder aligns with a peak in presynaptic machinery gene expression, whereas late-life risk for Alzheimers disease corresponds to heightened immune-related gene activity. Leveraging this principle of temporal convergence significantly improves the prioritization of disease genes. Our work establishes the developmental basis for the age of onset of complex diseases, providing a temporal roadmap for understanding disease mechanisms and developing age-appropriate therapeutic strategies.

genetics↗

bioRxiv2025

Discovery of Small Molecule CHI3L1 Inhibitors by SPR-Based High-Throughput Screening

Chitinase-3-like 1 (CHI3L1) is a secreted glycoprotein implicated in carcinogenesis and tumor immune evasion. Elevated CHI3L1 expression is frequently detected in cancer patients, highlighting it as a promising therapeutic target. To overcome the limited availability of small molecule CHI3L1 inhibitors, we established a surface plasmon resonance (SPR)-based high-throughput screening platform and applied it to a focused chemical library of small molecules. Primary screening identified seven hits, with compounds 1-4 and 1-7 validated as CHI3L1 binders (Kd = 10.4 {+/-} 1.0 M and 7.40 {+/-} 0.78 M, respectively). Both compounds disrupted the CHI3L1-galectin-3 interaction in AlphaLISA assays and engaged the CHI3L1 binding pocket in docking and molecular dynamics (MD) simulations. Importantly, functional evaluation in a multicellular 3D glioblastoma (GBM) spheroid model demonstrated that compound 1-7 potently reduced spheroid viability and inhibited STAT3 phosphorylation, outperforming both compound 1-4 and the known CHI3L1-STAT3 disruptor hygromycin B (HB). These findings validate SPR as a robust primary screening platform for CHI3L1 and demonstrate that the identified small molecule binders exert functional activity in a physiologically relevant multicellular GBM spheroid model.

pharmacology and toxicology↗

bioRxiv2025

Mycobacterium tuberculosis m4C DNA methyltransferase Rv3204 promotes mycobacteria survival under oxidative stress

Reactive oxygen species (ROS) inflict cellular damage yet are pivotal mediators of signaling pathways. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), persists as a major global health threat, partly due to its capacity to neutralize host-derived ROS toxicity. While DNA methylation deficiency is known to attenuate Mtb virulence, the specific role of DNA methyltransferases in mycobacterial survival under oxidative stress remains poorly defined. Here, we demonstrate that the mycobacterial protein Rv3204 functions as an N4-methylcytosine (m4C) DNA methyltransferase. Deletion of the Rv3204 homolog (Ms_1939) in Mycobacterium smegmatis significantly impaired bacterial survival upon rifampicin exposure. This phenotype was associated with heightened intracellular ROS accumulation and a failure to upregulate transcription of ROS detoxification genes. Furthermore, the mutant exhibited downregulated expression of DNA repair genes and increased susceptibility to fluoroquinolone antibiotics (norfloxacin, ofloxacin). Crucially, the Ms_1939 deletion strain displayed elevated levels of DNA damage. To our knowledge, this is the first study establishing a direct link between an m4C DNA methyltransferase and ROS homeostasis in mycobacteria. Our findings identify Rv3204 as a potential novel therapeutic target for modulating ROS sensitivity in M. tuberculosis. ImportanceMycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health threat due in part to its ability to withstand host-derived oxidative stress. This study identifies Rv3204 as a novel N4-methylcytosine DNA methyltransferase essential for mycobacterial survival under oxidative stress. We demonstrate that deletion of Rv3204 homolog in M. smegmatis leads to elevated intracellular ROS, impaired transcriptional activation of antioxidant and DNA repair genes, increased DNA damage, and heightened susceptibility to fluoroquinolone antibiotics. Our findings establish a critical link between epigenetic regulation via m4C methylation and ROS homeostasis in mycobacteria--a relationship previously unexplored. This work not only advances our understanding of bacterial epigenetic mechanisms in stress adaptation but also positions Rv3204 as a promising target for novel anti-tuberculosis strategies aimed at disrupting redox balance and enhancing antibiotic efficacy. DATA summaryRaw data files have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE139646 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139646).

genetics↗

bioRxiv2025

Profiling a large HIV-1 elite neutralizer cohort reveals remarkable CD4bs bNAb for HIV-1 prevention and therapy

Administration of HIV-1 neutralizing antibodies can suppress viremia and prevent infection in vivo. However, clinical use is challenged by broad envelope sequence diversity and rapid emergence of viral escape1-9. Here, we performed single B cell profiling of 32 top HIV-1 elite neutralizers to identify broadly neutralizing antibodies (bNAbs) with highest potency and breadth for clinical application. From 831 expressed monoclonal antibodies, we identified 04_A06, a new VH1-2-encoded CD4 binding site bNAb with remarkable breadth and potency against extended multiclade pseudovirus panels (GeoMean IC50 = 0.059 {micro}g/ml, breadth = 98.5%, 332 virus strains). Moreover, 04_A06 was not susceptible to classic viral CD4bs escape variants and maintained full viral suppression in HIV-1-infected humanized mice. Structural analyses revealed that antiviral activity is mediated by an unusually long 11-amino acid heavy chain insertion. This insertion facilitates inter-protomer contacts and interactions with highly conserved residues on the adjacent gp120 protomer. Finally, 04_A06 demonstrated high activity against contemporaneously circulating viruses from the Antibody Mediated Prevention (AMP) trials (GeoMean IC50 = 0.082 {micro}g/ml, breadth = 98.4%, 191 virus strains) and in silico modeling for 04_A06LS predicted HIV-1 prevention efficacy of >93%. Thus, 04_A06 will provide unique opportunities for effective treatment and prevention strategies of HIV-1 infection.

immunology↗

bioRxiv2025

CASP3-GSDME-mediated trophoblast pyroptosis contributes to systemic inflammation

Early-onset preeclampsia (EOPE) is associated with excessive apoptosis and inflammation, but the mechanistic link between these processes remains enigma. Here, we report elevated circulating pro-apoptotic proteins in EOPE patients at early pregnancy, along with concurrent CASP3 activation and GSDME cleavage in a subset of EOPE placentas. Using multiple trophoblast cell lines, we demonstrate that trophoblast cells, which highly express GSDME, undergo a shift from apoptosis to CASP3-dependent pyroptosis, driving inflammation. Notably, pyroptotic trophoblasts further induce pro-inflammatory macrophage polarization within placental villi organoids, establishing a feedback loop that amplifies both trophoblast pyroptosis and inflammatory responses in trophoblast organoids-macrophage assembloids. In vivo, CASP3-GSDME-mediated trophoblast pyroptosis contributes to systemic inflammation in wild-type pregnant mice but not in Gsdme-/- mice. Screening of EOPE prevention drugs reveals Vitamin D as a suppressor of GSDME activation and pyroptosis in trophoblast cells. These findings highlight the CASP3-GSDME axis as a promising therapeutic target for preeclampsia prevention.

cell biology↗

bioRxiv2025

40 Hz Audiovisual Stimulation Improves Sustained Attention and Related Brain Oscillations

Gamma oscillations (30-100 Hz) have long been theorized to play a key role in sensory processing and attention by coordinating neural firing across distributed neurons. Gamma oscillations can be generated internally by neural circuits during attention or exogenously by stimuli that turn on and off at gamma frequencies. However, it remains unknown if driving gamma activity via exogenous sensory stimulation affects attention. We tested the hypothesis that non-invasive audiovisual stimulation in the form of flashing lights and sounds (flicker) at 40 Hz improves attention in an attentional vigilance task and affects neural oscillations associated with attention. We recorded scalp EEG activity of healthy adults (n=62) during one hour of either 40 Hz audiovisual flicker, no flicker as control, or randomized flicker as sham stimulation, while subjects performed a psychomotor vigilance task. Participants exposed to 40 Hz flicker stimulation had better accuracy and faster reaction times than participants in the control groups. The 40 Hz group showed increased 40 Hz activity compared to the control groups in agreement with previous studies. Surprisingly, 40 Hz subjects had significantly lower delta power (2-4 Hz), which is associated with arousal, and higher functional connectivity in lower alpha (8-10 Hz), which is associated with attention processes. Furthermore, decreased delta power and increased lower alpha functional connectivity were correlated with better attention task performance. This study reveals how gamma audiovisual stimulation improves attention performance with potential implications for therapeutic interventions for attention disorders and cognitive enhancement.

neuroscience↗

bioRxiv2025

Identification and quantification of alternative polyadenylation sites in single cell RNA-seq data using scPAISO

AbstractsAlternative polyadenylation (APA) is a critical posttranscriptional mechanism that generates transcriptomic diversity through the production of mRNA isoforms with distinct 3 UTRs or coding sequences. Current APA analysis based on single-cell RNA sequencing (scRNA-seq) for establishing cell type-specific APA landscapes primarily rely on Read2 data, which lacks precise cleavage site (CS) information. This limitation restricts their ability to achieve precise de novo mapping of polyadenylation sites (PASs). Here, we present single-cell PolyAdenylation ISOform quantification (scPAISO), a computational pipeline designed for de novo identification of PAS and quantification of PAS isoforms in scRNA-seq data, by leveraging the often-discarded Read1 from 3 tag-based scRNA-seq protocols. Unlike existing tools, scPAISO directly captures mRNA 3 end cleavage sites, enabling superior performance in motif enrichment (stronger AAUAAA signal) and peak precision (sharper PAS peaks). Moreover, the smaller peak widths enhance the spatial resolution, enabling more accurate detection of closely spaced PASs in the genome. By integrating Read1 and Read2 data, scPAISO achieves isoform-level quantification with an assignment accuracy exceeding 95%. We demonstrate the robustness of scPAISO in identifying PASs and quantifying APA events across diverse biological contexts, including hematopoiesis, systemic sclerosis (SSc), and mouse tissues. We identified stage-specific 3 UTR lengthening in hematopoietic progenitors, global 3 UTR remodeling in SSc and tissue-specific polyadenylation (PA) preference along with RNA-binding proteins in mice. Overall, scPAISO represents a significant advancement in the analysis of APA at single-cell resolution and provides a powerful tool for exploring the regulatory landscape of APA, offering new insights into transcriptome complexity and gene regulation in both health and disease.

bioinformatics↗

bioRxiv2025

Mathematical Foundations of Beta Diversity: Why Common Metrics Fail in Microbiome Analysis

BackgroundIn microbiome studies, beta diversity quantifies dissimilarity between samples and is often visualized using ordination techniques. It enables researchers to characterize ecological structure, compare microbial communities, assess environmental or host-driven heterogeneity, and track longitudinal shifts over time. Although many diversity indices were originally developed with practical goals in mind, they lack a unified framework to ensure theoretical rigor and validity. This gap makes it challenging for researchers to evaluate and select appropriate beta diversity measures for microbiome analyses, potentially leading to biased analyses and invalid conclusions. ResultsTo bridge the persistent knowledge gaps, we systematically evaluate the commonly used beta diversity measures according to key mathematical properties, including whether they are true metrics, conform to Euclidean geometry, and satisfy conditional negative definiteness. We show that their violations can compromise downstream analyses such as PCoA, PERMANOVA, and kernel-based tests. In addition, drawing on mathematical consensus, we introduce a novel four-category classification of beta diversity measures: scale difference, difference scale, Hamming difference, and distribution difference. Complementing this framework, we build diagnostic tools for assessing Euclidean validity and develop remedial strategies that correct problematic dissimilarity matrices while preserving ordination structures. We demonstrate the effectiveness of these solutions using real-world microbiome datasets. ConclusionsThese results establish a unified framework for evaluating beta diversity in microbiome research, supported by an R package, interactive Shiny app, and step-by-step tutorials. The framework provides a clear roadmap for selecting and refining dissimilarity metrics, paving the way for future methodological advances.

bioinformatics↗

bioRxiv2025

Orthorhombic coronavirus main protease crystals provide a higher success rate in fragment screening

In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign.

biochemistry↗

bioRxiv2025

Human single-cell atlas analysis reveals heterogeneous endothelial signaling

Endothelial cells (ECs) play complex roles across tissues and vessel types. Yet, systematic investigations of EC heterogeneity in the combined context of vessel type and tissue microenvironment are still largely lacking. We integrated over three million single cells of scRNA-seq datasets in 15 human tissues and found that ECs in some tissues (e.g., heart and kidney) exhibited greater tissue specificity, while others displayed more substantial vessel specificity. We developed a computational pipeline to analyze cell-cell communications (CCC) mediated by metabolites or proteins to explore microenvironmental regulation. Interestingly, our results showed that CCC events involving ECs varied vastly across tissues, highlighting tissue-specific EC interactions. Using topic modeling, we identified CCC patterns, termed CCC topics, representing metabolite- and protein-mediated interactions between ECs and other tissue-resident cells. Most CCC topics exhibited high tissue specificity, potentially explaining microenvironmental regulations for EC heterogeneity. The work systematically investigates EC heterogeneity and provides insights into how EC heterogeneity was regulated across diverse tissue microenvironments.

bioinformatics↗

bioRxiv2025

Temperature-Related Intensity Change (TRIC)-based High Throughput Screening Platform for the Discovery of CHI3L1-Targeted Small Molecules

Chitinase-3-like 1 (CHI3L1) protein is a secreted glycoprotein involved in various normal physiological processes, while its abnormal elevation is closely associated to carcinogenesis. CHI3L1 promotes tumor progression by recruiting and polarizing immune cells to sustain an immunosuppressive microenvironment, while directly stimulating cancer cell proliferation and migration. Although CHI3L1 deletion has shown therapeutic potential in animal models, the development of small molecule modulators remains limited. To address this gap, we developed a TRIC-based high-throughput screening (HTS) platform for CHI3L1 binding and applied it to a 5,280-compound library, yielding 11 binders (hit rate: 0.21%). Of these, three compounds (9N05, 11C19, and 3C13) were validated by surface plasmon resonance (SPR). Among the validated hits, 9N05 showed the strongest binding affinity to CHI3L1 (Kd = 202.3 {+/-} 76.6 M) but only weak inhibition of CHI3L1/galectin-3 interaction. In contrast, 11C19 significantly disrupted this interaction (73.9 {+/-} 7.9% inhibition at 100 M; IC50 = 188.6 {+/-} 0.18 M). This proof-of-concept study establishes TRIC-based screening as a viable approach for identifying CHI3L1-targeted small molecules and supports its use in future modulator discovery.

biophysics↗

bioRxiv2025

Helicobacter pylori genome aggregation database reveals complex evolutionary forces shaping its genomic landscape and clinical impact in East Asia

We assembled HPgnomAD, a comprehensive global genome aggregation database of Helicobacter pylori (H. pylori) featuring 7,544 high-quality genomes from 278 populations, along with the first species-wide haplotype reference panel for genotype imputation. The panel provides high accuracy across diverse H. pylori populations, including those from low-coverage data, and is openly accessible with integrated analysis tools (https://www.hpgnomad.top/). Variant discovery revealed 1.82 million SNPs and 0.65 million InDels, with African strains exhibiting the most tremendous diversity and East Asian strains showing unexpectedly high novel variation. Fine-scale analysis of the hpEAsia lineage revealed six new sublineages shaped by altitude-, latitude-related divergence and region-specific admixture. Phylogenomic dating revealed two divergence waves in East Asia, paralleling Upper Paleolithic settlement and Neolithic human expansions, with highland-lowland separation at [~]18.8 kya, followed by the formation of complex geography-related population substructures. Genome-wide scans revealed adaptive loci related to metal acquisition, nitrogen metabolism, surface adhesion, and membrane transport, including altitude-associated highly differentiated variants linked to antibiotic resistance and gastric disease. By integrating genomic, evolutionary, environmental, and clinical data, HPgnomAD offers a framework for understanding H. pylori evolution, host-pathogen adaptation, and precision medicine worldwide.

evolutionary biology↗

bioRxiv2025

Spatial Transcriptomics Reveal Developmental Dynamics of the Human Cerebral Cortex and Striatum

The human fetal brain undergoes morphological changes that contribute to the development of regional functionalities. However, the features of structural development, the underlying molecular and cellular signatures in the fetal brain remain unclear. With spatial transcriptomics and snRNA-seq, we identified 25 forebrain regions and characterized the dynamic changes in the cortex and striatum during the late first and early second trimesters. In particular, we discovered that temporal lobe enriched NPY-expressing L2/3 EX neuron potentially interacted with L4 EX neurons during cortical expansion and arealization. Additionally, the gyrus and sulcus were developmental asynchronous, in which HOPX and SPARC genes were potentially involved. Further investigation on the striatum showed specific genes and cell types that enriched in patch and matrix compartments, and SST-positive interneurons potentially involved in the development of these structures. Together, our results give insights into the understanding of early fetal brain development.

bioinformatics↗

bioRxiv2025

Form Follows Function: A New Paradigm of Pancreatic Cancer Progression

Pancreatic ductal adenocarcinoma (PDAC) exhibits a distinctive propensity to invade nearby organs and infiltrate large blood vessels, even in the absence of distant metastasis. While the genetic and transcriptomic drivers of PDAC progression have been well studied, the mechanisms by which these molecular changes translate into functional, invasive behavior remain largely unknown. Here, we uncover a striking level of tissue organization, characterized by previously unrecognized spatial and geometric properties within and among tumor structures. Leveraging the first large-scale, AI-assisted, human-curated PDAC atlas from hematoxylin and eosin (H&E) images, we annotated, classified, and characterized 144,474 malignant and normal structures from treatment-naive (n=118) and neoadjuvant-treated PDAC patients (n=50). Additionally, we developed a new computational tool, SHAPE, to investigate PDAC aggressiveness through a comprehensive "geometrization" of cancer progression. Using traditional H&E-stained slides and three-dimensional (3D) tissue reconstruction experiments, we observed that invading tumor structures display an eccentric morphology with pronounced local angular coherence. These geometric and spatial properties revealed coherent architectural patterns, with invasive structures closely tracking vessels and nerves as they infiltrate surrounding tissue. Mechanistically, integration of morphological features from 39,045 annotated tumor structures with whole-genome and RNA sequencing data revealed that PDACs with numerous eccentric structures exhibit increased copy number alterations (CNAs), loss of heterozygosity (LoH) on the p-arm of chromosome 17, and a quasi-mesenchymal/basal-like molecular subtype. Spatial transcriptomic analysis of 1,650 tumor structures from six additional PDAC patients further confirmed upregulation of invasive cellular programs within highly eccentric structures, such as epithelial-to-mesenchymal transition (EMT), angiogenesis, coagulation, and complement pathways, underscoring their infiltrative nature. Finally, cross-validation of our AI-based method enabled a fully automated, highly interpretable computational approach to assist pathologists and clinicians in evaluating neoadjuvant chemotherapy response, predicting patient survival, and guiding chemotherapy in adjuvant settings. Collectively, these findings deepen our understanding of PDAC progression, identify a new hallmark of tumor architecture, and pave the way for full integration of AI-driven morphology-based approaches into clinical workflows to improve the management of PDAC patients.

cancer biology↗

bioRxiv2025

Structure-Guided Discovery of CHI3L1 Inhibitors from Ultralarge Chemical Spaces for Glioblastoma Therapy

Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival of approximately one year and limited therapeutic options. Chitinase 3-like 1 (CHI3L1) is increasingly recognized as a promising target in GBM due to its role in tumor progression and immune modulation. In this study, we employed an in-house structure-based screening strategy (SpaceDock) to explore a virtual chemical space of 377 billion compounds for potential CHI3L1 inhibitors. Using a reaction-aware ligand design approach, 60 top-scoring virtual hits were synthesized, and 45 were obtained with sufficient purity for experimental testing. Primary screening by microscale thermophoresis (MST) identified nine hits. Compounds showing dose-dependent binding were subsequently analyzed using surface plasmon resonance (SPR), leading to the identification of compound 9e with a dissociation constant (Kd) of 19.11 {micro}M. Importantly, 9e demonstrated robust, dose-dependent efficacy in a multicellular 3D GBM spheroid model, significantly reducing spheroid viability and downstream STAT3 signaling. These results highlight 9e as a promising drug candidate for modulating the CHI3L1-STAT3 axis and underscore the potential of structure-guided, reactivity-aware virtual screening of ultralarge chemical spaces to target non-enzymatic, conformationally dynamic proteins in complex cancer models.

pharmacology and toxicology↗

bioRxiv2025

Comparison and Dynamic interaction between Auditory Cortex and Prefrontal Cortex of Behaving Monkeys during Novelty Detection

The ability to detect deviations from expected sensory input is fundamental for adaptive behavior. We recorded electrocorticographic activity from the auditory (AC) and prefrontal (PFC) cortices of behaving macaques during an auditory oddball task to probe the cortical dynamics of predictive processing. Repetition of standard stimuli evoked suppression and facilitation in AC and strong low-frequency (2 Hz) enhancement in PFC, accompanied by bidirectional delta-band coupling indicative of a shared predictive state. Deviant stimuli triggered early AC responses followed by PFC activation and increased feedforward and feedback connectivity across delta, theta, and gamma bands. Behavioral engagement amplified both prediction and prediction error signals, strengthening cortical network coordination. Together, these findings reveal a hierarchical predictive network in which the AC encodes sensory regularities and violations, while the PFC integrates predictive context in a behaviorally dependent manner.

neuroscience↗

bioRxiv2025

Variation in virion phosphatidylserine content drives differential GAS6 binding among closely related flaviviruses

Many enveloped viruses engage phosphatidylserine (PS) receptors to enter cells, a phenomenon known as "apoptotic mimicry". We previously reported that Zika virus (ZIKV), but not closely-related West Nile virus (WNV) or dengue virus (DENV), utilized AXL to infect cells because only ZIKV could bind the AXL ligand GAS6, a PS-binding protein. In this study, we investigated the mechanisms underlying the differential ability of these viruses to bind GAS6. Although immature virions expose larger patches of the viral membrane than do mature ones, our data show that virion maturity levels did not contribute to GAS6 binding. Surprisingly, while ZIKV contains PS comparable to cellular membranes, PS on WNV and DENV is markedly reduced. These findings explain why only ZIKV can bind GAS6 and provide insights into a novel mechanism by which closely-related flaviviruses differentially utilize cellular entry factors. IMPORTANCEAmong flaviviruses, Zika virus uniquely causes microcephaly and congenital defects. While no flavivirus-specific entry receptors have been identified, they commonly take advantage of phosphatidylserine (PS) receptors to enter cells. Our previous studies revealed that Zika virus uniquely utilizes AXL, found in immune-privileged sites such as the brain and placenta, via binding to its ligand, GAS6. Our current study shows that despite being produced from the same cells, Zika virus has substantially higher PS content than closely related dengue and West Nile viruses, which likely explains Zika viruss unique ability to bind GAS6. These findings provide insight into how closely related flaviviruses can vary substantially in their use of cellular entry factors, potentially contributing to the distinct diseases they cause.

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↗