Biology subjects

Chen, B.

Publications and source records attributed to Chen, B..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

In vivo-compatible spatial multi-omics via hydrogen peroxide-independent APEX2 labeling

Proximity labeling (PL) technologies like APEX2 have transformed spatial multi-omics in live cells, but their long-standing dependence on hydrogen peroxide (H2O2) disrupts redox signaling and prevents use in live animals. Here we introduce H2O2-independent APEX2 (Hi-APEX), which uses a clickable tetrazine-phenol probe, requiring no enzyme engineering. We show that APEX2 directly catalyzes TP radical formation without H2O2 via a mechanism requiring the probes tetrazine group and a key histidine residue. We benchmarked Hi-APEX-based spatial multi-omics by mapping the mitochondrial matrix and dynamic secretomes. Hi-APEX significantly outperforms traditional APEX in capturing redox-sensitive processes such as stress response and ferroptosis, enabling discovering authentic stress granule components and protein interaction networks for mitochondria-localized GPx4. One mGPx4 interactor TRMT61B--known to regulate mitochondrial m{superscript 1}A modifications--promotes ferroptosis. Crucially, Hi-APEX achieves full in vivo compatibility, enabling direct PL in tumor xenografts and hippocampal neurons, thereby expanding PL-based spatial multi-omics from cellular systems to living organisms.

biochemistry↗

bioRxiv2026

Phosphorylation of a tumor-derived ASXL2 epitope remodels 1 peptide-HLA binding affinity and interaction dynamics

The expansion of cancer immunotherapy has made it increasingly important to identify tumor-specific HLA class I epitopes and to define the molecular determinants of peptide-HLA binding and stability. Post-translationally modified epitopes, particularly phosphorylation, can reflect oncogenic signaling states and offer highly tumor-selective targets. Despite their potential, the structural and dynamic mechanisms by which phosphorylation reshapes peptide-HLA affinity and stability remain unclear. Here, we investigate a phosphorylated ASXL2-derived epitope supported by confident MS/MS-based phosphosite localization and detected across multiple cancer types. We performed all-atom molecular dynamics simulations of peptide-HLA (pHLA) complexes to compare the phosphorylated peptide, its non-phosphorylated counterpart, and related variants. Our simulations indicate that phosphorylation reconfigures non-bonded interaction networks within the binding groove by introducing new local contacts, leading to an enhanced predicted binding affinity. Furthermore, principal component analysis revealed that phosphorylation increases the overall conformational flexibility and alters the collective backbone motions of the pHLA complex. Together, these findings provide a structural-dynamic basis for how phosphorylation can modulate pHLA stability and interaction dynamics, guiding the rational prioritization and targeting of cancer-specific pHLA complexes for immunotherapy.

bioinformatics↗

bioRxiv2026

Visualizing early Mycobacterium tuberculosis interactions with murine lung macrophages using intravital imaging

Intravital microscopy enables direct visualization of dynamic cellular processes within intact tissues, but its application to Mycobacterium tuberculosis (Mtb) has been limited by Biosafety Level 3 (BSL-3) containment requirements and the technical challenges of stabilizing the lung for high-resolution imaging. Here, we present a protocol that combines the thoracic Window for High-Resolution Imaging of the murine Lung (WHRIL) with a genetically defined, triple-auxotrophic Mtb strain (mc27902) approved for use under BSL-2 conditions. We describe the construction of a tdTomato-expressing derivative (mc28471) preparation of bacteria for intravenous infection and intravital imaging in reporter mice. This system enables visualization of rapid bacterial entry into the pulmonary vasculature, subsequent aggregation, and vascular occlusion, dissemination into the lung parenchyma, and macrophage uptake over three days post-infection. This protocol provides the first practical platform for real-time intravital imaging of mycobacteria in the lung and establishes a foundation for mechanistic studies of bacterial physiology, host recognition, and immune-mediated clearance using safe Mtb surrogates. SummaryThis protocol describes a biosafety level 2 (BSL-2)-compatible intravital imaging platform for visualizing Mycobacterium tuberculosis (Mtb) in the intact murine lung at single cell resolution. By combining the Window for High-Resolution Imaging of the murine Lung (WHRIL) with a fluorescently labeled, genetically defined triple auxotrophic Mtb strain (mc27902), this approach overcomes long-standing biosafety and technical barriers that have prevented real-time imaging of mycobacterial infection in vivo. The method enables direct visualization of early bacterial localization, aggregation, vascular interactions, and macrophage uptake during the initial hours to days following infection, providing a practical foundation for mechanistic studies of host-pathogen interactions under safe experimental conditions.

immunology↗

bioRxiv2026

Rab32/Rab38-positive Lysosome-Related Organelle degrades lipid droplet in hepatocytes by microautophagy

Rab32 and Rab38 are paralogous small GTPases involved in the biogenesis of lysosome-related organelles (LROs), yet their roles in hepatic lipid metabolism remain poorly defined. Here, Rab32 and Rab38 double-knockout (DKO) male mice exhibited an age-dependent increase in body weight accompanied by hepatic lipid accumulation, suggesting impaired hepatic lipid processing. In AML12 hepatocytes, Rab32 and Rab38 localized to ring-like, LAMP1-positive structures characteristic of LROs, whose size increased with cell confluence. Pharmacological inhibition of lysosomal acid lipase with orlistat led to the accumulation of lipid droplets (LDs) within Rab32/38-positive LROs, indicating that LD degradation occurs in these compartments. Additional treatment with bafilomycin A1 revealed invagination-like internal membrane structures within enlarged LROs. These processes were not affected by artificial inhibition of macroautophagy, highlighting the involvement of microautophagy. Ring-like signals positive for phosphatidylinositol 3-phosphate (PI3P) or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) were detected within or adjacent to LRO membranes, and LDs were frequently associated with these structures, suggesting a role for PI3P and PI(3,5)P2 in internal membrane formation. Vps4B was also required for efficient LD incorporation. Consistently, Rab32/38 double-knockdown (DKD) AML12 cells exhibited increased lipid accumulation, indicating impaired LD engulfment. Together, these findings identify Rab32/38-positive LROs as a structural platform for microautophagy-mediated lipid droplet degradation in hepatocytes.

cell biology↗

bioRxiv2026

Chronic Paraventricular OX1R Overexpression Induces Oxidative Stress and Hypertension in Rats

BackgroundHyperactivity of the orexin system has been implicated in hypertension, yet the long-term impact of orexin 1 receptor (OX1R) overexpression in the paraventricular nucleus (PVN), a key brain region involved in arterial blood pressure (ABP) regulation, remains unclear. This study examined whether chronic OX1R overexpression in the PVN alters cardiovascular, neuroendocrine, and oxidative functions. MethodsAdult male Sprague-Dawley rats received bilateral PVN injections of AAV2-OX1R or control virus AAV2-GFP. Their ABP and heart rate (HR) were monitored for eight weeks via radiotelemetry. Renal sympathetic nerve activity (RSNA) and ABP responses to PVN orexin A administration were assessed using in vivo recordings. Reactive oxygen species (ROS) were quantified using MitoProbe, and gene expression was analyzed by qPCR. Primary neuronal cultures were used to explore underlying mechanism involved in increased ROS production and neuronal activity. ResultsPVN OX1R overexpression increased mean ABP ([~]10 mmHg), water intake, and ROS levels in both the PVN and peripheral organs. In addition, orexin A evoked exaggerated RSNA (155% vs. 73%) and pressor responses (27 vs. 16 mmHg) in PVN OX1R overexpression rats compared to control rats, these effects were attenuated by the OX1R antagonist SB408124. Plasma vasopressin levels were elevated, and brain sections confirmed increased ROS in OX1R-positive neurons. In vitro experiments further demonstrated that orexin A upregulated ROS-generating enzymes, suppressed antioxidant genes, and enhanced neuronal excitability. ConclusionChronic PVN OX1R overexpression induces oxidative stress, sympathetic overactivity, and increased plasma vasopressin, contributing to hypertension development. Targeting PVN OX1R signaling may provide therapeutic benefit for hypertension.

physiology↗

bioRxiv2026

Injury-transduced SerpinOLs modulate neuroinflammation and glial activation in the diseased and non-diseased CNS

Oligodendroglial dysfunction is common in CNS diseases and injuries, but the molecular and functional responses of oligodendroglia to CNS pathologies remain poorly defined. Here, we report that oligodendrocytes (OLs) respond to demyelinating diseases by dysregulating serine protease inhibitor clade A member 3N (SERPINA3N). Homeostatic OLs transition to Serpina3n-expressing OLs (SerpinOLs) under other diseased conditions including stroke, endotoxicity, neurodegeneration, neurotrauma, and non-diseased healthy aging conditions. Mechanistically, general neuroinflammation or inflammatory mediators is insufficient for SerpinOL transition. Instead, oligodendrocyte damage/injury, even in the absence of neuroinflammation or glial activation, is sufficient for the transition. Phenotypically, SerpinOLs are characterized by molecular signatures of inflammatory and immune regulation and STAT3 signaling activation. Functionally, SerpinOLs exacerbate neuroinflammation and promote glial activation toward pro-inflammatory and neurodegenerative states. Together, our findings suggest SerpinOLs are a common population of injury-transduced OLs that amplify neuroinflammation and glial activation in the diseased and non-diseased CNS through SERPINA3N secretion. Our findings provide new insights into myelination-independent role of OLs in regulating CNS pathophysiology.

neuroscience↗

bioRxiv2026

Spatial Decoding of Tertiary Lymphoid Structure Maturation in Non-Small Cell Lung Cancer Using Deep Neural Networks

Understanding the role of tertiary lymphoid structures (TLS) is crucial in non-small cell lung cancer (NSCLC), as they are associated with patient prognosis and treatment outcomes. Specific cellular ecosystems that originate anti-tumor activity or predict immunotherapy response remain poorly characterized. To this end, we developed a high-resolution, multimodal spatial atlas jointly profiling transcriptomics, proteomics, and histology to characterize TLS maturation in NSCLC alongside secondary lymph organs as a baseline. Using this atlas, we proposed a pathologist-in-the-loop framework that combines a variational graph autoencoder (VGAE) with diffusion pseudotime to refine human expert annotations and characterize TLS maturation. These spatial molecular representations were extended to H&E whole-slide images via a vision transformer-based foundation model. Next, we resolved cellular composition, spatial organization, and cell-cell interactions within these data and defined two divergent spatial ecosystems. Clinical evidence suggests that these ecosystems are associated with distinct patient outcomes: a mature germinal center niche with favorable prognoses, and a tumor-macrophage-fibroblast niche with unfavorable prognoses. In summary, our work decodes key components of TLS heterogeneity, identifies hallmark spatial patterns involved in NSCLC adaptive immunity, and provides a framework for translating spatial omics insights into clinical applications.

genomics↗

bioRxiv2026

Unraveling Subcellular Ultrastructure with Cyclically Multiplexed Expansion Microscopy

Despite advances in fluorescence microscopy, spectral overlap and limited resolution hinder the dense mapping of the cellular ultrastructure. To overcome these challenges, we developed Cy-ExM, a high-plex imaging strategy that integrates optimized cryo-fixation for antigen preservation, expansion microscopy, and iterative immunofluorescence labeling. Using oblique plane microscopy, we perform three-dimensional super-resolution imaging of 20 biological targets encompassing the full cellular volume of individual mammalian cells.

cell biology↗

bioRxiv2026

CAR-T-drug conjugate against solid tumor

Chimeric antigen receptor (CAR)-T cell therapy has been clinically successful in hematologic cancers but faces challenges in solid tumors, primarily due to limited tumor infiltration, immunosuppressive tumor microenvironments (TME), and antigen heterogeneity. While combining CAR-T cell therapy with chemotherapy can enhance antitumor activity, this often leads to substantial systemic toxicity. In this study, we introduce CAR-T-drug conjugate (CAR-T-D-C), a new class of dual-functional therapeutics that effectively addresses these obstacles by integrating click chemistry for precise conjugation of cytotoxic agents onto antigen-specific CAR-T cells. By transforming the hostile TME into an ally, this approach facilitates localized delivery of cytotoxic payload directly to the tumor site, enhancing the overall effectiveness of CAR-T therapy in solid tumors. CAR-T-D-Cs with different CAR-T cell binders exhibit robust antitumor activity across diverse solid tumor models, including both human tumor xenografts and syngeneic models. Spatial transcriptomic studies reveals that CAR-T-D-C achieves improved CAR-T tumor infiltration and functional activation within the TME. Compared to conventional CAR-T therapy, CAR-T-D-C markedly enhances immune cell infiltration, augments effector functions, promotes antigen spreading, amplifies systemic immune responses, and improves overall anti-tumor immunity. CAR-T-D-C represents a versatile therapeutic concept that combines the potency of small molecule drugs and the specificity of CAR-T cells as a 2-in-1 immunochemotherapy for treatment of solid tumors.

immunology↗

bioRxiv2026

Fezf2 regulates differentiation of Aire-expressing and post-Aire mimetic epithelial populations maintaining thymic homeostasis

Fezf2 has been proposed to serve principally as a transcriptional regulator of broad self-antigen expression in medullary thymic epithelial cells (mTECs). Here, we demonstrate an additional function for Fezf2 as a developmental regulator of TEC lineage commitment. Fezf2 deficiency in the thymus led to a relative expansion of lung mimetic and Ccl21+ immature mTECs at the expense of MHCII-hi Aire-expressing and all other mimetic populations. Consistent with this, the disruption of mTEC development had functional consequences, including alterations in tuft-associated iNKT polarization and microfold-associated B-cell class switching. In addition, high-resolution transcriptomics revealed that Fezf2 and Aire regulate distinct transcriptional programs, with Fezf2 driving both activation and repression of a more limited set of tissue-restricted genes compared to the broad gene activation program observed for Aire. Pure transcriptional repression at Fezf2 target loci was sufficient to partially rescue mTEC development in global Fezf2-/- (tKO) mice, potentially through suppression of Lifr expression and modulation of downstream Stat3 signaling tone. These results expand our understanding of Fezf2 in mTEC biology, highlighting transcriptional repression as a required functional facet for adult steady-state lineage patterning across the mTEC compartment.

immunology↗

bioRxiv2025

The inactivation of DHHC7 in mouse liver promotes diet-induced obesity through a hepatic Prg4-GPR146 axis

The liver secretes hepatokines that coordinate whole-body metabolism. Posttranslational lipidation of signaling proteins by DHHC palmitoyltransferases controls membrane targeting and signaling, yet the role of hepatic palmitoylation in systemic metabolic regulation is largely unexplored. Using an inducible, liver-specific DHHC7 knockout (D7LKO) mouse, we report that loss of DHHC7 in hepatocytes potentiates adenylyl cyclase-PKA-CREB signaling through reducing palmitoylation of inhibitory G protein subunit (Gi), leading to transcriptional elevation and secretion of proteoglycan 4 (Prg4). Elevated circulating Prg4 acts on adipocytes by binding GPR146 via Prg4s N-terminal SMB domain, suppressing adipocyte PKA substrate phosphorylation and hormone-sensitive lipase (HSL) Ser563 phosphorylation, thereby impairing lipolysis. Under high-fat diet (HFD) feeding, D7LKO mice and mice with adenoviral hepatic Prg4 overexpression develop pronounced obesity characterized by increasing brown, subcutaneous, and visceral fat mass and adipocyte hypertrophy; strikingly, these animals show little impairment in glucose tolerance or circulating triglycerides but display elevated plasma cholesterol. Conditioned medium containing Prg4 recapitulates suppression of adipocyte HSL phosphorylation in vitro, and SMB-deleted Prg4 fails to bind GPR146 or inhibit HSL phosphorylation. Our findings define a liver palmitoylation- hepatokine axis that controls adipose lipolysis and predisposes to diet-induced fat accumulation, establishing Prg4-GPR146 as a mechanistic link between hepatic signaling and adipose energy mobilization.

physiology↗

bioRxiv2025

A scaffold attachment factor PHM-2 regulates synaptic transmission through SLO-2 potassium channel in C. elegans

Scaffold attachment factor B (SAFB) proteins are evolutionarily conserved DNA/RNA binding proteins that are involved in multiple processes of gene expression. These proteins are broadly expressed with particular high expression observed in the nervous system. However, their physiological roles in neurons are largely unclear. Here we show that PHM-2, the sole SAFB ortholog in C. elegans, regulates synaptic transmission at the neuromuscular junctions through an effect on SLO-2 potassium channel. We found that phm-2 knockout suppresses a sluggish phenotype of worms expressing a hyperactive SLO-2 channel, greatly reduces SLO-2-mediated neuronal whole-cell currents, and enhances neuromuscular synaptic transmission. In addition, we found that PHM-2 genetically interacts with another DNA/RNA binding protein, HRPU-2/hnRNP U, to control SLO-2 expression through a posttranscriptional mechanism. These results reveal a novel function of a SAFB protein in regulating neuronal activity, and may help understand the physiological roles of SAFB proteins in the nervous system of other species. Author SummaryProteins in the SAFB family are found in many species, and they help control how genes are expressed in cells. These proteins are commonly present in the nervous system, but their exact roles in nerve cells are not well understood. In this study, we examined the single SAFB-like protein called PHM-2 in the nematode C. elegans to learn how it affects the nervous system. We discovered that PHM-2 plays an important role in communication between nerve cells and muscles. Worms lacking PHM-2 were able to counteract the sluggish movement caused by a hyperactive potassium channel called SLO-2. Without PHM-2, nerve cells had much smaller electrical currents mediated by SLO-2 and a stronger signaling from nerves to muscles. We also found that PHM-2 works together with another genetic regulator, HRPU-2, to control the amount of SLO-2 protein made in nerve cells. These findings reveal a new role for SAFB proteins in shaping neuronal activity by regulating potassium channels. Understanding this process in worms may provide clues about how these proteins contribute to brain function in humans.

neuroscience↗

bioRxiv2025

The CCC complex directs phagosomal maturation and bactericidal activity in macrophages through PI(3)P regulation

The evolutionarily conserved COMMD/CCDC22/CCDC93 (CCC) complex regulates endolysosomal function and is required for effective antibacterial immunity. However, the mechanisms linking the CCC complex to macrophage function remain poorly understood. Using bone marrow-derived macrophages, we found that CCC deficiency impaired bacterial clearance and disrupted phagosome maturation, as evidenced by defective phagosomal membrane remodeling, reduced phagosome-endoplasmic reticulum contacts, and impaired phagosome-lysosome fusion. These defects were independent of the CCC complex interaction partner, Retriever. CCC-deficient macrophages exhibited excessive phosphatidylinositol 3-phosphate (PI(3)P) accumulation on phagosomal membranes due to markedly reduced recruitment of the PI(3)P phosphatase MTMR2. Restoration of normal PI(3)P levels rescued bactericidal activity, establishing aberrant PI(3)P regulation as the principal defect affecting CCC-deficient macrophages. These findings identify a previously unrecognized Retriever-independent function of the CCC complex in regulating PI(3)P dynamics during phagosome maturation, a process essential for efficient bacterial clearance and innate immune defense.

cell biology↗

bioRxiv2025

Efficient Identification of Short Tandem Repeats via Context-Aware Motif Discovery and Ultra-Fast Sequence Alignment

Tandem repeats (TRs) are highly polymorphic genomic elements, associated with diverse molecular traits and implicated in numerous human diseases. However, large-scale analysis of TRs has been limited by computational challenges, including motif recognition, detection in complex regions, and excessive computational cost. Here we present FastSTR, a computationally efficient tool for precise detection and characterization of TRs. FastSTR integrates a context-aware N-gram motif model with a segmented global alignment algorithm to enable accurate motif identification and boundary definition, even for repeat units up to 8 bp. Across 13 species, FastSTR achieved >90% recall and 99% precision, running several times faster than existing methods white outperforming them in both sensitivity and accuracy. Applied to the human genome, FastSTR uncovered previously unannotated HSATII elements, resolved population-specific TR demonstrate, and identified recurrent STR alterations in lung cancer. These results demonstrate FastSTR as a versatile framework for TR annotation and discovery, advancing studies of genome evolution, genetic diversity, and disease.

bioinformatics↗

bioRxiv2025

Deep-learning model guided discovery and characterization of bacterial unspecific peroxygenases

Unspecific peroxygenases (UPOs) are capable of catalyzing the selective oxidation of organic substrates under mild conditions, using hydrogen peroxide (H2O2) as the sole oxidant. This makes them one of the most promising biocatalysts for chemical synthesis. However, the major limitation restricting the application of UPOs to date is their difficulty in heterologous expression. Although more than 4,000 putative UPO enzymes have been recorded in databases, only about 50 of them can currently be heterologously expressed. All UPOs discovered so far originate from eukaryotes (mainly basidiomycetes and some ascomycetes), and they rely on the complex expression systems and post-translational modifications of their native hosts. This further exacerbates the challenges associated with heterologous expression of eukaryotic UPOs. In this work, we developed a deep-learning-based enzyme mining strategy and, for the first time, discovered novel UPO enzymes from bacteria, achieving successful heterologous expression in Escherichia coli. Bacterial UPOs differ greatly from fungal UPOs in sequence similarity, displaying completely distinct evolutionary trajectories. The discovery of bacterial UPOs advances our understanding of the catalytic mechanisms and expression characteristics within the UPO family, breaking the long-held assumption that UPOs can only originate from eukaryotes. Their excellent heterologous expression performance and broad catalytic versatility will further expand the application potential of UPOs.

bioinformatics↗

bioRxiv2025

A stress-dependent TRIM28-ALKBH2 feedback loop modulates chemotherapy response

Chemoresistance to DNA-damaging agents, including platinum and alkylating compounds, limits treatment efficacy in non-small cell lung cancer (NSCLC) and is frequently associated with elevated DNA repair activity. Here, we identify a stress-responsive feedback loop between the E3 ligase TRIM28 and the demethylase ALKBH2. TRIM28 binds ALKBH2 and promotes its K48-linked polyubiquitination and proteasomal degradation, whereas ALKBH2 enhances TRIM28 transcription and protein levels, forming a feedback loop. Notably, alkylation stress induces a biphasic response: acute MMS exposure enhances TRIM28-ALKBH2 association and accelerates ALKBH2 degradation, whereas prolonged exposure promotes TRIM28 degradation, leading to ALKBH2 stabilization and transcriptional upregulation. Clinically, ALKBH2 is frequently elevated in lung adenocarcinoma and is associated with worse survival, whereas TRIM28 exhibits prognostic value specifically in chemotherapy-treated patients. Functionally, MMS-cisplatin co-treatment increased DNA damage and reduced clonogenic survival by counteracting ALKBH2-dependent alkylation tolerance, linking the TRIM28-ALKBH2 loop to chemoresistance in NSCLC.

biochemistry↗

bioRxiv2025

A Global Discovery of Antimicrobial Peptides in Deep-Sea Microbiomes Driven by an ESM-2 and Transformer-based Dual-Engine Framework

The global crisis of multidrug-resistant pathogens necessitates innovative antimicrobial peptide (AMP) discovery. However, current AMP predictors are severely compromised by three overlooked systematic data biases, including sequence length imbalance, N-terminal methionine artifacts, and lack of microbial optimization. To overcome these fundamental bottlenecks, we developed XAMP, a dual-engine predictor integrating XAMP-E (ESM-2-based for high accuracy) and XAMP-T (a one-layer Transformer for high speed), trained on rigorously debiased datasets. Consequently, XAMP achieved a median AUC of 0.972, representing an improvement of up to 21.9% in AUC while operating 5 to 40 times faster. Beyond accurate classification, we found that AMPs inherently possess a significantly higher net charge (+3.9 vs. +0.5) despite similar hydrophobicity, along with distinct cooperative patterns such as W-K/R pairs. Applying this pipeline to 238 deep-sea metagenomes (>1000m depth), we identified 2,355 promising AMP candidates from microbial dark matter, and metaproteomic analysis confirmed in-situ expression for a subset of these candidates. Notably, these deep-sea AMPs exhibited a significant 1.53-to 2.93-fold enrichment in K/R/W/Y residues compared to known AMPs. Experimental validation demonstrated that six synthesized peptides exhibit potent, broad-spectrum activity against ESKAPE pathogens, with particular efficacy against Gram-negative bacteria that dominate deep-sea ecosystems and pose major clinical challenges. This study establishes that correcting algorithmic data biases is a prerequisite for reliable mining, providing a robust framework to unlock the therapeutic potential hidden within microbial dark matter, offering new avenues to combat the antibiotic resistance crisis.

bioinformatics↗

bioRxiv2025

Brain-wide Genome Editing via STEP-RNPs for Treatment of Angelman Syndrome

Brain-wide genome-editing remains a major hurdle for the treatment of neurogenetic disorders. Here, we report a non-viral, non-nanoparticle, chemical modification-based method, called Stimuli-Responsive Traceless Engineering Platform (STEP) that achieves highly efficient and brain-wide genome-editing of neurons in mice. Using cholesterol-based STEP as a lead, we show that a single administration of STEP-ribonucleoproteins (RNPs) results in functional genetic rescue with significant improvements across a battery of neurobehavioral domains in the Angelman syndrome (AS) mouse model. No significant off-target events or general toxicity effects are observed. STEP-RNPs are also highly efficient at editing human neurons and cortical brain organoids differentiated from AS patient-derived iPSCs. scRNA-seq analysis confirms functional genetic rescue via reactivation of Ube3a/UBE3A expression in human and mouse STEP-RNP-treated neuronal cells. Genome editing via STEP-RNPs has broad applications and the potential to treat many other neurogenetic disorders.

genetics↗