Biology subjects

Liu, M.

Publications and source records attributed to Liu, M..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Commensal taxa in gut microbiota limit antibiotic resistance during extended oral antibiotic use

Certain bacterial infections, such as those involving prosthetics, can require antimicrobial therapy over months to years, potentially increasing the burden of antimicrobial resistance. Here we longitudinally track the antimicrobial resistome in mice during continuous antibiotic dosing over 21 months. The burden of antibiotic resistance genes (ARGs) initially increases, but, surprisingly, declines in later months, approaching levels observed in untreated animals. ARG burden is regulated by taxonomy and declines as ARG-harboring taxa that initially bloom are replaced by commensals. Furthermore, we find that the dynamics of antibiotic-induced ARG burden are influenced by age-related differences in microbial taxonomy and can be removed by fecal microbiota transplantation. We show that commensals may regulate the resistome by limiting the growth of ARG-harboring taxa, thereby providing antimicrobial expansion resistance.

microbiology↗

bioRxiv2025

Subtype-specific neutralizing antibodies promote antigenic shift during influenza virus co-infection

Reassortment between different influenza strains occurs when they co-infect the same host cell. The emergence of a reassortant virus depends on both its intrinsic fitness and extrinsic factors, including pre-existing humoral immunity. The generation of pandemic strains, such as H2N2 and H3N2, and zoonotic influenza A viruses, like H5N6, H5N8, and H7N9, in birds is suggested to be the result of extensive selection by pre-existing antibodies. To further explore the role of humoral immunity in reassortment, we generated two divergent fluorescent protein-expressing viruses and used strain-specific and cross-reactive monoclonal antibodies (mAbs) to assess the impact of cross-immunity on reassortment. Our results indicate that all mAbs altered the genotypic diversity and significantly reduced the release of progeny virions in co-infected cells both in vitro and in vivo. Moreover, antibody transfer studies in mice revealed protection from challenge with divergent pathogenicity profiles. Notably, selection driven by a strain-specific mAb depended on its neutralizing specificity, whereas the selection driven by broadly reactive mAbs was independent of neutralization specificity. Our findings demonstrate that pre-existing neutralizing antibodies shape reassortment and that strain-specific neutralizing antibodies promote antigenic shift during co-infection, which is not the case for broadly cross-reactive antibodies that recognize influenza viruses from different subtypes.

microbiology↗

bioRxiv2025

scDIAGRAM: Detecting Chromatin Compartments from Individual Single-Cell Hi-C Matrix without Imputation or Reference Features

Single-cell Hi-C (scHi-C) provides unprecedented insight into 3D genome organization, but its sparse and noisy data pose challenges in accurately detecting A/B compartments, which are crucial for understanding chromatin structure and gene regulation. We presented scDIAGRAM, a data-driven method for annotating A/B compartments in single cells using direct statistical modeling and graph community detection. Unlike existing approaches, scDIAGRAM operates without relying on external information, such as the CpG density or imputation techniques, and preserves cell-to-cell heterogeneity. Accuracy and robustness of scDIAGRAM were illustrated through simulated scHi-C datasets and a human cell line. We applied scDIAGRAM to real scHi-C datasets from the mouse brain cortex, mouse embryonic development, and human acute myeloid leukemia (AML), demonstrating its ability to capture compartmental shifts associated with transcriptional variation. This robust framework offers new insights into the functional roles of chromatin compartments at single-cell resolution across various biological contexts.

bioinformatics↗

bioRxiv2025

RadD from Fusobacterium nucleatum Engages NKp46 to Promote Antitumor Cytotoxicity

Fusobacterium nucleatum, a Gram-negative bacterium implicated in periodontal disease, has emerged as a contributor to tumor progression in various cancers. Whether the presence of Fusobacterium nucleatum inhibits tumor progression is largely unknown. Here, we identify a subspecies-specific interaction between F. nucleatum and the natural killer (NK) cell receptor NKp46. Analysis of TCGA datasets revealed that the co-occurrence of F. nucleatum and high NKp46 expression correlates with improved survival in head and neck cancers but not in colorectal cancers. Using binding assays, we demonstrate that both human NKp46 and its murine ortholog, Ncr1, directly recognize the fusobacterial adhesin RadD. Genetic deletion of radD or blockade of NKp46 significantly impaired NK cell-mediated cytotoxicity in vitro and promoted tumor growth. In vivo infection with F. nucleatum accelerated tumor progression, with an exacerbated effect observed in the absence of RadD or NKp46. These findings highlight RadD as a critical ligand for NKp46 and establish the NKp46-RadD axis as a key interface in host-microbe-tumor interactions, offering a novel target for immunotherapeutic intervention in cancer influenced by microbial factors.

immunology↗

bioRxiv2025

CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer's Disease Susceptibility

The innate immune gene CD33, encoding a myeloid inhibitory sialic acid-binding receptor, is associated with Alzheimers disease (AD) susceptibility. The AD-associated rs3865444CC risk variant reduces splicing of the sialic acid-binding domain and increases expression of the full-length (sialic acid-binding) CD33 isoform seven-fold compared to the rs3865444AA protective genotype. Here we identify CD45 as an immune cell-specific sialic acid-dependent cis CD33 binding partner, whose phosphatase activity is inhibited by CD33. Overexpression of CD33 or loss of CD45 contributes to impaired microglial clearance of amyloid beta and amyloid beta-induced loss of dendritic spines in microglial-neuronal co-cultures, aligning with a detrimental effect of CD33-mediated inhibition of CD45. CD33-CD45 interaction frequency was increased in monocytes from individuals with the rs3865444CC risk variant compared to rs3865444AA, as well as in AD compared to controls, independent of genotype. Furthermore, an interaction between CD33 and PTPRC (encoding CD45) gene expression in human brain tissue was associated with a pathological diagnosis of AD and global burden of AD pathology. Our findings thus establish a functional interaction between CD33 and CD45 relevant to AD susceptibility and systemic myeloid dysfunction in this disease.

neuroscience↗

bioRxiv2025

CD33 and Clusterin Interact Biophysically and Genetically to Modulate Alzheimer Risk

We report the results of structural, functional and genetic studies on the CD33 sialic acid- binding receptor that reveal how non-coding variants in CD33 alter risk for Alzheimers disease (AD). The full-length CD33M isoform, whose expression is upregulated by non-coding AD-risk alleles, preferentially forms dimers at the cell surface, where they interact with AD-related proteins (clusterin and A{beta}). This interaction induces CD33M inhibitory signalling and downregulates protective microglial functions including phagocytic removal of amyloid plaques. Human brain expression quantitative trait loci (eQTL) and causal mediation analyses confirm that quantitative interactions between CLU and CD33 genotypes modulate AD phenotypes and suggest that genotypes at these loci might be used to personalise future therapeutic approaches. Our work also highlights several other unexpected aspects of CD33 biology, including a soluble shed extracellular fragment of CD33M and a similar soluble secreted product arising from a truncating mutation in the CD33 extracellular domain (CD33M{Delta}4bp).

neuroscience↗

bioRxiv2025

Integrated small and long RNA sequencing in single oocytes reveals piRNA-mediated transposon repression during human oogenesis

The piwi-interacting RNA (piRNA) pathway plays a pivotal role in controlling transposable element (TE) activity, which is crucial for the developmental competence of gametogenesis. Although piRNAs have been studied in golden hamsters and other representative mammals, little is known about the relationship between distinct piRNA populations and their regulatory effects on TEs in human oocytes. In this study, we simultaneously profiled small and long RNA transcriptomes in individual human oocytes across four developmental stages. piRNAs, especially PIWIL3-associated short piRNAs (short-piRNAs), are the predominant small non-coding RNAs during human oogenesis. A marked increase in short-piRNAs after the primordial follicle stage coincided with a global downregulation of TE expression, particularly LINE-1 (L1) and endogenous retroviruses (ERVs). On the other hand, PIWIL1- and PIWIL2-associated long piRNAs (long-piRNAs) were correlated with the silencing of certain specific ERV subfamilies. Genomic-context analyses revealed that highly productive piRNA clusters have evolved asymmetric antisense insertion bias toward L1 and ERVs, contributing to TE families-specific regulation. Our findings highlight the global effect of piRNA-mediated TEs repression, with short-piRNAs acting as the primary and broad-spectrum suppressors, and long-piRNAs providing coordinated ERV-specific silencing. In summary, this study provides a valuable dataset of small and long RNA co-expression landscapes in developing human oocytes and offers insights into the coordinated yet distinct roles of different PIWI/piRNA classes in repressing TEs during human oogenesis.

genomics↗

bioRxiv2025

PRISM: A platform for illuminating viral dark matter

The exploration of bacteriophage diversity remains constrained by reliance on conventional plaque assays, which bias discovery toward phages that form visible plaques and propagate efficiently. The discovery process, as well as measuring correct phage concentrations and phage resistance frequencies, are also time-consuming and labor intensive. Here, we present PRISM (Phage Recovery and Investigation in Single-droplet Microenvironments), a droplet microfluidics-based, plating-independent platform for the high-throughput isolation, quantification, and characterization of bacteriophages. By encapsulating phage-host interactions in water-in-oil microdroplets at single-phase resolution, and then coupling them with fluorescence-based detection and sorting at million-droplet scale, PRISM enables rapid identification of novel phages, including those that evade detection in conventional assays. We demonstrate PRISMs capabilities across several key applications: (i) recovery of both novel "plaquing" and "non-plaquing" Salmonella phages from a concentrated environmental sample, where PRISM recovered multiple representatives of a non-plaquing genus completely missed by conventional plating-based approaches; (ii) accurate titering of poor-plaquing phages such as Rhodococcus phage ReqiDocB7, for which PRISM reported a 2-fold higher titer than conventional plaque assays; and (iii) plating-free determination of phage resistance frequency, with PRISM yielding resistance and lysogeny frequencies (e.g., 1 in 4.4 x 105 cells for T7 Escherichia phage resistance and 1 in 21 cells for Escherichia Lambda phage lysogeny) that closely match conventional estimates. These data highlight PRISMs enhanced resolution and accuracy, as well as its ability to detect rare, non-plaquing, or slow-replicating phages, in a high speed and automated fashion, thereby underscoring the platforms power to access viral "dark matter" traditionally overlooked using conventional methods.

microbiology↗

bioRxiv2025

FLEXTAG: A Small and Self-renewable Protein Labeling System for Anti-fading Multi-color Super-resolution Imaging

Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (Fluorescent Labeling for Exchangeable, X-resilient Tagging in Advanced Generic Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12-18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports unprecedented durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed a novel protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.

bioengineering↗

bioRxiv2025

Genotype-Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

Spastic Paraplegia 4 (SPG4) is the most prevalent form of Hereditary Spastic Paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and debilitating gait impairment, primarily driven by axonal degeneration of corticospinal motor neurons (CSMNs). Caused by mutations in the SPAST gene encoding spastin, an AAA-ATPase involved in microtubule severing and intracellular organelle function, SPG4 accounts for 40-50% of autosomal dominant HSP cases, yet without effective treatments. Although reduced microtubule acetylation has emerged as a key pathological mechanism, whether and how distinct mutations lead to microtubule deacetylation and subsequent neurodegeneration remains unclear. To address this, we generated isogenic human induced pluripotent stem cell (hiPSC) lines with two distinct heterozygous SPAST mutations - SPASTWT/C448Y (missense) and SPASTWT/S245X (truncation). Employing an innovative differentiation protocol, we created human motor cortical organoids enriched in CSMNs, providing a robust platform to study SPG4 pathophysiology. These organoids revealed striking genotype-phenotype distinctions, with mutation-specific variations in CSMN loss, axonal degeneration and neuronal activities, mirroring clinical heterogeneity. Mechanistic studies identified aberrant activation of histone deacetylase 6 (HDAC6), a major neuronal microtubule deacetylase, as a key driver of SPG4 pathology. This dysregulation was specifically attributed to mutant M1-spastin, the longer isoform of spastin. Remarkably, pharmacological inhibition of HDAC6 with Tubastatin A restored microtubule acetylation status and mitigated axonal degeneration in both SPAST-mutant organoids, with corresponding improvements in corticospinal tract integrity and gait deficits validated in SPG4-transgenic mice. Collectively, our study establishes isogenic hiPSC-derived motor cortical organoids as a robust human model for corticospinal motor neuron degeneration and identifies HDAC6 hyperactivation as a central pathogenic mechanism and viable therapeutic target in SPG4.

neuroscience↗

bioRxiv2025

An Unexpected Role of Molecular Chaperones in Regulating the Excited States and Activities of Folded Enzymes

Molecular chaperones are recognized for assisting protein folding. Emerging evidence suggests that chaperones also interact with natively folded proteins. Yet their functional impact on folded proteins remains unclear. Here, we show that chaperones can directly modulate the conformational dynamics and catalytic efficiency of enzymes in their native states. Spy and Hsp70 increased lysozyme activity by altering conformational exchanges, and this effect extended across other chaperones and enzymes. Hsp70, Spy, Hsp104, and Hsp20 enhanced the catalytic activities of multiple folded enzymes, including alkaline phosphatase, DNA polymerase Pfu, endonucleases Cas12a/Cas13a, and xylanase. These findings uncover an unanticipated function of chaperones in regulating enzyme function expanding their mechanistic scope beyond folding assistance and suggests opportunities in enzyme engineering, diagnostics, and cellular regulation.

molecular biology↗

bioRxiv2025

Calbindin Stratifies Midbrain Dopaminergic Neurons Governing Distinct Aspects of Locomotion

Despite advances in delineating the molecular diversity and projection patterns of midbrain dopaminergic (DA) neurons, their specific contributions to locomotion and motor learning remain poorly defined. Here, we applied intersectional ablation and chemogenetic approaches to dissect the distinct roles of calbindin-expressing (CALB1+) and non-expressing (CALB1-) DA neurons in locomotion. Using newly engineered intersectional constructs, we ablated CALB1+ or CALB1- DA neurons in the mouse midbrain. Loss of either subtype led to pronounced deficits in the initiation and vigor of voluntary movements, as demonstrated by a reduction in peak speed, acceleration and deceleration of locomotor bouts. Notably, only CALB1- ablation disrupted locomotor learning. Beyond these functional effects, we observed that selective ablation of CALB1 DA neurons induced local microglial activation and was followed by a non-cell-autonomous loss of CALB1- DA neurons, suggesting that CALB1- neurons are more vulnerable to inflammation triggered by CALB1 neuron loss. We then confirmed these findings by performing acute inhibition of either population using inhibitory DREADD hM4Di. CALB1- DA neurons inhibition impaired the initial acquisition of locomotor learning, whereas inhibition of CALB1+ DA neurons disrupted the retention of acquired motor skills from previous days. Moreover, inhibition of CALB1+ DA neurons further impaired the initiation and amplitude of voluntary movements, as well as the velocity and acceleration/deceleration of locomotor bouts. Together, these findings provide causal evidence for functional specialization among molecularly distinct midbrain DA subtypes and reveal new aspects of mesostriatal circuit organization underlying locomotion and motor memory.

neuroscience↗

bioRxiv2025

Potent Efficacy of Computer-Aided Designed Peptide Degrader Drug on PCSK9-Mediated Hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein receptor (LDLR) degradation, which leads to increased levels of low-density lipoprotein cholesterol (LDL-C) and a heightened risk of hypercholesterolemia. Existing therapeutic strategies--such as monoclonal antibodies and gene-editing approaches--are limited by high production costs, potential safety concerns, and limited effectiveness in targeting intracellular PCSK9. Targeted protein degradation (TPD) represents a promising alternative capable of overcoming these limitations. In this study, we utilized computer-aided drug design (CADD) to develop a peptide-based degrader, Cadd4, aimed at selectively reducing PCSK9 levels. Through molecular docking and structural refinement, a peptide sequence with strong binding affinity to PCSK9 was identified. Functional evaluation was conducted both in vitro using human LX-2 cells and in vivo in high-fat diet (HFD)-induced hypercholesterolemic mouse models. Cellular uptake, PCSK9 degradation, and LDLR restoration were assessed via confocal microscopy, western blotting, and biochemical analysis. Safety was evaluated by monitoring liver enzyme levels and general physiological parameters. Cadd4 exhibited efficient intracellular delivery and selective PCSK9 degradation in LX-2 cell. In HFD-fed mice, hepatic PCSK9 expression was reduced by 38%, accompanied by a marked increase in LDLR levels. These changes translated into a 25% reduction in total plasma cholesterol and a 29% decrease in LDL-C concentrations. Biodistribution studies indicated liver-specific accumulation of Cadd4, consistent with its intended site of action. Experiments using human liver tissue further confirmed its ability to decrease PCSK9 and enhance LDLR levels. No signs of liver toxicity or adverse systemic effects were observed throughout the study. This work introduces Cadd4 as a CADD-designed peptide-based degrader capable of lowering plasma cholesterol by promoting LDLR recovery via PCSK9 degradation. Its intracellular mechanism of action addresses key gaps in current PCSK9-targeted therapies and supports the broader utility of TPD platforms in managing hypercholesterolemia. These findings offer a foundation for developing cost-effective, scalable therapies within the context of precision cardiovascular medicine.

pharmacology and toxicology↗

bioRxiv2025

Tumor Cells Enriched for Interferon and Inflammatory Programs Pre-Exist in High Grade Serous Ovarian Cancer and are Proportionately Significantly Increased Post Chemotherapy

Drug-tolerant, high-grade serous ovarian cancer (HGSOC) cells that persist after first-line chemotherapy and subsequently relapse often retain sensitivity to secondary treatment, suggesting a therapeutic window before stable chemoresistance emerges. We performed single-cell RNA sequencing (scRNA-seq) on seven matched pairs of tumors, collected pre- and post-chemotherapy, to define vulnerabilities in these reversibly-resistant cells. Treatment induced a marked enrichment of tumor and stromal cell populations expressing correlated interferon (IFN) and inflammatory (IFM) gene signatures, with a concurrent depletion of proliferation-related and MYC-associated states in the tumor cells. Cross-cohort single cell sequencing analysis of >130 treatment-naive tumors revealed heterogeneity in the abundance of IFN/IFM-expressing cells. Multiplex immunofluorescence imaging of IFN-stimulated gene (ISG) products confirmed the presence of spatially clustered ISG-positive tumor cells in all cases, as well as in serous tubal intraepithelial carcinomas (STIC lesions), the presumptive HGSOC precursors. ISG expression correlated strongly with ORF1P, a protein encoded by the endogenous retrotransposon LINE1. These data suggest that early oncogenic events drive LINE1 derepression and innate immune activation, establishing an IFN-rich transcriptional state that persists in tumor subpopulations and is strongly enhanced by chemotherapy.

cancer biology↗

bioRxiv2025

The bifunctional dynamin-like GTPase switch DynAB modulates both vegetative and sporulation cell division in Streptomyces.

As part of their complex developmental lifecycle, the multicellular filamentous bacteria Streptomyces have evolved two modes of cell division distinct from binary fission. In vegetative cell division, irregularly spaced cross-walls form within vegetative hyphae, whereas in sporogenic hyphae, regularly spaced sporulation septa form synchronously, leading to spore formation. However, the mechanisms controlling these processes largely remain unknown. The bacterial dynamin-like protein pair DynAB was previously reported to be a component of sporulation septa. Here, we reveal that DynA and DynB have critical roles in both cell division modes, regulating the formation of sporulation septa and cross-walls, with the Streptomyces cell division protein SsgB modulating their activities. dynAB deletion and overexpression, respectively, increased and abrogated cross-wall formation in vegetative hyphae, and Streptomyces DynAB also inhibited cell division in Escherichia coli, resulting in formation of unicellular filamentous cells and suggesting an ancient and conserved function for these proteins. Notably, SsgB could relieve the inhibition of cell division by DynAB in both Streptomyces and E. coli. In contrast to DynAB, SsgB overexpression generated spore-like compartments in vegetative hyphae, a phenomenon that required disruption of the DynAB complex. Fluorescent tags revealed dynamic localization of DynAB during development, and further analyses indicated that, in sporogenic hyphae, the timing of DynAB expression, their GTP-binding activity, and interaction with SsgB were associated with the synchronous initiation of sporulation septation. Our findings establish DynAB as an integrator of spatiotemporal cues in bacterial multicellularity and provide insights into the evolution of complex cell cycle regulation in prokaryotes. SignificanceThe evolution of multicellularity in bacteria involved the development of complex mechanisms to spatially and temporally coordinate cell division. Streptomyces, renowned for their production of bioactive secondary metabolites, exemplify bacterial complexity. This study reveals how Streptomyces employ the dynamin-like proteins DynAB as a bifunctional molecular switch, enabling transition between vegetative and reproductive growth through integrated interactions with developmentally regulated proteins and the availability of the energy supplier GTP. Importantly, DynAB could repress cell division in both Streptomyces and Escherichia coli, suggesting an evolutionary origin of these proteins prior to bacterial multicellularity. Our work reveals the dual function and switch mechanism of DynAB in Streptomyces, providing insights into the emergence of dynamic cell cycle control in prokaryotes.

microbiology↗

bioRxiv2025

Plasma Membrane Damage by Environmental Materials Enhances Cell-Cell Fusion and Impairs Immune Functions of Macrophages

Macrophages are the most abundant phagocytes and play an essential role in host defense. Previous studies have shown that many environmental materials can activate macrophages and trigger inflammatory responses. However, whether these exposures alter macrophage function in host defense remains unclear. This study found that many environmental materials (such as carbon nanotubes, tungsten carbide [WC], and detergents) can damage the plasma membranes of macrophages. This damage leads to decreased reactive oxygen species (ROS) production and phagocytosis but elevated cell-cell fusion. In vivo, airway exposure to laundry detergent impaired the recruitment of macrophages and other myeloid cells to the lung and dramatically dampened protective TH1 and TH17 cell responses, leading to increased susceptibility to Candida infection in mice. Overall, our data indicate that exposure to environmental materials compromises macrophage membrane integrity and impairs host defense. These findings may aid in the development of effective preventive and therapeutic strategies.

immunology↗

bioRxiv2025

UniCure: A Foundation Model for Predicting Personalized Cancer Therapy Response

Predicting drug efficacy across diverse patient contexts remains a major challenge in oncology, as models trained on cancer cell lines often fail to capture patient-specific biology. Emerging biological foundation models and patient-derived technologies offer a promising solution. Here, we present UniCure, the first pre-trained foundation model integrating both omics and chemical foundation models (UCE and Uni-mol) to predict transcriptomic responses to drugs across diverse cellular and tissue contexts, enabling personalized cancer therapy and drug prioritization at the individual level. Rather than encoder/decoder used in traditional models, UniCure utilizes parameter-efficient fine-tuning (PEFT) techniques for optimizing the training process, a novel FlexPert module for modeling flexible drug-cell interactions, and a Maximum Mean Discrepancy (MMD) loss for learning unpaired data. Trained on over 1.8 million perturbation RNA-seq profiles over 22,000 compounds, 166 cell types, and 24 tissues, UniCure achieves high accuracy in predicting both dose-dependent responses and drug combination effects, demonstrating strong generalization across bulk and single-cell transcriptomic data. In particular, fine-tuning on our patient-derived tumor-like clusters and real-world data of 800 profiles enables UniCure to generate individualized therapeutic predictions on patients tissue samples. In addition, UniCure enables patient stratification based on the predicted drug responses, providing a new way for subtyping patients. UniCures drug prioritization was validated across over 1000 patients from pan-cancer cohorts and supported by experiments of candidate therapeutics. By enabling the potential to screen millions of compounds per patient at scale, UniCure represents a biologically grounded tool that could advance personalized precision oncology and accelerate drug discovery.

bioinformatics↗

bioRxiv2025

Modeling Adeno-Associated Viral Vector 6-mediated In Vivo Gene Delivery to Expanded Non-Mobilized Haemopoietic Stem Cells from Transfusion dependent Thalassemia Patients in a Humanized Mouse

Hematopoietic stem cells (HSC) are important targets for gene modification therapies (GMT) as they originate several serious genetic conditions including the {beta}-haemoglobinopathies. Potentially curative ex vivo GMT pose the barriers of accessibility, myeloablation-associated morbidity and prohibitive cost. In vivo GMT using non-integrating single-strand adeno-associated viral vectors (ssAAV) are a promising alternative that address these challenges directly, although the small ssAAV payload limits the capacity to package much larger gene or base editors. We investigated the feasibility of targeting human HSC in vivo with a dual-ssAAV6 strategy, which in future may be useful to deliver split-intein editing tools to overcome this limitation. We engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ humice with human hCD45+CD34+ HSC from transfusion-dependent {beta}-thalassemic patients to test in vivo targeting of hCD45 cells with ssAAV6, then administered 5E+12 genomes/kg of ssAAV6-GFP/ssAAV6-mCherry. Humice showed peak single-transgene expression (GFP+ or mCh+) of 1.96-10.17%, and dual-transgene expression (GFP+mCh+) of 31.77% in circulating hCD45+ cells. Nested hCD45+ from liver, spleen and bone marrow showed single-and dual-transgene expression of 36.13-68.14% and 21.91-59.44% respectively. Secondary transplantation experiments demonstrated long-term persistence of AAV6-transduced hCD45 cells showing single-and dual-transgene expression of 9.19-60.72% and 7.15-9.19% respectively, with significant increase in expression from circulating cells. Minimal pro-inflammatory cytokine expression was observed following ssAAV6 administration in thalassemia humice compared with humice carrying non-thalassemia HSC. Our model demonstrates the efficiency of in vivo ssAAV6-mediated targeting of thalassaemia HSC, potential long-term survivability of transduced cells, and feasibility of a dual-AAV strategy for gene editing, which offers a promising alternative to ex vivo GMT for {beta}-haemoglobinopathies.

molecular biology↗