Biology subjects

Chen, B.

Publications and source records attributed to Chen, B..

At least 91 records · Page 5Linked to original sources

bioRxiv2025

Tissue architecture dynamics underlying immune development and decline in the thymus

The age-associated decline in adaptive immune function, widely observed in vertebrates, has been attributed to thymic involution. To gain insights into the structural and transcriptional changes underlying this phenomenon, we employed high-resolution spatial transcriptomics and T-cell receptor (TCR) sequencing in mice. By analyzing 21 thymus samples spanning mouse lifespan, we uncovered significant alterations in thymic organization, including disrupted T cell development and the emergence of B cell aggregates. We also observed age- related changes in cell-cell interactions, marked by increased antigen-presenting cell presence in thymic medullary regions and a shift from inflammatory to suppressive macrophages, fostering an immunosuppressive niche. Furthermore, aged thymus tissues exhibited an abundance of regions with reduced TCR diversity, accompanied by distinct changes to gene expression profiles. Our study establishes a valuable reference for understanding aging-related alterations in adaptive immunity, revealing mechanisms underlying age-induced immunological decline.

immunology↗

bioRxiv2025

Competing Programs Shape Cortical Sensorimotor-Association Axis Development

The neocortex is organized along a dominant sensorimotor-to-association (S-A) axis, anchored by modality-specific primary sensorimotor areas at one end and transmodal association areas that form distributed networks supporting abstract cognition at the other. The developmental mechanisms shaping this axis remain elusive. Here, we present converging multispecies evidence supporting the Multinodal Induction-Exclusion in Network Development (MIND) model, in which S-A patterning is governed by competing processes of induction and exclusion, driven by opposing transcriptomically-defined identity programs emerging from different nodes. Key molecular and connectional features of association cortices arise through pericentral programs, originating around fronto-temporal poles and partially regulated by retinoic acid. They progress inward toward central territories of the naive neocortex along fronto-temporally polarized trajectories. Central programs are induced through interactions between topographically separated first-order sensorimotor thalamocortical inputs and the neocortex, promoting the formation of primary areas while excluding pericentral programs. Influenced by SATB2 and ZBTB18, these evolutionarily conserved programs compete for the same territory and create spatial compartmentalization of axon guidance, cell-cell adhesion, retinoic acid signaling, synaptogenesis, Wnt signaling, and autism risk genes. Notably, PLXNC1 and SEMA7A exhibit anti-correlated expression and repulsive functions in shaping cortico-cortical connectivity along the S-A axis. These processes of induction and exclusion establish an S-A equilibrium and topography in which primary sensorimotor areas emerge as focal islands within the broader ocean of distributed associative networks. The MIND model provides a unifying framework for understanding experimental, evolutionary, and clinical phenomena, revealing induction and exclusion as antagonistic complementary principles shaping the S-A axis and processing hierarchies.

neuroscience↗

bioRxiv2025

Toxoplasma effector TgWIP hijacks dendritic cell actin and motility via Nck1/Grb2 and the WAVE complex

The intracellular parasite Toxoplasma gondii enhances its dissemination to distant organs by hijacking infected leukocytes via a Trojan Horse mechanism. Upon infecting dendritic cells (DCs), Toxoplasma induces a hypermigratory phenotype characterized by podosome dissolution and formation of F-actin stress fibers. We previously showed that these cytoskeletal changes depend on the effector protein Toxoplasma WAVE complex-interacting protein (TgWIP) secreted from parasites to infected leukocytes. Here, we identify the host adaptor proteins Non-catalytic region of tyrosine kinase adaptor protein 1 and 2 (Nck1/2) and Growth factor receptor-bound protein 2 (Grb2) as direct TgWIP interactors. TgWIP mainly uses two distinct proline-rich regions (PRRs) to interact with Nck1 and Grb2. Mutating these PRRs abrogates TgWIP binding to Nck1 and Grb2 and diminishes podosome dissolution and DC hypermotility. Furthermore, we show that TgWIP directly interacts with the actin nucleation promoting factor WAVE Regulatory Complex (WRC) via a WRC-interacting receptor sequence (WIRS). Disrupting this interaction also influences actin cytoskeletal remodeling and DC hypermotility. Collectively, our data reveal that TgWIP directly interacts with multiple actin regulators, including Nck1, Grb2, and the WRC, to remodel the actin cytoskeleton of the host cells, elucidating a key mechanism that Toxoplasma exploits to enhance host cell migration and dissemination.

microbiology↗

bioRxiv2025

CellVoyager: AI CompBio Agent Generates New Insights by Autonomously Analyzing Biological Data

Modern biology increasingly relies on complex, high dimensional datasets such as single-cell RNA sequencing (scRNA-seq). However, the richness of such data means that conventional analyses may only scratch its surface. Extracting meaningful insights from these datasets often requires advanced computational methods and domain expertise. Current AI agents for biology are primarily focused on executing user-specified commands and are therefore limited by the users creativity and familiarity with which kinds of analyses are useful. Furthermore, these agents do not account for prior analyses already attempted by researchers, reducing their ability to build upon existing work. To address these limitations, we introduce CellVoyager, an AI agent that autonomously explores scRNA-seq datasets in novel directions conditioned on prior user-ran analyses. Built on large language models, CellVoyager ingests both the dataset and a record of prior analyses to generate and test new hypotheses within a Jupyter notebook environment. We evaluate CellVoyager on CellBench, a new benchmark based on 50 published scRNA-seq studies encompassing 483 analyses. Given only the background sections of these papers, CellVoyager outperformed GPT-4o and o3-mini by up to 20% in predicting which analyses the authors eventually conducted. We then carried out three in-depth case studies where CellVoyager is given previously published papers with their scRNA-seq datasets and conducts analyses to generate new findings. The original authors of each study evaluated these findings and consistently rated them as creative and sound; 80% of the agents hypotheses were deemed scientifically interesting. For example, in one case study, the agent found that CD8+ T cells in COVID-19 infection are more primed for pyroptosis, which was not explored by the original researchers. CellVoyager also reanalyzed a brain aging dataset to discover a previously unreported association between increased transcriptional noise and aging in the subventricular zone of the brain. These results demonstrate that CellVoyager can act both autonomously and collaboratively to accelerate hypothesis generation and computational biology. It also highlights the potential of agents like CellVoyager to unlock new biological insights by reanalyzing the vast existing biological data at scale.

bioinformatics↗

bioRxiv2025

Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromona fuliginea and its applications in functional genomics

Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is inefficient and time-consuming. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we introduced the CRISPR/Cas9 system into Pseudoalteromonas for the first time, and conducted an extensive investigation into the application of the Type II CRISPR/Cas9 system for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets and confirmed the gene editing effects through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3xflag and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70% (range: 73.3%-95.8%), which is significantly higher than the traditional homology-based approach (less than 0.1%). In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications. IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in Pseudoalteromonas fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.

molecular biology↗

bioRxiv2025

A lipid metabolism defect is an underlying contributor to Diamond Blackfan anemia syndrome

Analysis of neither Diamond Blackfan anemia syndrome (DBAS) cohorts nor animal models has revealed a potential mechanism for the variable anemia phenotype, a key feature of this disease. Here, we utilized an established Rpl5Skax23-Jus/+ murine DBAS model in order to study this dynamic erythropoiesis deficiency. These haploinsufficient mice exhibit variably penetrant craniofacial and cardiac defects mimicking the phenotypes of DBAS patients bearing RPL5 mutations. We additionally discovered that this specific heterozygous splicing mutation is pathogenic and leads to partial intron retention. By examining the transcriptome of fetal liver erythroid progenitors at E12.5, we demonstrate that the downregulation of erythroid differentiation pathways is consistent with the DBAS phenotype. We also identified dysregulated transcription of lipid metabolism genes with significant reduction in the abundance of Scd1 in a subset of E12.5 mutant embryos at risk for erythroid failure. SCD1, a key enzyme that converts saturated to monounsaturated fatty acids, has not been previously linked to erythropoiesis or DBAS. When anemia was induced in adult mice, pretreatment with an SCD1 inhibitor resulted in improved erythropoiesis. This analysis suggests a key role of lipid metabolism in the variable anemia penetrance in DBAS and highlights a previously unappreciated pathway that may serve as a potential target for drug development. Key PointsO_LIThe variable anemia in Rpl5Skax23-Jus/+mice is triggered by intrinsic/extrinsic stress C_LIO_LIRpl5 haploinsufficient murine and human erythroid progenitors exhibit a lipid metabolism signature with downregulation of Scd1/SCD. C_LI

developmental biology↗

bioRxiv2025

Probing the CaV1.2 interactome in heart failure identifies a positive modulator of inotropy and lusitropy

BackgroundMaladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical sub-cellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy. MethodsUsing two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, CaV1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography. ResultsFrom mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic CaV1.2 1C channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to CaV1.2 {beta}2B-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of CaV1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban. ConclusionsUsing proximity proteomics to examine the effects of HF in vivo, we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.

cell biology↗

bioRxiv2025

Dictionary of human intestinal organoid responses to secreted niche factors at single cell resolution

The intestinal epithelium is often a site of pathology, such as in inflammatory bowel disease (IBD), and its maintenance is highly modulated by interactions with the microenvironment. However, a systematic understanding of how the myriad of niche cues impact distinct epithelial cell types in a diseased context is still lacking. To address this gap, we first benchmarked diverse human colonic organoid injury models against IBD tissue, and established a disease-relevant model of epithelial inflammation using TNF, IFN{gamma}, and IL1{beta}. Using this system, we built a dictionary of epithelial responses to 81 secreted niche factors at single cell resolution via donor-pooled, multiplexed single cell RNA-sequencing (scRNA-seq). The comprehensive nature of our atlas allowed us to map relationships between perturbations, infer the function of less well-characterized ligands, and identify cell type-specific perturbed pathways. Finally, we established the relevance of organoid-derived gene programs by mapping them to single cell and spatial atlases of human IBD tissue. Our resource offers a global view of epithelial responses to microenvironmental cues in a physiologically relevant disease context and generates new hypotheses for signaling factors that may be involved in epithelial homeostasis and repair.

bioengineering↗

bioRxiv2025

Metabolic modulation of intratumoral cholesterol with gut microbiota for the treatment of colorectal cancer

Excess cholesterol is positively correlated with colorectal cancer (CRC). Current therapeutic strategies for modulating cholesterol levels in CRC are limited and often come with complications. Here, we demonstrated that microbiome shunting of intestinal cholesterol to anticancer metabolites is a safe and applicable therapeutic approach for CRC. By screening major microbial metabolic products of cholesterol, we found that 4-cholesten-3-one (4-C-3) was selectively depleted in fecal samples and tumor tissues of patients with CRC. 4-C-3 exhibits strong antitumor effects on human CRC cell lines, patient-derived organoids, and patient-derived xenograft (PDX) models. Mechanistically, 4-C-3 suppresses CRC tumorigenesis by dually targeting the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homologue (KRAS). 4-C-3 directly binds to EGFR, blocking its signal transduction by inhibiting the binding of EGFR ligands. Additionally, 4-C-3 inhibits oncogenic KRAS variants, such as KRASG12D, by suppressing nucleotide exchange activity and effector engagement. By targeting both EGFR and KRAS, 4-C-3 reduces primary resistance to anti-EGFR therapies caused by KRAS mutations in CRC. As a proof-of-concept study, we showed that delivery of 4-C-3 by Oscillibacter ruminantium, a 4-C-3- producing commensal bacterium that is reduced in CRC patients, or a nonpathogenic Escherichia coli strain engineered to specifically convert intestinal cholesterol into 4-C-3, removed intratumoral cholesterol and led to rapid tumor regression in multiple models of CRC. These results suggest that microbial therapies for restoring intestinal cholesterol homeostasis represent a new therapeutic avenue for CRC.

cancer biology↗

bioRxiv2025

Differential Regulation of Hepatic Macrophage Fate by Chi3l1 in MASLD

Metabolic dysfunction-associated steatotic liver disease (MASLD) progression involves the replacement of protective embryo-derived Kupffer cells (KCs) by inflammatory monocyte-derived macrophages (MoMFs), yet the regulatory mechanisms remain unclear. Here, we identify chitinase 3-like 1 (Chi3l1/YKL-40) as a critical metabolic regulator of hepatic macrophage fate. We observed high expression of Chi3l1 in both KCs and MoMFs during MASLD development. Genetic deletion of Chi3l1 specifically in KCs significantly exacerbated MASLD severity and metabolic dysfunction, whereas MoMF-specific Chi3l1 deletion showed minimal metabolic effects. Mechanistic studies revealed that this cell type-specific regulation arises from differential metabolic requirements: KCs display elevated glucose metabolism compared to MoMFs. Chi3l1 directly interacts with glucose to inhibit its cellular uptake, thereby selectively protecting glucose-dependent KCs from metabolic stress-induced cell death while having negligible effects on less glucose-dependent MoMFs. These findings uncover a novel Chi3l1-mediated metabolic checkpoint that preferentially maintains KCs populations through glucose metabolism modulation, providing important new insights into the pathogenesis of MASLD and potential therapeutic strategies targeting macrophage-specific metabolic pathways.

molecular biology↗

bioRxiv2025

PIBE: Parallel ion beam etching of sections collected on wafer for ultra large-scale connectomics

We developed a parallel ion beam etching device that simultaneously thins hundreds of sections on a whole 4-inch wafer at 20 nm steps, enabling sub-centimeter volumetric imaging with high axial resolution. Compared with traditional volume electron microscopy, PIB preserves in-situ continuity within sections and simplifies inter-section registration. Its high axial resolution facilitates reliable neuron segmentation and tracing with reduced proofreading, providing an efficient route toward ultra-large-scale connectomics, such as whole-mouse-brain reconstruction.

neuroscience↗

bioRxiv2025

Large-scale single-neuron recording using the uFINE array in human cortex

Monitoring neural population activity at single-cell resolution is essential for driving fundamental research and clinical innovations. However, translating advanced recording techniques from animal models to humans remains a significant challenge. Flexible neural electrodes have recently emerged as powerful tools for large-scale single-unit recordings due to their superior biocompatibility and high recording density. Here, we demonstrate reliable, high-density single-unit recordings during intraoperative procedures in human patients using ultra-Flexible Implantable Neural Electrode (uFINE) arrays. The uFINE array exhibited sufficient mechanical robustness to maintain structural integrity throughout surgical operations. We successfully recorded 616 single units from 10 patients, with up to 135 single units simultaneously recorded. The flexibility of uFINE array minimized signal disturbances from brain pulsations, enabling stable and continuous single-unit detection. Stimulus and response tuning were observed at the level of individual neurons in awake patients. This uFINE-based recording approach offers unique opportunities to investigate human-specific cognitive functions and develop next-generation brain-machine interfaces.

bioengineering↗

bioRxiv2025

Integration of the hammerhead ribozyme into structured RNAs to measure ligand-binding events for riboswitch candidates and aptamers

Some structured RNAs, such as riboswitches and aptamers, can bind their cognate ligands and have been used in biosensors and gene expression control elements. However, current methods for detecting ligand binding to structured RNAs are either severely limited or inconvenient. In this study, we design a multi-base pair bridge to integrate a hammerhead ribozyme into structured RNAs to detect ligand binding events. The experimental results demonstrate that the length of the bridge has a significant effect on the cleavage of the ribozyme; optimal cleavage can be achieved with three to six base pairs in the bridge. The dissociation constant (KD) values obtained through this method are in agreement with those determined by in-line probing techniques, and 1 pmol of allosteric ribozyme RNA is sufficient for measurement. We apply this method to evaluate the binding affinity of the riboswitch candidate Motif_9307. Our findings indicate that this motif has no binding affinity for S-adenosylmethionine or several other tested ligands, which is consistent with the results of the in-line probing experiments. Notably, our method reveals an increase in cleavage activity when yeast extract is added as a mixture of ligands, suggesting that the ligand of Motif_9307 is present in the extract. In conclusion, we develop an alternative approach for measuring ligand binding events associated with riboswitch candidates and aptamers.

molecular biology↗

bioRxiv2025

SpatialAgent: An Autonomous AI Agent for Spatial Biology

Advances in AI are transforming scientific discovery, yet spatial biology, a field that deciphers the molecular organization within tissues, remains constrained by labor-intensive workflows. Here, we present SpatialAgent, an autonomous AI agent for spatial biology research. SpatialAgent couples large language models with a Plan-Act-Conclude architecture, dynamic tool and skill retrieval, multimodal interpretation, and verification modules that audit generated claims. It supports the full discovery loop, from gene-panel design and multimodal annotation to trajectory inference, cell-cell communication analysis, imputation, and hypothesis generation. Across human and mouse brain, heart, tonsil, colon, and prostate datasets, SpatialAgent outperformed established computational baselines and matched or surpassed expert scientists in key tasks. In open-ended case studies, it recovered known tissue organization and generated spatially grounded hypotheses. In a prospective mouse prostate cancer Xenium study, it designed a compact 100-gene add-on panel that profiled 4.2 million cells across 21 samples, improved cell-type and malignant-state prediction, and captured spatially structured tumor and microenvironment programs. SpatialAgent establishes a framework for autonomous and collaborative discovery in spatial biology.

bioinformatics↗

bioRxiv2025

Effect of the S2' site cleavage on SARS-CoV-2 spike

SARS-CoV-2 initiates infection of host cells by fusing its envelope lipid bilayer with the cell membrane. To overcome kinetic barriers for membrane fusion, the virus-encoded spike (S) protein refolds from a metastable prefusion state to a lower energy, stable postfusion conformation. The protein is first split into S1 and S2 fragments at a proteolytic site after synthesis, and presumably further cleaved at a second site, known as the S2 site, before membrane fusion can occur. We report here a cryo-EM structure of S2 fragment after the S2 cleavage, possibly representing a late fusion intermediate conformation, in which the fusion peptide and transmembrane segment have yet to pack together, distinct from the final, postfusion state. Functional assays demonstrate that the S2 cleavage accelerates membrane fusion, probably by stabilizing fusion intermediates. These results advance our understanding of SARS-CoV-2 entry and may guide intervention strategies against pathogenetic coronaviruses.

biochemistry↗

bioRxiv2025

Machine-Guided Dual-Objective Protein Engineering for Deimmunization and Therapeutic Functions

Cell and gene therapies often rely on the expression of exogenous proteins derived from nonhuman organisms. An emerging consensus is to reduce the potential immunogenicity of such therapies by instead using human protein domains. However, as we engineer these human-derived proteins, we create nonhuman peptides at the linkers or junctions between domains and at mutated residues within them, which still pose a risk of immunogenicity that has largely been left unaddressed. Here, we present a modular workflow to simultaneously optimize the functions of proteins and minimize their immunogenic risk using existing machine learning models that predict protein function and nonhuman peptide immunogenicity from their sequences. We first applied this workflow to existing transcriptional activation and bio-orthogonal RNA binding domains. Then we generated a set of small molecule-controllable transcription factors with human-derived zinc finger DNA-binding domains for targeting orthogonal non-genomic DNA sequences. Finally, we established a workflow for creating deimmunized zinc finger arrays to target arbitrary genomic DNA sequences and used it to upregulate expression of two therapeutically relevant genes, UTRN and SCN1A. Our future-proof, modular workflow offers a proof of principle for making cell and gene therapies safer and more efficacious through dual-objective protein optimization using state-of-the-art algorithms.

bioengineering↗

bioRxiv2025

Development and Characterization of a Sf-1-Flp Mouse Model

The use of genetically engineered tools, including combinations of Cre-LoxP and Flp-FRT systems, enable the interrogation of complex biology. Steroidogenic factor-1 (SF-1) is expressed in the ventromedial hypothalamic nucleus (VMH). Development of genetic tools, such as mice expressing Flp recombinase (Flp) in SF-1 neurons (Sf-1-Flp), will be useful for future studies that unravel the complex physiology regulated by the VMH. Here, we developed and characterized Sf-1-Flp mice and demonstrated its utility. Flp sequence was inserted into Sf-1 locus with P2A. This insertion did not affect Sf-1 mRNA expression levels and Sf-1-Flp mice do not have any visible phenotypes. They are fertile and metabolically comparable to wild-type littermate mice. Optogenetic stimulation using adeno-associated virus (AAV)-bearing Flp-dependent channelrhodopsin-2 (ChR2) increased blood glucose and skeletal muscle PGC-1 in Sf-1-Flp mice. This was similar to SF-1 neuronal activation using Sf-1-BAC-Cre and AAV-bearing Cre-dependent ChR2. Finally, we generated Sf-1-Flp mice that lack {beta}2-adrenergic receptors (Adr{beta}2) only in skeletal muscle with a combination of Cre/LoxP technology (Sf-1-Flp::SKM{Delta}Adr{beta}2). Optogenetic stimulation of SF-1 neurons failed to increase skeletal muscle PGC-1 in Sf-1-Flp::SKM{Delta}Adr{beta}2 mice, suggesting that Adr{beta}2 in skeletal muscle is required for augmented skeletal muscle PGC-1 by SF-1 neuronal activation. Our data demonstrate that Sf-1-Flp mice are useful for interrogating complex physiology.

neuroscience↗

bioRxiv2025

Molecular Mechanisms Limiting the Therapeutic Window of AAV Gene Therapy in Mouse Models of Blue Cone Monochromacy

Blue cone monochromacy (BCM) is an X-linked retinal disorder caused by mutations in the OPN1LW/OPN1MW gene locus, resulting in impaired cone function and structural degeneration. We conducted a comparative analysis of AAV-mediated gene therapy in Opn1lw/Opn1mw double knockout (DKO) and Opn1mwC198R/Opn1sw-/- (C198R) BCM mouse models and evaluated the therapeutic window, efficacy, and longevity. Our results demonstrate that the AAV8-Y733F capsid achieved superior cone rescue compared to AAV5. While both DKO and C198R models showed similar therapeutic windows and rescue longevity, treatment efficacy decreased markedly in older mutant mice. Structural analysis revealed that aged cones in both models displayed degenerative changes, including mislocalized mitochondria and compromised connecting cilia. At the molecular level, we observed reduced AAV-mediated transgene expression in DKO and C198R older cones, which may result from decreased transduction efficiency, decreased circular episome stability, genome-wide transcription/translation downregulation, targeted mRNA/protein degradation, or overall cone degeneration. Notably, the cone-specific promoters for Pde6c and Cngb3 maintained robust activity in degenerating cones. These findings suggest that combining an efficient AAV serotype with an optimized cone promoter could be a viable approach to extend the therapeutic window and enhance treatment longevity for BCM patients.

molecular biology↗