Biology subjects

Yang, Z.

Publications and source records attributed to Yang, Z..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Bivalent mRNA booster encoding virus-like particles elicits potent polyclass RBD antibodies in pre-vaccinated mice

mRNA vaccines emerged as a leading vaccine technology during the COVID-19 pandemic. However, their sustained protective efficacies were limited by relatively short-lived antibody responses and the emergence of SARS-CoV-2 variants, necessitating frequent and variant-updated boosters. We recently developed the ESCRT- and ALIX-binding region (EABR) mRNA vaccine platform, which encodes engineered immunogens that induce budding of enveloped virus-like particles (eVLPs) from the plasma membrane, thereby resulting in presentation of immunogens on cell surfaces and eVLPs. Prior studies showed that spike (S)-EABR mRNA-LNP immunizations elicited enhanced neutralizing antibody responses against ancestral and variant SARS-CoV-2 compared with conventional S mRNA-LNP in naive mice, but the effectiveness of S-EABR mRNA-LNP boosters in the context of pre-existing immunity has not been investigated. Here, we evaluated monovalent Wuhan-Hu-1 (Wu1) and bivalent (Wu1/BA.5) S-EABR mRNA-LNP boosters in mice pre-vaccinated with conventional Wu1 S mRNA-LNP. Compared to conventional S mRNA-LNP boosters, the EABR approach enhanced monovalent and bivalent mRNA-LNP booster-induced neutralizing responses against Omicron subvariants BA.1, BA.5, BQ.1.1, and XBB.1, with bivalent S-EABR mRNA-LNP consistently eliciting the highest titers. Epitope mapping of polyclonal antisera by deep mutational scanning revealed that bivalent S-EABR mRNA-LNP boosted diverse "polyclass" anti-receptor-binding domain (RBD) responses, suggesting balanced targeting of multiple RBD epitope classes. In contrast, monovalent S, bivalent S, and monovalent S-EABR mRNA-LNP boosters elicited less diverse polyclonal serum responses primarily targeting immunodominant RBD epitopes. Cryo-EM structures demonstrated that bivalent mRNA immunizations promote S heterotrimer formation, potentially enhancing bivalent S-EABR mRNA-LNP booster-induced antibody breadth and polyclass epitope targeting by activating cross-reactive B cells through intra-S crosslinking. These findings support the future design of bivalent or multivalent S-EABR mRNA-LNP boosters as a promising strategy to confer broader, and therefore potentially more durable, protection against emerging SARS-CoV-2 variants and other rapidly evolving viruses.

immunology↗

bioRxiv2025

Population trajectory analysis reveals divergent state-space geometries across three cortical excitatory cell types

Cortical population activity unfolds along structured trajectories in state space, reflecting underlying dynamics of the neural networks involved. However, how such geometry differs across neural cell types remains poorly understood. Using a large-scale two-photon calcium imaging dataset from the Allen Brain Observatory, we characterized the population trajectories of three cortical excitatory subtypes (Cux2, Emx1, and Slc17a7). These cells constitute most of the layer 2/3 excitatory populations, span distinct projection and functional hierarchies, and collectively orchestrate cortical integration and excitatory signaling. While viewing drifting gratings, Cux2 neurons exhibited compact and stable trajectories, whereas Emx1 and Slc17a7 showed broader and more distributed dynamics, implying their functional divergence in visual processes. Such geometric distinctions can be mechanistically accounted for within a low-dimensional control space where the dominant axes correspond to the effective integration timescale and recurrent gain. This geometric-mechanistic framework provides a generalizable route to bridging cortical dynamics with fundamental, cellular identity.

neuroscience↗

bioRxiv2025

Tracking the Evolutionary Patterns of RNA Modifications from Bryophyte to Flowering Plants by Mass Spectrometry

RNA modifications are critical for the regulation of gene expression. Comprehensive profiling of RNA modifications is a prerequisite for unravelling their identities and functions. The plant hormone auxin plays pivotal roles in regulating plant growth, development, and environmental responses. In contrast to the extensive studies in animals, there is a paucity of knowledge regarding the existence and functions of RNA modifications in plants. This is due to the low abundance of modified ribonucleosides and the lack of accurate analytical methods. This study employs a state-of-the-art hydrophilic interaction liquid chromatography-tandem-mass spectrometry (HILIC-MS/MS) platform to achieve the sensitive detection of 12 nucleosides at the pM to nM level. Utilizing this platform, we were able to identify N4,2-O-dimethylcytidine (m4Cm) in plants by LC-MS/MS for the first time, which provided direct evidence for the widespread of this modification in plants. Moreover, a comprehensive investigation was undertaken to examine the 12 types of RNA modifications and their response patterns to auxin in a range of prototype plants, spanning from bryophyte (Marchantia polymorpha) to flowering plants (Arabidopsis thaliana, Oryza sativa, and Zea mays). The evolutionary patterns of the 12 RNA modifications were depicted from bryophyte to flowering plants, and their potential functions in plant growth, development, and auxin responses were revealed.

biochemistry↗

bioRxiv2025

An open-source pipeline for calcium imaging and all-optical physiology in human stem cell-derived neurons

High-throughput profiling of neuronal activity at single-cell resolution is essential for advancing our understanding of brain function, enabling large-scale functional screens, and modeling neurological disorders. However, existing approaches are limited by scalability, manual data processing, and variability, thus restricting their ability to detect disease-associated phenotypes. Here, we present a scalable, open-source platform that integrates optogenetic stimulation, calcium imaging, automated data acquisition, and a fully integrated analysis pipeline. By combining spontaneous and evoked activity profiling, the system enables robust quantification of dynamic neuronal responses across hundreds of stem cell-derived human neurons and multiple timepoints, facilitating phenotyping at both cellular and network levels. We validated the platform by recapitulating established activity phenotypes in neurodevelopmental disorders including CDKL5 Deficiency Disorder and SSADH deficiency. In addition, we generated CRISPR-Cas9 knock-in human induced pluripotent stem cell (hiPSC) lines stably expressing the genetically encoded calcium indicator GCaMP6s to model network dysfunction in Tuberous Sclerosis Complex (TSC). Using this system, we further demonstrated functional rescue of the altered neuronal activity observed in the TSC following pharmacological intervention. By linking single-cell dynamics to population-level phenotypes, this framework provides a powerful and broadly applicable tool for disease modeling, mechanistic studies, and therapeutic screening across a range of neurological disorders.

neuroscience↗

bioRxiv2025

Functional screening of TCR-like antibodies for targeted cancer immunotherapy

Although hybridomas and display technologies have long been used in antibody discovery, identifying TCR-like antibodies that recognize major histocompatibility (MHC)-presented neoantigens remains an inefficient process. Here, we present a functional screening approach for antibody discovery based on T cell-specific functionality. By constructing Synthetic T cell receptor and Antigen Receptor (STAR)-T cells expressing antibody libraries and co-culturing them with target cells, we observed distinct antigen-specific STAR endocytosis and T cell activation. These two indicators are combined into an E-A functional index, which we demonstrate as an effective screening strategy for identifying antibodies with strict antigen sensitivity and specificity. Using this E-A functional screening method, we successfully discovered antibodies or nanobodies targeting cell-surface tumor antigens and neoantigens presented by human leukocyte antigen (HLA). These antibodies exhibited potent anti-tumor efficacy both in vitro and in vivo. Moreover, converting these antibodies into chimeric antigen receptors (CARs) and bispecific antibodies also induced T cell activation and tumor killing. In summary, our study establishes a robust functional screening strategy for identifying antibodies or TCR-like nanobodies targeting tumor-specific neoantigens, offering significant potential for advancing targeted cancer therapies.

immunology↗

bioRxiv2025

Ciclopirox suppresses poxvirus replication by targeting iron metabolism

Poxviruses remain a significant global health concern, necessitating the development of novel antiviral strategies. Through high-throughput screening, we previously identified ciclopirox (CPX), an FDA-approved antifungal, as a hit that inhibits vaccinia virus (VACV) replication. Here, we further characterized its antiviral activity and mechanism of action using human primary fibroblasts. CPX significantly reduced VACV titers without reducing host cell viability, with an EC50 in the sub-micromolar range and a CC50 >500 M. Rescue experiments demonstrated that CPX inhibits viral replication primarily through chelation of intracellular Fe3+ and, to a lesser extent, Fe2+, as evidenced by partial restoration of viral replication with ferric ammonium citrate supplementation. Furthermore, overexpression of the iron-dependent enzymes RRM2 and the VACV-encoded F4L reduced the inhibitory effect of CPX, indicating that these host and viral proteins are affected by CPX treatment. Moreover, CPX treatment also suppressed cowpox virus and monkeypox (mpox) virus replication in vitro. It also reduced VACV titers in ex vivo mouse lung tissue. These findings highlight host iron metabolism as a critical determinant of poxvirus replication and support repurposing CPX as a broad-spectrum orthopoxvirus antiviral candidate.

microbiology↗

bioRxiv2025

Temperate phages enhance host fitness via RNA-guided flagellar remodeling

Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FR{varphi}), here we show that FR{varphi} exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FR{varphi} improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.

microbiology↗

bioRxiv2025

MolUNet++: Adaptive-grained Explicit Substructure and Interaction Aware Molecular Representation Learning

Molecular representation learning is a critical task in AI-driven drug development. While graph neural networks (GNNs) have demonstrated strong performance and gained widespread adoption in this field, efficiently extracting and explicitly analyzing functional groups remains a challenge. To address this issue, we propose MolUNet++, a novel model that employs Molecular Edge Shrinkage Pooling (MESPool) for hierarchical substructure extraction, utilizes a Nested UNet framework for multi-granularity feature integration, and incorporates a substructure masking explainer for quantitative fragment analysis. We evaluated MolUNet++ on tasks including molecular property prediction, drug-drug interaction (DDI) prediction, and drug-target interaction (DTI) prediction. Experimental results demonstrate that MolUNet++ not only outperforms traditional GNN models in predictive performance but also exhibits explicit, intuitive, and chemically logical interpretability. This capability provides valuable insights and tools for researchers in drug design and optimization.

bioinformatics↗

bioRxiv2025

Dual Targeting of BRAFV600E and Ferroptosis Results in Synergistic Anticancer Activity via Iron Overload and Enhanced Oxidative Stress

PurposeWhile combination BRAF and MEK inhibitor treatment in BRAFV600E-mutant cancers results in a response, treatment resistance and toxicity are common. Ferroptosis is an iron-dependent form of non-apoptotic cell death. BRAF inhibition has been associated with increased sensitivity to ferroptosis that is dependent on Glutathione Peroxidase 4 (GPX4). Experimental DesignIn vitro, ex vivo, and in vivo models of anaplastic thyroid cancer (ATC) were used to evaluate the anticancer activity of combination BRAF inhibition and ferroptosis induction. ResultsTargeting key regulators of ferroptosis--GPX4, using RSL3 and ML162, and system Xc-, using erastin--induced significant cell death in all ATC cell lines. Combination of dabrafenib and RSL3 synergistically increased cell death in BRAFV600E-mutant ATC cells, and significantly inhibited cellular migration and colony formation. Mechanistically, lipid peroxidation, reactive oxygen species levels, and intracellular Fe2+ increased significantly with combination treatment compared with each agent alone. Analysis of cell membrane iron importers and exporters showed significantly lower expression of ferroportin-1 (an iron exporter), suggesting the synergistic anticancer activity was due to increased iron accumulation and oxidative stress, leading to enhanced ferroptotic cell death. BRAFV600E-mutant ATC cell spheroids showed synergistic cell death with dabrafenib and RSL3 treatment. In vivo, combination dabrafenib and ferroptosis induction (by targeting GPX4 using C18, and system Xc- with IKE) significantly inhibited tumor growth in an orthotopic ATC mouse model. Additionally, dabrafenib-resistant BRAFV600E-mutant ATC cells were more sensitive to ferroptosis induction than parental cells. ConclusionsDual targeting of BRAFV600E and ferroptosis results in synergistic anticancer activity and overcomes resistance to BRAF inhibition.

cancer biology↗

bioRxiv2025

Pentose Sugars Encode Sequence-Dependent DNA-RNA Segregation for Biomimetic Multiphase Condensates

The nucleus segregates DNA and RNA with high precision to safeguard genome stability and gene regulation despite their homologous structures, yet the underlying molecular principles have long evaded both understanding and synthetic translation. Here we show that a single atomic difference between the pentose sugars, a 2-OH in RNA versus a 2-H in DNA, suffices to drive their phase segregation with cationic peptides. This subtle chemical distinction makes RNA bind peptides more strongly than sequence-identical DNA, generating an asymmetry that, when modulated by sequence-encoded homotypic interactions, drives the formation of core-shell multiphase condensates. Using a supervised machine-learning approach, we distill these molecular principles into a predictive design rule and harness it to program a library of oligonucleotide pairs, termed SEGREGamers, that self-assemble into droplets with coexisting DNA- and RNA-rich domains. These synthetic condensates recapitulate nuclear compartment functions, including selective molecular partitioning and enhanced RNA catalysis. Our results establish a chemically encoded platform for engineering synthetic nuclear mimics and programmable biomolecular condensates, and suggest that sugar identity may have served as an ancient physical mechanism for organizing nucleic acids.

biophysics↗

bioRxiv2025

VAMPIRE: Analyzing variation and motif pattern in tandem repeats

Tandem repeats (TRs) are pervasive in eukaryotic genomes and play key roles in genome organization, evolution, and function, particularly in complex regions such as centromeres and subtelomeres. Although long-read sequencing technologies have improved the resolution of these regions, existing methods remain limited in their ability to systematically and accurately characterize large-scale TRs. Here, we introduce VAMPIRE, a k-mer-based computational tool for comprehensive TR discovery, annotation, and quantification. Unlike previous methods, VAMPIRE enables reference-free, fine-grained decomposition of both simple and complex TRs, capturing motif variation in sequence, length, and structure with high sensitivity and scalability. Applied to complete telomere-to-telomere (T2T) human and nonhuman primate (NHP) genome assemblies, VAMPIRE reveals previously unrecognized high-order repeat inversions within human centromeres--an underappreciated evolutionary mechanism contributing to centromere diversity. Additionally, the tool identifies lineage-specific and expanded TRs, including human-specific STR/VNTR expansions and NHP-specific subtelomeric heterochromatin (e.g., pCht/StSat), underscoring their dynamic turnover and structural complexity. VAMPIRE provides a robust and scalable framework for TR analysis in the era of long-read sequencing, with broad utility across human genetics, evolutionary biology, and the study of complex TRs in non-model organisms.

genomics↗

bioRxiv2025

Trpv4 mediates temperature induced sex change in ricefield eel

The ricefield eel (Monopterus albus), an economically important aquaculture species in China, is a freshwater teleost fish that exhibits protogynous hermaphroditism. Although progress has been made in understanding the sex determination and differentiation of this species, the underlying mechanisms remain unclear. Here we show that warm temperature promotes gonadal transformation by up-regulating testicular differentiation genes such as dmrt1/sox9a in ovaries. Trpv4, a Ca2+-permeable cation channel expressed in gonadal somatic cells, is highly sensitive to ambient temperature and links environmental temperature to testicular differentiation in ricefield eel. In female fish reared at cool temperature, injection of Trpv4 agonist into the ovaries leads to a significant up-regulation of male pathway genes, and in female fish exposed to warm temperature, Trpv4 inhibition or trpv4 siRNA knockdown suppresses warm temperature-induced male gene expression. pStat3 signaling is downstream of Trpv4 and transduces Trpv4-controlled calcium signaling into the sex determination cascades. Inhibition of pStat3 activity prevents the up-regulation of testicular differentiation genes by warm temperature treatment and ovarian injection of Trpv4 agonist, whereas activation of pStat3 is sufficient to induce the expression of male genes, in the presence of Trpv4 antagonist. pStat3 binds and activates jmjd3/kdm6b, an activator of the dmrt1 gene. Consistently, ovarian injection of Kdm6b inhibitor blocks the up-regulation of testicular differentiation genes by warm temperature exposure. We propose that environmental factors, such as temperature, promote gonadal transformation of ricefield eel by inducing the expression of male pathway genes in ovaries via the Trpv4-pStat3-Kdm6b-dmrt1 axis. Our results provide new insights into the molecular mechanism underlying natural sex change of ricefield eel, which will be useful for sex control in aquaculture. HighlightsO_LIWarm temperature promotes gonadal transformation of ricefield eel C_LIO_LITrpv4 links environmental temperature and the sex determination pathway C_LIO_LIpStat3 is downstream of Trpv4-controlled calcium signaling C_LIO_LIpStat3 binds and activates kdm6b/jmjd3 C_LI

developmental biology↗

bioRxiv2025

Structural Basis for Cas9-Directed Spacer Acquisition in Type II-A CRISPR-Cas Systems

CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas92-Csn28-Cas18-Cas24) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

molecular biology↗

bioRxiv2025

Features fusion or not: harnessing multiple pathological foundation models using Meta-Encoder for downstream tasks fine-tuning

The emergence of diverse pathological foundation models has empowered computational pathology tasks, including tumor classification, biomarker prediction, and RNA expression prediction. However, variations in model architecture and data sources lead to inconsistent downstream performance and complicate centralized training. Specifically, the lack of data sharing makes retraining foundation models with pooled data infeasible. Alternatively, the release of model parameters enables combining multiple models during fine-tuning. Inspired by the meta-analysis method, we propose the Meta-Encoder framework, which integrates features from multiple foundation models to generate a comprehensive representation, improving downstream fine-tuning task performance. Comparative experiments demonstrate that Meta-Encoder is more effective than individual foundation models, with its strengths more pronounced in handling complex tasks. While single models may perform sufficiently well for simple tasks, Meta-Encoder can match or even surpass the best-performing single model, alleviating concerns over model selection. Moderately challenging tasks benefit from Meta-Encoders concatenation or self-attention strategies, with the latter demonstrating superior performance in more challenging scenarios. For highly complex tasks, such as high-dimensional gene expression prediction, self-attention proves to be the most effective Meta-Encoder strategy, balancing feature integration and computational efficiency. For three patch-level spatial gene expression prediction tasks (HEST-Benchmark, CRC-inhouse, and Her2ST), the self-attention strategy improved the Pearson correlation by 38.58%, 26.06%, and 20.39%, respectively, compared to the average performance of three patch-level single models. Similarly, for the TCGA-BRCA, TCGA-NSCLC, and TCGA-CRC WSI-level bulk gene expression prediction tasks, the Pearson correlation increased by 14.36%, 9.27%, and 42.55%, respectively, compared to the average performance of two WSI-level single models. By leveraging multiple pathological foundation models using Meta-Encoder, it can further improve molecular characterization in pathology images to advance precision oncology.

bioinformatics↗

bioRxiv2025

Proteomic characterization of major fish allergy responsive protein parvalbumins in Hilsa (Tenualosa ilisha): A commercially important fish in Southeast Asia

The protein parvalbumin (PRV)-beta (PRVB) is the primary cause of food allergies to bony fish. Although PRVB is a well-characterized protein in many bony fishes, little is known about the Hilsa, an anadromous fish with great economic importance and predominantly found in Southeast Asia. In this study, we characterized the Hilsa PRV utilizing various proteomic approaches in response to two major riverine habitats and developmental stages. Unique peptide sets corresponding to three PRV isoforms were identified in Hilsa muscle tissues. Label-free quantitative proteomic analysis coupled with ELISA revealed higher levels of PRVB in young fish compared to adults, irrespective of their riverine habitats. A comparative quantitative analysis of PRVB further demonstrated that Hilsa had less PRVB than other commonly consumed freshwater fish species. Multiple reaction monitoring (MRM)-based targeted proteomic approach showed the potential of PRV as a marker protein for allergen quantitation and authenticating the presence of Hilsa in a complex freshwater fish mixture. Our findings collectively offer fundamental knowledge on Hilsa PRVs for further investigation on the food safety and quality evaluation of Hilsa fish.

biochemistry↗

bioRxiv2025

CNAttention: an attention-based deep multiple-instance method for uncovering CNA signatures across cancers

Somatic copy number aberrations (CNAs) represent a distinct class of genomic mutations associated with oncogenetic effects. Over the past three decades, significant volumes of CNA data have been generated through molecular-cytogenetic and genome sequencing-based techniques. This data has been pivotal in identifying cancer-related genes and advancing research on the relationship between CNAs and histopathologically defined cancer types. However, comprehensive studies of CNA landscapes and disease parameters are challenging due to the vast diagnostic and genomic heterogeneity encountered in "pan-cancer" approaches. In this study, we introduce CNAttention, an attention-based deep multiple instance learning method designed to comprehensively analyze CNAs across different cancers and uncover specific CNA patterns within integrated gene-level CNA profiles of 30 cancer types. CNAttention effectively learns CNA features unique to each cancer type and generates CNA signatures for 30 cancer types using attention mechanisms, highlighting the distinctiveness of their CNA landscapes. CNAttention demonstrates high accuracy and exhibits stable performance even with the incorporation of external datasets or parameter adjustments, underscoring its effectiveness in tumor identification. Expanding these signatures to cancer classification trees reveals common patterns not only among physiologically related cancer types but also among clinico-pathologically distant types, such as different cancers originating from neural crest derived cells. Additionally, detected signatures also uncover genomic heterogeneity in individual cancer types, for instance in brain lower-grade glioma. Additional experiments with classification models underscore the efficacy of these signatures in representing various cancer types and their potential utility in clinical diagnosis.

bioinformatics↗

bioRxiv2025

Identification and Rescue of Congenital Hyperinsulinism-Associated ABCC8 Mutations that Impair KATP Channel Trafficking

ATP-sensitive potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) couple glucose metabolism with insulin secretion in pancreatic {beta}-cells and are vital to glucose homeostasis. Loss-of-function mutations in SUR1 and Kir6.2, encoded by ABCC8 and KCNJ11, respectively are the commonest causes of severe persistent hypoglycemia in infants and children seen in the rare disease congenital hyperinsulinism (HI). The N-terminal transmembrane domain, TMD0, and the linker immediately C-terminal to TMD0, L0, of SUR1 (TMD0/L0) forms direct contact with Kir6.2 in KATP channels. Mutations in SUR1-TMD0 often impair KATP channel trafficking to the plasma membrane, causing severe disease unresponsive to treatment by the KATP activator diazoxide; however, surface expression and function of many such mutant channels can be rescued by reversible KATP inhibitor pharmacochaperones. Here, we identified seven new SUR1 missense mutations in TMD0/L0 from HI patients unresponsive to diazoxide and investigated their effects on KATP channel expression, function, and response to pharmacochaperones. All seven mutations, N32K, Y124F, P133R, W143R, L171P, G228D, and Y230C, reduced channel function in Rb+ efflux assays. Further characterization by immunoblotting, immunostaining and electrophysiology revealed that Y124F primarily causes defective channel gating, while the others impair channel trafficking to different extents. The trafficking mutations showed varied response to surface expression and function rescue by the reversible KATP inhibitor pharmacochaperones, tolbutamide and Aekatperone. The study underscores the critical role of SUR1-TMD0/L0 in KATP expression and gating. It further highlights the importance of detailed biochemical and functional studies of mutant channels in understanding their pathogenic roles and response to potential pharmacological therapies.

biochemistry↗

bioRxiv2025

Engineering novel AAV capsids by global de-targeting and subsequent muscle-specific tropism in mice and NHPs

Recombinant adeno-associated viral (rAAV) vectors are a potent tool, but their clinical application is restricted by insufficient target tissue transduction and liver toxicity. We employed a novel two-step engineering strategy to create novel rAAV capsids with global tissue de-targeting, then produced strong tissue-specific expression by adding a peptide sequence. We created a novel capsid, AAV.Zero1, with globally de-targeted transduction by loop swapping domains from AAV9 into AAV2. Making an R585A substitution (AAV.Zero2) re-targeted tissues but deleting residues 585-587 (AAV.Zero3) abrogated transduction. Inserting a myogenic peptide into AAV.Zero3 produced a novel capsid (AAV.eM) with strong muscle-specific transgene expression while maintaining minimal off-target expression, including in liver, which was conserved in two mouse strains and non-human primates. AAV.eM showed similar expression as the leading myotropic vector MyoAAV.4A but had a more favorable safety profile. Importantly, AAV.eM was able to functionally rescue a mouse model of Duchenne Muscular Dystrophy following systemic delivery of a micro-dystrophin gene. Thus, AAV.eM is an improved myotropic rAAV capsid that de-targets other tissues, especially the liver, and proof-of-concept for a platform to create capsids with specific properties that translate across species by addition of peptides onto low transduction backbones.

bioengineering↗