Biology subjects

Luo, L.

Publications and source records attributed to Luo, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Smartphone-integrated RPA-CRISPR-Cas12a Detection System with Microneedle Sampling for Point-of-Care Diagnosis of Potato Late Blight in Early Stage

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most devastating diseases affecting potato crops in the history. Although conventional detection methods of plant diseases such as PCR and LAMP are highly sensitive and specific, they rely on bulky and expensive laboratory equipment and involve complex operations, making them impracticable for point-of care diagnosis in the field. Here in this study, we report a portable RPA-CRISPR based diagnosis system for plant disease, integrating smartphone for acquisition and analysis of fluorescent images. A polyvinyl alcohol (PVA) microneedle patch was employed for sample extraction on the plant leaves within one minute, the DNA extraction efficiency achieved 56 g/mg, which is [~]3 times to the traditional CTAB methods (18 g/mg). The system of RPA-CRISPR-Cas12a isothermal assay was established to specifically target P. infestans with no cross-reactivity observed against closely-related species (P. sojae, P. capsici). The system demonstrated a detection limit of 2 pg/L for P. infestans genomic DNA, offering sensitivity comparable to that of benchtop laboratory equipment. The system demonstrates the early-stage diagnosis capability by achieving a [~]80% and 100% detection rate on the third and fourth day post-inoculation respectively, before visible symptoms observed on the leaves. The smartphone-based "sample-to-result" system decouples the limitations of traditional methods that rely heavily on specialized equipment, offering a promising way for early-stage plant disease detection and control in the field.

molecular biology↗

bioRxiv2025

A Multimodal Adaptive Optical Microscope For In Vivo Imaging from Molecules to Organisms

Understanding biological systems requires observing features and processes across vast spatial and temporal scales, spanning nanometers to centimeters and milliseconds to days, often using multiple imaging modalities within complex native microenvironments. Yet, achieving this comprehensive view is challenging because microscopes optimized for specific tasks typically lack versatility due to inherent optical and sample handling trade-offs, and frequently suffer performance degradation from sample-induced optical aberrations in multicellular contexts. Here, we present MOSAIC, a reconfigurable microscope that integrates multiple advanced imaging techniques including light-sheet, label-free, super-resolution, and multi-photon, all equipped with adaptive optics. MOSAIC enables non-invasive imaging of subcellular dynamics in both cultured cells and live multicellular organisms, nanoscale mapping of molecular architectures across millimeter-scale expanded tissues, and structural/functional neural imaging within live mice. MOSAIC facilitates correlative studies across biological scales within the same specimen, providing an integrated platform for broad biological investigation.

cell biology↗

bioRxiv2025

Cell-surface proteomic profiling identifies CD72 as a regulator of microglial tiling

Microglial tiling--the phenomenon of consistent cell-to-cell distances and non-overlapping processes--is regarded as a qualitative indicator of homeostasis, but mechanisms of microglial tiling are unknown. We used cell-surface proximity labeling and mass spectrometry to profile the microglial cell-surface proteome in an in vitro model of homeostatic glial physiology and used single-cell RNA sequencing and public databases to identify candidate cell-surface proteins that might modulate tiling. We designed an image-based functional assay which measures six morphological/spatial readouts to screen these proteins for modulation of tiling. CD72, a coreceptor to the B cell receptor that is expressed by microglia, disrupted tiling; we validated its effects in vitro and in situ in organotypic hippocampal brain slices. Phosphoproteomic studies revealed that CD72 modulates pathways associated with cell adhesion, repulsive receptors, microglial activation, and cytoskeletal organization. These results lay the groundwork for further investigation of the functional roles of tiling in homeostasis and disease.

neuroscience↗

bioRxiv2025

NUSAP1 regulates mitotic processes via KIF2C interaction and AURKA phosphorylation in primary microcephaly

Primary microcephaly (PM) is a neurodevelopmental disorder characterized by a significantly smaller head than normal. Despite the identification of several genes associated with PM subtypes, the etiology remains unclear in a significant proportion of patients. Here, we reported two de novo nonsense mutations in the NUSAP1 gene identified in two independent PM families: c.1209 C>G (p.Tyr403*) and c.1213C>T (p.Gln405*). Nusap1-edited mouse models, designed to mimic Human NUSAP1 mutations, replicated the microcephaly phenotype, characterized by a small brain and thinner cerebral cortex. We demonstrated that Nusap1 was specifically expressed in neural stem/progenitor cells (NSPCs), and the number of NSPCs was significantly reduced in Nusap1-edited mice. In vitro experiments revealed that NUSAP1 mutations disrupted mitotic metaphase chromosome alignment, increased polyploidy, induced cell cycle arrest, and promoted apoptosis. Co-IP confirmed direct interactions between NUSAP1 and KIF2C, and showed that truncating mutations impaired their binding. Furthermore, NUSAP1 rescued the cell cycle arrest and increased apoptosis caused by KIF2C overexpression in NUSAP1 KO HEK293T cells, whereas the mutant truncated protein failed to exert such a rescue effect. We also uncovered an interaction between NUSAP1 and Aurora kinase A (AURKA). Intriguingly, subsequent experiments revealed that AURKA-mediated phosphorylation of NUSAP1 modulates its binding affinity to KIF2C. In conclusion, our findings established NUSAP1 as a novel pathogenic gene for PM. We proposed a model in which AURKA-mediated phosphorylation of NUSAP1 regulates its interaction with KIF2C, balancing spindle microtubule stability and depolymerization to ensure proper sister chromatid segregation. Disruptions in this pathway impair NSPCs mitosis, thereby affecting neocortical development.

genetics↗

bioRxiv2025

A New Model of Heart Failure with Preserved Ejection Fraction Induced by Metabolic Syndrome in Ossabaw Miniature Swine

A major obstacle to progress in heart failure with preserved ejection fraction (HFpEF) is the paucity of clinically relevant animal models. We developed a large, translationally relevant model in Ossabaw minipigs, which are genetically predisposed to the metabolic syndrome (MetS). Pigs were fed a "Western diet" high in calories, fructose, fat, cholesterol, and salt and received 1-2 deoxy-corticosterone acetate (DOCA) depots (n=10). After 6 months, they exhibited liver function abnormalities and marked increases in body weight, arterial blood pressure, serum cholesterol and triglycerides, and plasma glucose and insulin levels (glucose tolerance test), indicating the development of a full MetS. Echocardiography demonstrated no change in LV ejection fraction but progressive concentric LV hypertrophy and left atrial dilatation. Doppler echocardiography showed increased E/e ratio and increased peak early (E) and peak late atrial (A) transmitral inflow velocities, with no change in E/A ratio. Right heart catheterization demonstrated increased central venous pressure, pulmonary arterial systolic pressure, and pulmonary capillary wedge pressure. Clinically, pigs exhibited impaired exercise capacity, assessed by treadmill tests, associated with chronotropic incompetence. Pathologic examination showed significant myocardial fibrosis, myocyte hypertrophy, and liver fibrosis. In contrast, lean pigs fed a standard diet (n=3) did not show any changes at 6 months. The Ossabaw porcine model described herein is unique in that it recapitulates the entire constellation of major multiorgan comorbidities and hemodynamic, clinical, and metabolic features of MetS-driven human HFpEF: obesity, arterial hypertension, hyperlipidemia, glucose intolerance, insulin resistance, liver fibrosis and dysfunction, pulmonary hypertension, increased LV filling pressures, concentric LV hypertrophy, LV diastolic dysfunction with preserved systolic function, and impaired exercise capacity. Because of its high clinical relevance, this model is well-suited for exploring the pathophysiology of MetS-driven HFpEF and the efficacy of new therapies.

developmental biology↗

bioRxiv2025

Mitochondrial heterogeneity disrupts osteoclast differentiation and bone resorption by impairing respiratory complex I

Mitochondrial heteroplasmy, the co-existence of different mitochondrial genomes within a cell, is linked to aging and disease. Patients with heteroplasmy due to mitochondrial mutations experience multiple organ complications, particularly poor bone health and bone structure defects. However, the mechanisms involved are generally unknown, due largely to the difficulty of manipulating mtDNA in vivo. To overcome this, we leveraged a heteroplasmic mouse model and discovered that mitochondrial heteroplasmy affects a fundamental developmental process. Specifically, the differentiation of osteoclasts, which resorb bone tissue and maintain bone homeostasis. Mechanistically, there was a reduced localization of specifically respiratory complex I subunits in mitochondria in heteroplasmic mice, disrupting ATP production and osteoclast differentiation. In addition, autophagic flux is exhausted, and the autophagy inducer spermidine restores mitochondrial health and rescues osteoclast activity, both in mice and in cells from patients with primary mitochondrial disease. Together, we identify the mechanisms by which mitochondrial heteroplasmy impacts osteoclastogenesis and discover spermidine as a modulator of this process, which presents a potential treatment for human heteroplasmic conditions such as mitochondrial diseases, which are largely untreatable. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/651799v4_ufig1.gif" ALT="Figure 1"> View larger version (94K): org.highwire.dtl.DTLVardef@131d3f6org.highwire.dtl.DTLVardef@52bf2aorg.highwire.dtl.DTLVardef@848bb3org.highwire.dtl.DTLVardef@1efa572_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Comparative phenotypic, genomic and transcriptomic characterisation of two Salmonella Typhimurium strains for a first-in-human challenge model

BackgroundInvasive non-typhoidal Salmonella (iNTS) disease remains a major public health challenge in sub-Saharan Africa. Salmonella Typhimurium is responsible for the majority of cases, with specific lineages being associated with increased risk of bloodstream infection. We have recently developed a Salmonella Typhimurium controlled human infection model (CHIM) to better understand disease pathogenesis and to provide a platform to test candidate vaccines. Selecting appropriate challenge strainsis a central design consideration in developing challenge model protocols. We describe the rationale, manufacture, and detailed characterisation of the two Salmonella Typhimurium strains used in the first-in-human NTS CHIM. MethodsTwo Salmonella Typhimurium strains, 4/74 (ST19) and D23580 (ST313), were selected from the UK Health Security Agency National Collection of Type Cultures and manufactured under Good Manufacturing Practice (GMP) conditions. Challenge agent stocks underwent microbial limits testing, viability and stability assessments, and phenotypic characterisation including growth kinetics, motility, acid sensitivity, and antibiotic susceptibility. Whole-genome sequencing was performed to confirm genetic stability post-manufacture. The effect of pre-challenge handling conditions was assessed in saline and sodium bicarbonate buffers, and bacterial survival was evaluated under simulated gastric and intestinal conditions using a modified Rossett-Rice model. Transcriptomic profiling was undertaken to determine whether sodium bicarbonate exposure altered expression of key virulence genes. ResultsBoth strains retained their expected phenotypic characteristics, including reduced motility and melibiose utilisation in D23580. GMP stocks remained pure, viable, and stable post-manufacture, with no unexpected genomic mutations detected. Both strains were susceptible to clinically relevant antibiotics used in the study. Survival was maintained in neutral and mildly acidic conditions, with significant reduction below pH 3.5. Stability was preserved for up to one hour in saline buffer and during simulated gastric transit in the Rossett-Rice model. Transcriptomic analysis showed no significant changes in Salmonella pathogenicity island-1 or 2, or flagellar gene expression following sodium bicarbonate exposure ConclusionsThese findings confirm that the Salmonella Typhimurium 4/74 and D23580 strains retained genomic and phenotypic integrity post-manufacture. The resulting challenge stocks provide the foundation for an ongoing NTS CHIM that aims to advance understanding of NTS and iNTS pathogenesis and support candidate vaccine testing.

microbiology↗

bioRxiv2025

The leading region of many conjugative plasmids is depleted in restriction-modification targets

To successfully transfer into a recipient, conjugative plasmids must overcome the new cells defense mechanisms. The leading region of many plasmids - the first part to enter the recipient - is highly enriched in anti-defense genes. Here, we investigate whether selective pressure from defense systems has also affected its sequence composition. We consider two possibilities: greater target depletion (to avoid triggering target-based defense systems) or greater codon adaptation (to more quickly express anti-defense genes). We analyse the leading region of 1,751 conjugative plasmids belonging to 13 plasmid taxonomic units (PTUs) present in the bacterial order Enterobacterales. First, we investigate GC content and possible depletion of short palindromic motifs (4-8bp) which could be a signature of selective pressure from Type II restriction-modification systems, and find evidence of depletion in certain PTUs. Then we use the codon adaptation index (CAI) to assess codon usage and find that different conjugative PTUs shown different patterns. We show that the leading region of plasmids with MOB-F and MOB-P relaxases is more adapted to optimal host codon usage, consistent with a selective pressure for translational efficiency in anti-defense genes that are transcribed while still single-stranded DNA. In particular, anti-restriction genes in the leading region have high CAI, and we use this fact to identify a single-stranded promoter in the F plasmid. Our findings uncover a new facet of plasmid evolution and emphasise the intensity of the selective pressure that plasmids face from defense systems.

microbiology↗

bioRxiv2025

Proteomic and structural comparison between cilia from primary ciliary dyskinesia patients with a DNAH5 defect

Primary ciliary dyskinesia (PCD) is a genetic disorder that affects the motile cilia in various organs, leading to recurrent respiratory infections, subfertility, and laterality abnormalities. Traditional diagnostic methods include high-speed video microscopy, immunofluorescence staining, electron microscopy, and genetic screening. However, how different disease-causing variants in the same PCD gene affect clinical presentation, as well as ciliary composition and structure, is not well understood. We investigated the effect of various mutations in DNAH5, an axonemal dynein heavy chain, using mass spectrometry and cryo-electron tomography. We demonstrated differences in the axonemal composition for patients with dnah5 mutations. Furthermore, we showed that reductions in some ciliary components are patient specific. Some of them, KIAA1430, CFAP97, and DTHD1, were not previously recognized as components of human respiratory motile cilia. Finally, we demonstrated that some differences in protein abundance between wild-type and PCD samples can be observed in the 96-nm repeated unit of the axoneme. Our results suggest that many axonemal components are affected in the DNAH5-defective samples. Additionally, our results highlight that disparate mutations have seemingly distinctive consequences for the axonemal composition.

cell biology↗

bioRxiv2025

A single amino acid variant in the variable region I of AAV capsid confers liver detargeting

AAV capsid serotypes isolated from nature have been widely used in gene delivery and gene therapy. Recently, more than 1,000 distinct AAV capsids were identified from human clinical samples by high-throughput, long-read DNA sequencing. In this study, we tap into this broad natural biodiversity of AAV capsids to develop liver-tropic AAV capsids. We initially screened a subset of variants derived from AAV8 (n=159) for packaging efficiency. The high-yielding variants were subjected to a barcoded vector library screen in mice and ferrets for their ability to mediate liver gene transfer. Although no variant surpassed AAV8 for liver targeting, several exhibited a liver detargeting phenotype. Among these, we focused on the N271D variant (AAV8 VP1 numbering), located in the variable region I (VR-1), which has been previously implicated in influencing liver tropism. The liver detargeting phenotype of AAV8.N271D was confirmed by single vector administration in mice. Additionally, we grafted the N271D variant onto AAV9 and MyoAAV capsids (N270D by AAV9 VP1 numbering). The AAV9.N270D and MyoAAV.N270D vectors showed a similar liver-detargeting phenotype, although muscle targeting was moderately reduced. Although we did not identify any capsid variants that outperform AAV8 in liver transduction, this study reinforces the important role of VR-1 in modulating liver tropism and highlights the potential of engineering VR-1 residues to reduce liver gene transfer and associated toxicity observed in several gene therapy studies.

molecular biology↗

bioRxiv2025

Repulsive interactions instruct synaptic partner matching in an olfactory circuit

Neurons exhibit extraordinary precision in selecting synaptic partners. Although cell-surface proteins (CSPs) mediating attractive interactions between developing axons and dendrites have been shown to instruct synaptic partner matching1,2, the degree to which repulsive interactions play a role is less clear. Here, using a genetic screen guided by single-cell transcriptomes3,4, we identified three CSP pairs--Toll2-Ptp10D, Fili-Kek1, and Hbs/Sns- Kirre--in mediating repulsive interactions between non-partner olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites in the developing Drosophila olfactory circuit. Each CSP pair exhibits inverse expression patterns in the select ORN-PN partners. Loss of each CSP in ORNs led to similar synaptic partner matching deficits as the loss of its partner CSP in PNs, and mistargeting phenotypes caused by overexpressing one CSP could be suppressed by loss of its partner CSP. All CSP pairs are also differentially expressed in other brain regions. Together, our data reveal that multiple repulsive CSP pairs work together to ensure precise synaptic partner matching during development by preventing neurons from forming connections with non-cognate partners.

neuroscience↗

bioRxiv2025

Rewiring an olfactory circuit by altering the combinatorial code of cell-surface proteins

Proper brain function requires the precise assembly of neural circuits during development. Despite the identification of many cell-surface proteins (CSPs) that help guide axons to their targets1,2, it remains largely unknown how multiple CSPs work together to assemble a functional circuit. Here, we used synaptic partner matching in the Drosophila olfactory circuit3,4 to address this question. By systematically altering the combination of differentially expressed CSPs in a single olfactory receptor neuron (ORN) type, which senses a male pheromone that inhibits male-male courtship, we switched its connection from its endogenous postsynaptic projection neuron (PN) type nearly completely to a new PN type that promotes courtship. From this switch, we deduced a combinatorial code including CSPs that mediate both attraction between synaptic partners and repulsion between non-partners5,6. The anatomical switch changed the odor response of the new PN partner and markedly increased male-male courtship. We generalized three manipulation strategies from this rewiring-- increasing repulsion with the old partner, decreasing repulsion with the new partner, and matching attraction with the new partner--to successfully rewire a second ORN type to multiple distinct PN types. This work demonstrates that manipulating a small set of CSPs is sufficient to respecify synaptic connections, paving ways to explore how neural systems evolve through changes of circuit connectivity.

neuroscience↗

bioRxiv2025

Tissue-resident skeletal muscle macrophages promote recovery from viral pneumonia-induced sarcopenia in normal aging.

Sarcopenia, which diminishes lifespan and healthspan in the elderly, is commonly exacerbated by viral pneumonia, including influenza and COVID-19. In a study of influenza A pneumonia in mice, young mice fully recovered from sarcopenia, while older mice did not. We identified a population of tissue-resident skeletal muscle macrophages that form a spatial niche with satellite cells and myofibers in young mice but are lost with age. Mice with a gain-of-function mutation in the Mertk receptor maintained this macrophage-myofiber interaction during aging and fully recovered from influenza-induced sarcopenia. In contrast, deletion of Mertk in macrophages or loss of Cx3cr1 disrupted this niche, preventing muscle regeneration. Heterochronic parabiosis did not restore the niche in old mice. These findings suggest that age-related loss of Mertk in muscle tissue-resident macrophages disrupts the cellular signaling necessary for muscle regeneration after viral pneumonia, offering a potential target to mitigate sarcopenia in aging.

immunology↗

bioRxiv2025

Title: Cell-autonomous and non-cell-autonomous effects of TRIM72 on ALS disease progression

Dysfunction of RNA-binding proteins, including TDP-43 and FUS, has been associated with amyotrophic lateral sclerosis (ALS); however, the underlying mechanisms are largely unknown. Here, we reported that a neuronal upregulation of TRIM72 (Tripartite Motif Containing 72) in FUS mutation knockin ALS models slows disease progression. TRIM72 interacts with Commander, a protein complex for recycling of membrane proteins, facilitating membrane repair and antioxidation. Exosomal TRIM72 is detected in ALS patient cerebrospinal fluid (CSF) and extracellular application of exosomal TRIM72 protects cell from membrane damage. In a sporadic ALS cohort, CSF TRIM72 level associates ALS disease progression. AAV-mediated neuronal expression of TRIM72 slows down the disease progressions in ALS models and in an ALS patient without adverse effects over a year treatment course. Taken together, our results suggest a universal neuronal protection of a TRIM family protein in cell-autonomous and non-cell-autonomous manners in ALS.

neuroscience↗

bioRxiv2024

Deciphering the thiolactonization mechanism in thiolactomycin biosynthesis

Thiolactomycin (1), which features a unique{gamma} -thiolactone ring, is a promising antibiotic candidate that specifically targets bacterial type II fatty acid synthase. Despite extensive studies on its pharmacological activities, modes of action, and chemical synthesis, the enzymatic processes responsible for forming the activity-determining{gamma} -thiolactone ring have remained largely unknown. Here, we resolve this problem by revealing that the condensation and heterocyclization (Cy) domain of the nonribosomal peptide synthetase (NRPS) TlnC (TlnCCy), along with the cytochrome P450 enzyme TlnA, cooperatively enable the{gamma} -thiolactone assembly. TlnCCy mediates an unusual sulfurtransfer reaction to sulfurate the polyketide intermediate, generating a thiocarboxylate intermediate. Subsequently, TlnA acts as a{gamma} -thiolactone synthase, converting the linear thiocarboxylate intermediate into 1 via a distal radical-based cyclization mechanism. These findings not only expand the functional and catalytic repertoires of NRPS Cy domains and P450 enzymes, but also highlight a special enzymatic strategy for{gamma} -thiolactone biosynthesis in nature. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/629141v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@7e0ad2org.highwire.dtl.DTLVardef@f86b80org.highwire.dtl.DTLVardef@159d136org.highwire.dtl.DTLVardef@1b633ff_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Macrophage migration-dependent retraction fibers and migrasomes - a structural basis for spatiotemporal cytokine secretion.

Migrating cells leave behind trails of matrix-bound retraction fibers and migrasomes, but their cell-specific physiological functions are largely unknown. We comprehensively characterize retraction fibers and migrasomes of migrating macrophages in mouse and zebrafish in vitro and in vivo models. These structures share common and macrophage-centric components of tetraspanin enriched microdomains (TEMs) including tetraspanin 4 (TSPAN4), the integrin CD11b and the pTRAP, SCIMP; like TSPAN4, we show that SCIMP expression modulates retraction fiber and migrasome formation. In immune-activated migrating macrophages, rear-positioned recycling endosomes regulate the secretory trafficking of newly synthesized inflammatory cytokines into retraction fibers and migrasomes. Transmembrane TNF is delivered via SNARE-mediated carriers to the cell surface and to the surfaces of both retraction fibers and migrasomes, from where it can be released. Retraction fibers are thus newly identified here as cytokine secretion sites, and along with migrasomes they allow migrating macrophages to secrete more cytokine compared to stationary cells. Whereas cytokine secretion has been viewed as a process of ad hoc diffusion, instead, we reveal a new mechanism of migration-dependent, localized deposition and spatiotemporal release of bioactive cytokines from RFs and migrasomes. Cytokine deployment is thus organized in a constrained manner facilitating direct influence on local inflammatory responses.

cell biology↗

bioRxiv2024

The sparse driver system for in vivo single-cell labeling and manipulation in Drosophila

In this protocol, we introduce a sparse driver system for cell-type specific single-cell labeling and manipulation in Drosophila, enabling complete and simultaneous expression of multiple transgenes in the same cells. The system precisely controls expression probability and sparsity via mutant FRT sites with reduced recombination efficiency and tunable FLP levels adjusted by heat-shock durations. We demonstrate that this generalizable toolkit enables tunable sparsity, multi-color staining, single-cell trans-synaptic tracing, single-cell manipulation, and in vivo analysis of cell-autonomous gene function. For details on the use and execution of this protocol, please refer to Xu et al. 2024. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/626507v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1a53b9org.highwire.dtl.DTLVardef@6eaa3aorg.highwire.dtl.DTLVardef@169cb2forg.highwire.dtl.DTLVardef@9aafb3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Cardiac Reprogramming with Drug Resistance Alleviates Doxorubicin-induced Cardiotoxicity in Mice and Pigs

Doxorubicin (Dox)-induced cardiotoxicity represents a significant clinical adverse effect associated with cancer chemotherapy treatment. The systematic toxicity associated with the currently available drug dexrazoxane has limited its clinical utility. Inspired by cancer drug resistance mechanisms, we propose a novel strategy termed Transient overexpression of p-glycoprotein (P-gp) for Cardiac reprogramming (TopCare) to induce cardiac drug resistance as a treatment for cardiotoxicity. This approach involves reprogramming cardiomyocytes by delivering lipid nanoparticles (LNPs)-based mRNA therapeutics to induce temporary P-gp overexpression, which in turn reduces intracellular Dox levels and suppresses cytotoxic effects. Our results demonstrated that LNPs with P-gp mRNAs (P-gp LNPs) enhance P-gp expression in H9c2 cells, significantly lowering the cytotoxicity of both Dox and paclitaxel (PTX). In a mouse model with Dox-induced cardiotoxicity, it was observed that P-gp LNPs effectively promoted P-gp overexpression in cardiomyocytes, with a time-dependent decline in P-gp protein levels. This TopCare strategy resulted in improved survival rates, restored cardiac function, and reduced myocardial fibrosis and structural cardiac alterations. Furthermore, studies in large animals have shown that intrapericardial (iPC) injection of P-gp LNPs effectively mitigates adverse effects and restores cardiac function in pig models of Dox-induced cardiotoxicity. The significant cardioprotective effects achieved through cardiac drug resistance highlight the safety, efficacy, and clinical potential of the TopCare strategy for alleviating doxorubicin-induced cardiotoxicity.

bioengineering↗