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Chen, X.

Publications and source records attributed to Chen, X..

At least 217 records · Page 12Linked to original sources

Scattering-enabled epi-quantitative phase imaging reveals subcellular detail in organoids and deep mouse brains

Imaging subcellular structures deep within thick, turbid biological tissues remains fundamentally limited by light scattering, which distorts optical wavefronts and degrades contrast, resolution, and sensitivity. These limitations hinder quantitative interrogation of complex biological systems where resolving dynamic microenvironments at subcellular resolution is critical. Here, we introduce scattering-enabled epi-quantitative phase imaging (SEEQPI), a label-free method that leverages tissue scattering and provides subcellular spatial resolution, nanometer-scale spatiotemporal phase sensitivity, and millimeter-scale imaging depth in murine brains. SEEQPI is enabled by common-path phase-shifting confocal epi-interferometry with near-infrared illumination and the scattering-enabled phase reconstruction algorithm. SEEQPI requires low illumination power, minimizing tissue damage while enabling high-speed imaging of biological dynamics. We demonstrate simultaneous, colocalized imaging of subcellular structures with SEEQPI, third-harmonic generation, and three-photon fluorescence microscopy in liver cancer spheroids and in vivo mouse brains. SEEQPI enables quantitative, longitudinal studies of dry mass dynamics in intact, living biological systems.

bioengineering↗

Phosphatidylserine exposure by developing astrocytes initiates microglia-mediated developmental cell death

Developmental cell death is classically attributed to apoptosis, yet in mammalian retina, large numbers of developing astrocytes die non-apoptotically during a defined developmental window. Astrocyte death is important for patterning a cellular template that guides angiogenesis, but the underlying mechanism remains unknown. Here we show that healthy developing astrocytes initiate their own elimination by recruiting microglia via regulated exposure of the membrane lipid phosphatidylserine. Experimentally increasing phosphatidylserine exposure in astrocytes, but not neurons, accelerates their removal by microglia without changing how many astrocytes ultimately survive. This acceleration causes profound vascular defects resembling pathological features of retinopathy of prematurity. Genetic disruption of MFGE8, a phosphatidylserine-binding protein, suppresses microglia-mediated astrocyte killing and prevents vascular pathology despite continued phosphatidylserine exposure. This mechanism extends beyond the retina, because phosphatidylserine also initiates astrocyte death in developing cerebral cortex. Together, these findings identify phosphatidylserine exposure as a developmental signal that times microglia-mediated astrocyte elimination, with essential consequences for neurovascular development.

neuroscience↗

Hierarchical Reconfiguration of Neurocognitive Task Set Representations Mediates Cognitive Flexibility

Cognitive control organizes abstract contexts, stimuli, and actions into hierarchically structured representations. This organization supports flexible behavior but requires updating at multiple levels of the hierarchy, a process reflected in task switch costs. However, it remains unclear how updating differs across levels of abstraction and how these differences relate to behavior and neural representations. Here, we investigated the behavioral and neural sources of switch costs using fMRI and behavioral data from healthy adult participants (male and female). We employed a hierarchical control task that dissociates abstract context reconfiguration from more concrete task-set reconfiguration of stimulus-response mappings. We predicted that task sets, which incorporate sensory-motor mappings, would be more strongly influenced by feedforward inputs than higher-level contextual goals, which are more abstracted from immediate perceptual and motor demands. As predicted, subordinate rule switches were faster and were more strongly influenced by task-irrelevant perceptual changes, whereas context switches were slower and relatively insensitive to such interference. To characterize the neural basis of these effects, we quantified trial-to-trial reconfiguration of multivoxel activity patterns. Across the brain, larger pattern shifts predicted larger RT switch costs, linking representational reorganization to behavioral performance. Importantly, representational reconfiguration differed across hierarchical levels and anatomical systems. Subordinate rule updating was modulated by task-irrelevant perceptual features and expressed in distributed perceptual and motor networks, whereas context reconfiguration engaged the mid-lateral frontal cortex and was comparatively insulated from interference. Our results reveal how the hierarchical structure of neural representations supports flexible updating with interference-shielded contextual representations subserving behavioral control. SignificanceDaily activities often require simultaneously updating different tasks and thoughts. Driving requires maintaining a stable destination goal while rapidly updating motor plans (brake, accelerate, turn, etc.) in response to changing perceptual information (traffic lights, pedestrians, etc.). Although people perform such tasks with ease, it remains unclear how neural and cognitive representations are structured to respond to these flexibility demands. The present study suggests that sensory-motor plans prioritize flexibility by allowing greater influence from sensory inputs, which can create interference across brain networks when that input is task-irrelevant. Contextual information is insulated from this interference by representing contexts as distinctly as possible in the lateral prefrontal cortex, resulting in slower but more stable context switching.

neuroscience↗

Identification of an epigenetically and phenotypically distinct peritumoral glioblastoma cell population linked to inferior patient outcome

Glioblastoma (GB) is an aggressive and therapy-resistant primary brain tumor with dismal prognosis. Lethality is in most patients caused by a local peritumoral (Edge) relapse near the resection cavity. This region is insufficiently studied and experimental models are scarce. We have analyzed matched tissue samples and cell cultures from bulk tumors and Edge regions of 11 GB patients. Genomic profiling displayed similar genetic alterations and subclonal distributions of matched bulk and Edge regions. Functional, phenotypic and combined single nucleus (sn) RNA-seq and ATAC-seq analyses showed distinct differences between bulk and Edge GB cells with similar shifts across patients. Sn multiomics uncovered a subset of Edge cells defined by unique chromatin accessibility signatures and elevated mesenchymal and immune gene expression. Integrated computational and functional investigations converged on microglia-derived Oncostatin-M as a driver of the Edge-unique phenotype, and high expression of Edge-related signatures correlated with inferior patient outcome in independent GB cohorts.

cancer biology↗

Climate drives plasticity and genetic adaptation in apple trees

BackgroundPerennial crops respond to climate change through phenotypic plasticity and local adaptation, yet how these responses are orchestrated at the molecular level and reshaped by domestication remains poorly understood. ResultsWe grew 1,249 seedlings from five populations of M. domestica, M. orientalis, and M. sylvestris under four simulated European climates, quantified 12 phenotypic traits, performed RNA sequencing on 96 individuals, and integrated these data with previously published genome-wide polymorphism data from M. sylvestris to disentangle conserved climate responses from local adaptation. Climate was the dominant driver of phenotypic and transcriptional variation, revealing 344 conserved climate-responsive genes enriched for stress responses, nutrient metabolism, and cell wall biosynthesis. These genes are under strong purifying selection and carry fewer deleterious mutations. In contrast, genome-environment association analyses identified 217 loci associated with temperature and precipitation gradients, only a subset of which showed climate-responsive expression and elevated genomic differentiation. Population-level expression divergence closely mirrored neutral genetic differentiation, indicating that most transcriptomic divergence reflects demographic history rather than adaptive regulatory evolution. Domestication reshaped molecular diversity and mutation load without disrupting the conserved climate-response program. ConclusionsClimate responses in apple are primarily mediated by an evolutionarily conserved transcriptional program maintained by strong purifying selection, whereas local adaptation relies on largely distinct, population-specific loci. These findings reveal that conserved plasticity constitutes the dominant molecular response to climatic variation, whereas local adaptation contributes a secondary, population-specific layer of evolutionary change.

evolutionary biology↗

Reliability and validity of multi-band multi-echo fMRI

Precision mapping of individual differences in human brain spontaneous activity is critical for characterizing brain function and guiding clinical translation. Multi-echo resting-state functional MRI (ME-rfMRI) has the potential to provide more reliable mapping of inter-individual differences in intrinsic brain function than conventional single-echo rfMRI (SE-rfMRI). However, the psychometric benefits of ME-rfMRI for mapping individual differences in spontaneous brain activity (SBA) have not been systematically investigated, which is fundamental for understanding the human brain in health and disease. To quantify the psychometric performance of ME-rfMRI in precisely mapping inter-individual variations in SBA, we employed a psychometric design. We scanned 27 healthy adults with both ME-rfMRI and SE-rfMRI to assess short-term (minutes) test-retest reliability, as well as validity across eyes-open (EO) and eyes-closed (EC) resting states. Our results demonstrate that ME-rfMRI improves reliability relative to SE-rfMRI by 10.8% for amplitude of low-frequency fluctuation (ALFF), 4.4% for regional homogeneity (ReHo), and 8.0% for voxel-mirrored homotopic connectivity (VMHC) in cortical regions, and by 12.5% for ALFF, 8.3% for ReHo, and 4.0% for VMHC in subcortical regions. This enhancement is achieved by increasing inter-individual variability while reducing intra-individual variability. Notably, ME-rfMRI accentuates inter-individual variability in the unimodal cortex and clearly delineates two parallel systems with distinct variability patterns within the motor cortex. Concurrently, ME-rfMRI increases effect sizes (4%-8%) for detecting individual differences between EO and EC resting states. Interestingly, ME-rfMRI reveals differential effects of eye closure on two intertwined systems in the primary motor and sensory cortices, extending previous findings. Computational simulations indicate that, compared to SE-rfMRI, ME-rfMRI facilitates experimental designs with reduced costs (i.e., 4%-11% smaller sample sizes) for precise mapping of individual differences. These findings establish, for the first time, the psychometric performance of ME-rfMRI measurements, supporting its potential utility in personalized neuroscience applications, including neurodevelopment and brain disorders.

neuroscience↗

Advancing Protein Design via Multi-Agent Reinforcement Learning with Pareto-Based Collaborative Optimization

Protein design is revolutionizing biotechnology, yet existing approaches struggle to balance structural foldability with functional performance. Structure-based models excel at generating stable protein backbones but often overlook critical functional properties, while protein language models capture evolutionary and functional signals but frequently predict sequences lacking structural stability. Integrating these complementary approaches remains challenging due to their inherently conflicting objectives. We present MAProt, a multiagent framework that synergistically combines structure-based and protein language model-based methods for protein design. Each agent specializes in a distinct aspect of the design objective: the structure-based agent (e.g., ProteinMPNN) ensures compatibility with the target backbone, while protein language model-based agents (e.g., ESM, SaProt) capture evolutionary plausibility and functional potential. To reconcile conflicts and achieve optimal trade-offs, we introduce a Pareto-based negotiation module that enables effective multi-objective coordination and consensus among agents. Extensive experiments on benchmark datasets demonstrate that MAProt achieves a remarkable improvement over state-of-the-art baselines, and generalizes robustly across a range of tasks, including thermodynamic folding stability design, functional protein design, and high-affinity antibody design. These results highlight the power of collaborative optimization for advancing rational protein engineering. Codehttps://github.com/biomed-AI/MAProt

bioinformatics↗

caRBP-Pred: Deep Learning-based Prediction of Chromatin-Associated RNA-Binding Proteins Using Short Peptide Sequences

RNA-binding proteins (RBPs) are pivotal in cellular processes ranging from RNA metabolism to 3D genome organization. A distinct subset, chromatin-associated RBPs (caRBPs), binds directly to chromatin to function as transcriptional regulators. However, identifying caRBPs via traditional methods like Chromatin Immunoprecipitation Sequencing (ChIP-seq) and Mass Spectrometry (MS) is labor-intensive and costly. While computational tools for DNA- and RNA-binding protein (DRBP) prediction exist, they often rely on outdated Gene Ontology annotations and fail to capture the unique characteristics of chromatin association. Here, we introduce caRBP-Pred, a novel deep learning approach combining Convolutional Neural Networks (CNN) and Bidirectional Long Short-Term Memory networks (BiLSTM). Unlike previous methods utilizing full-length sequences, our model is trained on chromatin-contact peptides derived from mouse embryonic stem cells (mESCs). caRBP-Pred achieves a superior Area Under the Curve (AUC) of 0.81 using peptide sequence information alone, significantly outperforming existing DRBP predictors which exhibit low recall for caRBPs. We further predicted 52 potential caRBPs in mice. Notably, validation against human homologs confirmed that our model accurately predicts candidates with experimentally verified chromatin-binding capabilities. Collectively, caRBP-Pred is the first tool specifically designed to predict caRBPs based on chromatin-contact peptides, offering a valuable resource for investigating regulatory roles of caRBPs on transcription.

bioinformatics↗

A hypothalamo-septo-hippocampal circuit for REM sleep-dependent consolidation of social memory

REM sleep constitutes a critical window for memory consolidation, yet the brain circuits orchestrating this process remain incompletely defined. Here, we identify a lateral supramammillary nucleus (SuM)-medial septum (MS) projection as a REM sleep-specialized pathway essential for hippocampal memory consolidation. Fiber photometry and optrode recordings revealed that lateral SuM-MS projecting neurons were selectively active during REM sleep. REM-specific optogenetic silencing of this projection impaired consolidation of both social and contextual fear memories. Crucially, silencing of its downstream target, the MS-CA2 pathway, during REM sleep selectively disrupted social memory while sparing contextual fear memory. This functional dissection establishes a hypothalamo-septo-hippocampal circuit (lateral SuM-MS-CA2) dedicated to social memory processing, in parallel to the recently-described direct SuM-CA2 pathway. These results also position the SuM as a REM sleep-hub that routes information via parallel septal pathways to consolidate distinct memory modalities.

neuroscience↗

ANKS1B in the Nucleus Accumbens controls escalated cocaine self-administration via regulating CBP-FoxO3 complex

The transition from controlled to escalated drug intake is a core feature of cocaine use disorder (CUD), yet the molecular mechanisms underlying this behavioral escalation remain poorly defined. Our prior genome-wide association study (GWAS) identified ANKS1B as a significant shared genetic risk factor for heroin, methamphetamine, and alcohol dependence, suggesting a broad role in addiction vulnerability. However, the specific function of ANKS1B in cocaine addiction and its associated neural mechanisms were unknown. Here, we found that ANKS1B expression level in the Nucleus Accumbens (NAc) was downregulated after extended cocaine use, and manipulating ANKS1B could selectively influence the escalation of cocaine intake and the subsequent cocaine-seeking behavior in the long-access cocaine self-administration rat model. Molecular experiments reveal that ANKS1B interacts with the histone acetyltransferase CBP to control H3K27 acetylation and extended cocaine intake, via epigenetically repressing the transcription factor FoxO3. Overall, these findings suggest that ANKS1B is a crucial factor influencing the escalation of cocaine use. The ANKS1B-CBP-FoxO3 signaling pathway presents a promising target for potential therapeutic interventions for controlling extended cocaine use.

neuroscience↗

Python metabolomics uncovers a conserved postprandial metabolite and gut-brain feeding pathway

Most mammals consume small and frequent meals. By contrast, pythons are ambush predators that exhibit extreme feeding and fasting patterns and provide a unique model for uncovering molecular mediators of the postprandial response1-3. Using untargeted metabolomics, here we show that circulating levels of the metabolite para-tyramine-O-sulfate (pTOS) are increased >1,000-fold in pythons after a single meal. In pythons, pTOS production occurs in a microbiome-dependent manner via sequential decarboxylation and sulfation of dietary tyrosine. In both pythons and mice, pTOS administration activates a neural population in the ventromedial hypothalamus (VMH). In mice, these VMH neurons are required for the anorexigenic effects of pTOS. Chronic administration of pTOS to diet-induced obese male mice suppresses food intake and body weight. pTOS is also present in human blood, where its levels are increased after a meal. Together, these data uncover a conserved postprandial anorexigenic metabolite that links nutrient intake to energy balance.

physiology↗

RodZ acts through MreBCD to activate the elongasome in Escherichia coli

Most bacteria are surrounded by a peptidoglycan (PG) matrix that maintains cell shape and provides protection against turgor pressure. In many rod-shaped bacteria, synthesis of PG along the cell cylinder is organized by the elongasome, also called the Rod complex, which consists of six highly conserved proteins, including the actin-like MreB, the PG synthase complex RodA-PBP2 and three regulatory membrane proteins, MreC, MreD and RodZ. However, how these proteins interact with each other to form the elongasome and synthesize lateral PG remains elusive. In this study, by characterizing MreC mutations affecting elongasome activity in Escherichia coli, we provide evidence that MreC alternates between an active and an inactive state and these mutations affect MreC interaction with PBP2. We also find that RodZ interacts with MreC via a periplasmic region and disruption of this interaction compromises elongasome function. Additionally, we show that the cytoplasmic region of RodZ, which interacts with MreB, work synergistically with activating MreC mutations to promote rod shape formation. These results indicate that RodZ activates the elongasome by initiating two signaling cascades, one in the periplasm via MreCD, and another in the cytoplasm through MreB. This role of RodZ in regulating elongasome activity is analogous to that of FtsN in activating the divisome, suggesting that the mechanisms governing the activation of PG synthesis in cell elongation and division are similar in E. coli. ImportanceThe elongasome, or Rod complex, mediates lateral peptidoglycan synthesis during cell elongation in many rod-shaped bacteria. It consists of the cytoskeletal protein MreB, the peptidoglycan synthase RodA-PBP2, and three regulatory proteins MreCD and RodZ. Although it has been extensively studied, how its activity is controlled remains incompletely understood. Here, we reveal the roles of MreCD and RodZ in regulating elongasome activity in Escherichia coli. Our results indicate that RodZ triggers lateral peptidoglycan synthesis by interacting with MreB in the cytoplasm and MreCD in the periplasm. These interactions likely switch MreBCD to the active state such that they can stimulate the activity of the RodA-PBP2 complex. This regulatory mechanism resembles the activation mechanism of the divisome that mediates septal peptidoglycan synthesis.

microbiology↗

Catestatin ameliorates tauopathy and amyloidogenesis via adrenergic inhibition

Neurodegenerative disorders like Alzheimers disease (AD), Corticobasal Degeneration (CBD), and Progressive Supranuclear Palsy (PSP) are characterized by Tau aggregation, synaptic dysfunction, neuroinflammation, and progressive cognitive decline. Although metabolic dysregulation and neuropeptide imbalance have been linked to these disorders, the functional consequences of such imbalance and its potential for therapeutic reversal remain poorly understood. Our previous work identified chromogranin A (CgA), which encodes a pro-hormone for several metabolic peptides, as a key regulator of Tau pathology. Here, we investigate Catestatin (CST), a CgA-derived peptide that is a potent inhibitor of catecholamine release and has been shown to increase insulin sensitivity and lower peripheral blood pressure. We report significant reductions in CST levels in the hippocampus and cortex of AD brains, as well as in the frontal cortex of CBD and the basal ganglia of PSP. Supplementing CST in cortical neuronal cultures and organotypic slice cultures (OTSC) decreased Tau phosphorylation and aggregation. In vivo, CST administration in PS19 Tauopathy mice reduced pathological Tau species, attenuated gliosis, and improved cognitive function. CST treatment also lowered amyloid plaque burden and neuroinflammation in 5xFAD mice. Mechanistically, CST decreased epinephrine (EPI) levels in both PS19 and 5xFAD mice and suppressed downstream protein kinase A (PKA) hyperactivation in PS19 and OTSC. These findings reveal a previously unrecognized neuropeptidergic mechanism linking CST deficiency to elevated adrenergic receptor (ADR)-EPI-PKA stress signaling and Tauopathy-driven neurodegeneration, suggesting CST replacement as a promising therapeutic approach.

neuroscience↗

Microglia stabilize sleep homeostasis via adenosine A3 receptor signaling

Sleep homeostasis maintains the sleep-wake balance through sleep pressure, a process orchestrated by the accumulation of extracellular adenosine (eADO). Microglial Ca{superscript 2} activity has been implicated in sleep regulation, but the mechanism whereby microglia sense sleep pressure remains unclear. Here we show that microglia regulate sleep homeostasis through brain state-dependent Ca2+ activity driven by adenosine A3 receptor (A3R) signaling. Using miniaturized two-photon microscopy (mTPM) in freely behaving mice, we demonstrate that microglial Ca{superscript 2} activity is rapidly altered by brain-state transitions. Pharmacological experiments reveal that microglial Ca2+ dynamics are predominantly mediated by A3R in response to brain state-dependent eADO oscillations. Microglia-specific deletion of A3R attenuates these state-dependent Ca2+ dynamics, impairs microglial morphological plasticity across sleep-wake cycles, and leads to sleep fragmentation by increasing transitions between wakefulness and non-rapid eye movement (NREM) sleep. Together, these findings establish that microglia regulate sleep homeostasis by stabilizing both wakefulness and NREM sleep, a process critically dependent on eADO-A3R signaling.

neuroscience↗

Macrophage-Dendritic Cell-T-Cell Tetrads Orchestrate Antitumor Immunity and Response to Checkpoint Blockade

Immune checkpoint inhibitors (ICIs) elicit durable responses in only a subset of patients with solid tumors, underscoring the need to define the cellular architectures that govern effective antitumor immunity. Here we identify a spatially organized multicellular immune unit comprising macrophages, cDC1s, CD4 T-cells, and CD8 T-cells that emerges in response to anti-CTLA-4 or dual checkpoint blockade. We term these structures tetrads. Using multiplexed imaging and spatial transcriptomics in mouse and human tumors, we show that tetrads assemble early during immune priming, depend on the ICOS-ICOSL pathway, and are enriched for ICOS Th1-like CD4 T cells and ICOSLhigh cDC1s. CD8 T-cells within tetrads exhibit an activated, non-terminally differentiated state, while tetrad-associated macrophages display an interferon-{gamma}-responsive program that sustains CD8 T-cell function and prevents dysfunction. Functionally, ICOSL cDC1s are required for tumor eradication in vivo. In patients with bladder cancer treated with neoadjuvant dual checkpoint blockade, tetrad, but not triad or dyad formation correlates with clinical response. These findings establish tetrads as a fundamental cellular unit coordinating antitumor immunity and responsiveness to ICIs.

immunology↗

PtdIns(3,5)P2 Is an Endogenous Ligand of STING in Innate Immune Signaling

Cytosolic DNA exposure triggers innate immune responses through cyclic GMP-AMP (cGAMP) synthase (cGAS)1-3. Upon binding to DNA, cGAS is activated to produce cGAMP, which functions as a second messenger that binds to stimulator of interferon genes (STING), an endoplasmic reticulum (ER)-localized signaling adaptor3-5. STING then traffics from the ER to the Golgi, leading to activation of the kinases TBK1 and IKK and subsequent induction of interferons and other cytokines6-10. Here we show that phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] is an endogenous ligand of STING that functions together with cGAMP to induce STING activation. Proteomics analysis identified a constitutive interaction between STING and PIKfyve, an enzyme that produces PtdIns(3,5)P2 in mammalian cells. Deletion of PIKfyve blocked STING trafficking from the ER and TBK1 activation. In vitro reconstitution revealed a strong and selective effect of PtdIns(3,5)P2 on STING activation. Purified STING bound directly to PtdIns(3,5)P2 in a fluorescence resonance energy transfer (FRET) assay. Consistently, PtdIns(3,5)P2 promoted cGAMP-induced STING oligomerization by binding to a groove between STING dimers as revealed by cryo-EM (Li et al., co-submitted). Similar to PIKfyve depletion, mutation of the PtdIns(3,5)P2-binding residues in STING blocked its trafficking and downstream signaling. These results reveal PtdIns(3,5)P2 as a lipid ligand of STING with essential roles in innate immunity.

biochemistry↗

A gastric microbial chromatin remodeler drives gastric cancer progression and immune evasion by reprogramming the host epigenome

Emerging evidence highlights the crucial role of microbial communities in modulating cancer development and progression. In gastric cancer (GC), we identify Streptococcus anginosus (SA) as a tumor-resident oncobacterium that drives both tumor progression and immune evasion. We define a novel mechanism in which SA secretes extracellular vesicles (saEVs) that translocate the bacterial chromatin remodeler saSNF2 into host cell. There, saSNF2 partners with host transcription factor TEAD1--through its ATPase activity and participation in BAF complex assembly--to coordinately activate oncogenic transcription. This transkingdom interaction upregulates the palmitoyltransferase ZDHHC11, which in turn stabilizes PD-L1 via palmitoylation to establish an immunosuppressive niche, while also amplifying other TEAD1 target genes to fuel tumor progression. Functionally, saEVs promote tumor growth and limit CD8+ T-cell infiltration in vivo. Strikingly, pharmacological inhibition of ZDHHC11 reverses immune evasion and synergizes with anti-PD-1 checkpoint blockade. Our results establish SA as a multifaceted driver of GC and reveal the saSNF2-ZDHHC11 axis as a promising target to potentiate immunotherapy.

cancer biology↗

Decoupling Efficacy from Toxicity: Engineering Spatial Control in AAV-Mediated Gene Therapy

Hepatotoxicity poses a critical safety challenge for AAV-mediated gene therapy. To mitigate this, we evaluated strategies to minimize off-target hepatic transduction using an antibody expression model. We compared (i) muscle-restricted wild-type AAV9 expression and (ii) a novel myotropic capsid variant, AAV.eM. In humanized B-NDG mice bearing Raji-Luc lymphomas, intravenous administration of AAV9-MHCK7 encoding an CD19CD3 bispecific T-cell engager failed to reduce tumor burden. Conversely AAV.eM-MHCK7-CD19CD3 substantially alleviated tumor burden and achieved lymphoma clearance. By leveraging tissue-specific microRNAs, precise restriction of AAV.eM-mediated transgene expression to skeletal or cardiac muscle was achieved. Incorporating a heart-specific miR-208a binding site into the transgenes 3UTR did not compromise therapeutic efficacy when delivered via AAV.eM-MHCK7. Intramuscular delivery of AAV9-MHCK7-CD19CD3 or AAV.eM-MHCK7-CD19CD3 both cleared Raji-Luc tumors at a dose of 5 x 1012 vg/kg, underscoring the advantage of localized and targeted rAAV delivery over systemic administration. Notably, only AAV.eM-MHCK7-CD19CD3 achieved tumor eradication at a tenfold lower intramuscular dose (5 x 1011 vg/kg), reducing manufacturing costs and risks of dose-dependent immunogenicity and toxicity. Our findings demonstrate that combining tissue-specific targeting--via engineered capsids or tissue-selective promoters--with local delivery robustly reduces off-target hepatic expression, providing a strategic framework for enhancing the safety of AAV-based gene therapies.

cancer biology↗