Experimental & Molecular Medicine

Immune checkpoint inhibitors (ICIs), especially CTLA-4 inhibitors (CTLA-4), exhibit a high incidence of colitis as an immune-related adverse event (irAE) during cancer treatment, severely limiting patient benefit. Clinically, both treatment interruption and existing intervention drugs for ICI-mediated colitis may compromise antitumor efficacy. However, there is inadequate research on the pathogenesis of ICI-mediated colitis, with findings often conflicting. Here, we first established multiple clinically relevant animal models, including an immuno-humanized ICI-mediated colitis model. Through time-series transcriptomics, we discovered that CTLA-4-induced colonic toxicity exhibits characteristics ranging from early metabolic reprogramming represented by glycolysis to later immune disorders represented by Th17 responses. By targeting colonic CTLA-4+ T cells, CTLA-4 blocked CD80/CD86-CTLA-4 interaction, thereby activating the PI3K-AKT-mTOR pathway. Subsequently, mTOR mediated metabolic reprogramming in T cells, shifting them from Treg-biased oxidative phosphorylation to Th17-biased glycolysis. The colonic toxicity of CTLA-4 has also been demonstrated to depend on the PI3K-AKT-mTOR pathway, glycolysis, and Th17 responses. Notably, metformin significantly relieved ICI-mediated colitis by inhibiting mTOR without impeding antitumor efficacy. Collectively, these findings highlighted the metabolic-immune axis in the colonic toxicity of ICI and provided a clinically superior intervention strategy.

Autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD) is one of the leading hereditary kidney diseases. Currently there is no targeted treatment. To illuminate human relevance of mesencephalic astrocyte-derived neurotrophic factor (MANF)-based therapy, we have established patient induced pluripotent stem cell (iPSC)-derived kidney organoid model carrying UMOD p.H177-R185del, the leading mutation causing ADTKD. We have discovered that MANF can directly bind and repress ER calcium release channel IP3R1, thus enhancing AMPK-induced autophagy in a TRIB3-dependent manner. The therapeutic implication of this finding may well be extended to other protein misfolding diseases.

Vancomycin-resistant enterococci (VRE) is one of the serious threat to global public health, with the diminishing effectiveness of antibiotics. There is an urgent need for novel strategies to control this multidrug-resistant bacterial infections. Here, our findings demonstrate that sublancin, a bacteriocin produced by Bacillus subtilis, exhibits intrinsic antibacterial activity and, more importantly, potentiates vancomycin, therapy restoring its efficacy against VRE. Using a series of in vitro assays, including fractional inhibitory concentration index, time-killing analysis, and resistance development assays, we show that sublancin significantly enhances the bactericidal effectiveness of vancomycin. In vivo, the sublancin-vancomycin combination therapy markedly reduced bacterial loads, improved svrvival rates in a Galleria mellonella model and enhanced bacterial clearance rates in a mouse model. Mechanistic studies using RT-PCR revealed that sublancin down-regulates the expression of the vanA resistance gene cluster. All in all, these findings make vancomycin and this antibiotic peptide combination as a promising candidatese to enhance vancomycin efficacy and overcome VRE infections, potentially through inteference with resistance gene expression.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is fundamental to tissue repair and regeneration but also underlies pathological conditions such as cancer progression. Targeting angiogenesis has thus become a promising approach for developing novel cancer therapeutics. While various phytochemicals have demonstrated anti-angiogenic effects, the role of 2-5(H)-Furanone, a naturally occurring lactone found in various plants and marine sources with diverse biological activities, remains insufficiently explored. In this study, we systematically evaluate the anti-angiogenic potential of 2-5(H)-Furanone using Human Umbilical Vein Endothelial Cells (HUVECs) as an in vitro model and zebrafish embryos as an in vivo model. Experimental findings demonstrated that treatment of HUVECs with increasing concentrations of 2-5(H)-Furanone led to significant, dose-dependent reductions in proliferation, invasion, migration, and tube formation. Analyses of gene expression revealed marked downregulation of key pro-angiogenic mediators, VEGF, and HIF-1. Complementing these in vitro results, in vivo studies in zebrafish embryos showed robust, dose-dependent inhibition of intersegmental vessel (ISV) formation, accompanied by suppression of critical angiogenesis-related genes. Molecular docking further supported these observations by indicating stable binding of 2-5(H)-Furanone to major angiogenic targets, including VEGFR2, MMP2, HIF-1, and PIK3CA. Collectively, our data demonstrate that 2-5(H)-Furanone potently inhibits angiogenesis, as evidenced in both HUVEC and zebrafish models, through functional and molecular mechanisms. These findings support the further development of 2-5(H)-Furanone as a promising anti-angiogenic therapy candidate.

Congenital anomalies of the kidney and urinary tract (CAKUT) are the primary cause of pediatric kidney failure, yet the genetic etiologies remain elusive for most affected individuals. Reanalysis of trio exome sequencing data from 80 Chinese CAKUT patients identified 32 rare, predicted deleterious variants. Replication in unrelated families from a national multicenter database prioritized four novel candidate genes--DOCK11, MIB1, TENM2, and TNS1. These candidates are involved in both well-characterized developmental pathways and more under-explored biological processes relevant to renal and ureteric morphogenesis. CRISPR-Cas9-mediated zebrafish knockout studies were employed to validate the potential association of these genes with kidney abnormalities including significant pericardial edema, malformed renal tubules, and impaired glomerular filtration. These findings offer potential genetic diagnoses for 10% of CAKUT probands, and demonstrate that exome reanalysis can substantially improve diagnostic yield and inform personalized clinical management. Overall, this study expands the known genetic landscape of CAKUT.

Chronic pain affects a significant portion of the global population and imposes substantial clinical and socioeconomic burdens. Current treatments mainly rely on opioid analgesics, which carry serious risks of dependence and misuse, underscoring the urgent need for alternative therapeutic strategies. Galanin receptors (GALR1-3) are known to be involved in modulating pain, yet their specific roles remain poorly understood due to the lack of receptor subtype-selective ligands. Recently, spexin has been identified as an endogenous peptide that selectively activates GALR2 and GALR3, offering a new scaffold for developing pharmacological tools targeting these receptor subtypes. In this study, we report the design and characterization of a modified spexin analog, LIT-01-144, engineered through N-terminal functionalization with a fluorocarbon chain to improve metabolic stability while preserving receptor selectivity. In vitro assays showed that LIT-01-144 has high potency at GALR2 and GALR3, with minimal activity at GALR1. Pharmacokinetic studies revealed a significantly longer plasma half-life compared to native spexin and no central nervous system penetration. In mice, intracerebroventricular administration of LIT-01-144 produced strong antinociceptive effects at doses ten times lower than spexin. While systemic administration showed no notable antinociception in naive animals, LIT-01-144 significantly reduced pain responses in a mouse model of persistent inflammatory pain induced by complete Freunds adjuvant (CFA). This antinociceptive activity was specifically mediated through GALR2 and was independent of opioid receptor pathways. In situ hybridization further showed an increase in Galr2-positive neurons in dorsal root ganglia of inflamed mice. Overall, these findings highlight GALR2 as a promising peripheral target for developing non-opioid analgesics and demonstrate the potential of LIT-01-144 as a valuable tool for understanding GALR2-mediated mechanisms of pain modulation.

Until now, most genetic risk for vascular dementia (VD) remains unknown. Here, we firstly performed the largest cross-ancestry genome-wide association study meta-analysis comprising 5,886 VD and 1,027,883 controls of European, East Asian, South Asian, African, and Admixed American ancestry. We identified 37 genome-wide significant loci including CLU and APOE tagged by common variants and 35 loci tagged by rare variants, and demonstrated enrichment of VD heritability in lung and genetic association between VD and lung function traits. We further conducted a cross-trait of VD and Alzheimers disease, and identified 13 genome-wide significant loci including CR1, BIN1, GRM7, HLA-DRA, TREM2, CLU, ECHDC3, AGBL2, MS4A4E, PICALM, SLC24A4, ABCA7, and APOE. A multi-omics integrative analysis identified 619 genes. 241 genes were significantly differentially expressed in VD cells and 21 exhibited strong evidence of interaction with FDA-approved drugs. Collectively, our findings provide valuable insights into the potential underlying mechanisms of VD.

Stimulator of interferon genes (STING) agonists and derivative molecules have been extensively developed for tumor immunotherapy. However, systemic exposure toxicity risks have constrained clinical trial progression and even threatened patient lives. Currently, systematic toxicity assessments for STING agonists remain lacking, with the mode of action for major organ injury yet to be elucidated. Here, we focused on STING agonist-induced lung injury, revealing that systemic administration of STING agonists caused pulmonary hemorrhage, inflammatory alterations, and respiratory dysfunction. Through single-cell RNA sequencing and immune deletion studies, we found that lung endothelial cells could be stimulated by STING agonists and then secreted chemokines and IL-15 to recruit and activate NK cells. NK cells could induce endothelial cell apoptosis via IFN-{gamma}. Tbx21+ NK subpopulations, which activated by endothelial cells, could produce chemokines to recruit neutrophils. Neutrophils secreted IL-1{beta} through positive feedback pathways and form neutrophil extracellular traps during lung injury. This study elucidates the critical role of the endothelial cell-NK cell-neutrophil axis in mediating STING agonist-associated pneumonia, offering insights for developing intervention strategies for STING agonist toxicity.

The hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 protein forms a complex with SMCR8 and WDR41 (CSW), which acts as a GTPase-activating protein (GAP) regulating small GTPases like ARF1 and RABs involved in intracellular trafficking. Although these findings implicated the ARF1 dysregulation in ALS/FTD and the critical need for validation of its inhibition as potential intervention, small molecules that target the interactions between CSW and ARF1 are lacking. In this study, we showed that the tyrosine-phosphorylated form (Tyr-782) of ASAP1, an ARF-GAP that inactivates ARF1, is increased in the motor cortex of both sporadic ALS and ALS with C9orf72 mutations. Overexpression of C9orf72 led to Golgi disorganization, partially mimicking the effects of ARF1 inhibitor brefeldin A on dispersion of Golgi apparatus. To identify a better strategy to enhance C9orf72 and ARF1 interactions, we applied rational design and virtual screening of a 40-million compound library of small molecules targeting the ARF1-CSW interface. Molecular docking, MM-GBSA binding energy, ADME/Tox profiles, and interaction analysis established MCULE-5095997944 as a top candidate for ARF1 modulation. MCULE-5095997944 demonstrated strong binding to ARF1 in the nanomolar range, reduced GTP-bound ARF1 levels upon ARF1 activation, and altered ARF1-dependent Golgi organization. These studies identified the first small molecule targeting ARF1-CSW interaction and further support ARF1 modulation as a potential therapeutic approach for ALS/FTD.

Efficient and cell-specific gene delivery to cochlear inner hair cells (IHCs) remains a major challenge for inner ear gene therapy. Here, we identify and characterize a novel AAV2-derived capsid, AAV-WM04, that enables highly efficient and selective IHC transduction at low doses. Using an in vivo-directed evolution strategy, we generated a randomized AAV2 capsid library with 9-amino acid insertions and performed iterative selection in the adult mouse cochlea. Next-generation sequencing revealed enrichment of several variants, among which AAV-WM04 exhibited superior packaging efficiency and pronounced IHC tropism. AAV-WM04 achieved near-complete IHC transduction throughout the cochlear axis in adult mice, outperforming clinically relevant vectors with minimal off-target expression and no detectable ototoxicity. Robust and exclusive IHC transduction was further validated in non-human primates following round window membrane delivery, underscoring translational potential. Therapeutically, AAV-WM04 enabled efficient dual-AAV trans-splicing delivery of the large OTOF gene, resulting in uniform full-length otoferlin expression in IHCs. In a humanized Otof Q829X/Q829X mouse model, AAV-WM04 restored auditory function across a broad frequency range at relatively low doses and achieved durable hearing recovery. Collectively, these findings establish AAV-WM04 as a next-generation IHC-targeted vector with high efficiency, safety, and cross-species applicability for precision gene therapy of hereditary hearing loss.

The precise integration of large DNA fragments into the human genome holds significant therapeutic potential. Here, we demonstrate that combining engineered piggyBac (PB) transposase with CRISPR/Cas9 enables targeted integration of PB transposons into specified genomic loci. Our engineered PB transposase (PBase) retains high excision activity while substantially reducing endogenous integration activity. In the developed Cas9-PBase fusion system, PBase excises the transposon to generate linear DNA fragments, while Cas9 introduces site-specific double-strand breaks (DSBs), facilitating insertion of the excised fragment at the target locus. The optimized tool achieves 6.1-7.3 kb transposon integration at multiple genomic sites with 10-15% efficiency, demonstrating 60-80% targeted integration specificity. As a proof of concept, we inserted a 7.1 kb transposon encoding three genes into the {beta}2M locus of human induced pluripotent stem cells (iPSCs), conferring protection against allogeneic natural killer (NK) cell-mediated cytotoxicity in derived iNK cells. These results establish Cas9-PBase as a precise and programmable platform for large DNA sequence insertion with potential clinical applications.

The evolutionary trajectory of lung squamous cell carcinoma (LUSC) remains poorly defined, hindering the development of effective therapies. By integrating genomic and transcriptomic sequencing from human LUSC specimens, we delineated a polyclonal-to-monoclonal evolutionary trajectory during LUSC progression. This evolutionary pattern was corroborated by single-cell RNA sequencing, which revealed consistent tumor cell heterogeneity. Specifically, the SBS5 mutational signature was enriched and correlated with poor prognosis independent of tumor stage. By further utilizing the spontaneous LUSC mouse model to identify key genomic and genetic events in LUSC progression, we observed that the JNK pathway was inhibited and that cytoskeleton-related pathways were dysregulated during LUSC development, and identified the mutations in the JNK pathway (e.g., DACT1) and cytoskeletal regulators (e.g., KIF26A). Collectively, these findings established a polyclonal-monoclonal evolution paradigm for LUSC, potentially regulated by JNK pathways, which could benefit LUSC precision therapeutics.

Excessive osteoclast formation is a key contributor to pathological bone loss in disorders such as osteoporosis and rheumatoid arthritis. Asarinin, a natural lignan, has not previously been examined in the context of osteoclast differentiation. Here, we investigated the anti-osteoclastogenic effects of asarinin using RANKL-stimulated RAW264.7 cells. Asarinin significantly suppressed TRAP-positive multinucleated osteoclast formation under the tested conditions. Mechanistically, asarinin selectively inhibited RANKL-induced phosphorylation of p38 and ERK MAPKs, leading to reduced c-Fos expression and inhibition of NFATc1 nuclear translocation. In addition, asarinin disrupted actin ring formation in mature osteoclasts. Collectively, these findings identify asarinin as a pathway-selective inhibitor of osteoclast differentiation, targeting the p38/ERK-c-Fos-NFATc1 axis while sparing parallel signaling pathways.

Single-cell expression quantitative trait loci (sc-eQTL) analyses are powerful in identifying context-specific susceptibility genes from genome-wide association studies (GWAS) loci. However, few studies have comprehensively investigated cells of lung cancer origin in non-European populations. Here, we built a lung sc-eQTL dataset from 129 Korean women never-smokers with epithelial cell enrichment. eQTL mapping identified 2,229 genes with an eQTL in 33 cell types, including East Asian-specific findings when compared to predominantly European datasets. Integration with single-cell chromatin accessibility data demonstrated an enrichment of cell-type specific eQTLs in cell-type matched candidate enhancers, while shared eQTLs were more frequently found near promoters. Colocalization and transcriptome-wide association study unveiled 36 susceptibility genes from 22 cell types in 22 lung cancer loci, including 10 loci not achieving genome-wide significance in prior GWAS. Around 47% of these genes were from cells of the alveoli, underscoring their importance, especially in lung adenocarcinoma (LUAD) susceptibility. Focusing on the trajectory of alveolar epithelial cell regeneration, we detected 785 cell-state-interacting QTLs, which overlapped with 28% (10) of the identified susceptibility genes. Finally, we experimentally validated East Asian-and alveolar type 2 cell-specific eQTL of TCF7L2 underlying East Asian LUAD locus, 10q25.2. Consistent with its role as a Wnt/{beta}-catenin effector, TCF7L2 displayed significant effect on lung adenocarcinoma cell growth. Our data highlighted context-specific susceptibility genes, especially from alveolar cells of lung, contributing to lung cancer etiology.

Pharmacogenomics is an essential component of precision medicine; however, most existing knowledge has been derived from populations of European ancestry, limiting the understanding of pharmacogenomic diversity in East Asian populations. In this study, we applied genotype imputation to the Korea Biobank Array v2.0 using a reference panel of 8,062 Korean whole-genome sequencing (WGS) samples and analyzed pharmacogenomic variants and phenotypes in 14,490 Korean individuals. To assess the accuracy of imputation-based variant detection, we compared imputed genotypes with matched WGS data from 735 individuals and with genotypes obtained from the commercial PangenomiX Plus Array Kit for an additional 137 individuals, demonstrating high concordance. When extended to the full cohort, all individuals were found to carry at least one pharmacogenomic variant, with high frequencies observed in key pharmacogenes including CYP2C19, SLCO1B1, CYP3A5, and VKORC1. Phenotype distributions were broadly consistent with previous WGS-based studies in East Asians but showed notable differences compared with European populations. Overall, this population-specific, large-scale analysis provides a comprehensive pharmacogenomic landscape in Koreans and highlights the importance of ancestry-tailored data for equitable precision medicine.

Methylmalonic acidemia (MMA) is a recessive genetic disease caused by variants in the MMUT (mitochondrial enzyme methylmalonyl-CoA mutase) gene or by defects in transport or metabolism of MMUT cofactor (5 deoxyadenosylcobalamin), including variants in the MMAB gene. For the most recurrent pathogenic MMAB variant, c.556C>T (R186W), we identified a corrective editing strategy using adenine base editing. Deploying an adenine base editor mRNA and optimized hybrid guide RNA with lipid nanoparticles, we observed efficient in vitro corrective editing of the variant to wild-type, with minimized bystander editing and off-target editing in hepatocytes. These observations lay the groundwork for a gene editing therapy for patients with MMA resulting from at least one copy of the MMAB c.556C>T (R186W) variant, as well as a platform of similar therapies for patients with MMA caused by other variants amenable to adenine base editing.

Mesial (a.k.a., medial) temporal lobe epilepsy (MTLE) is the most common focal epilepsy1,2 and, in drug-resistant cases, is treated by surgical removal of the anterior temporal lobe, which often shows neuronal loss and gliosis consistent with hippocampal sclerosis (HS)2. MTLE with HS has minimal contribution from germline genetic variation3, and is associated with prior precipitating insults such as prolonged childhood seizures and head trauma4-6. Somatic variants in Ras-MAPK pathway genes were recently reported in a few MTLE surgical specimens7,8, but their prevalence, clinical relevance, and underlying biological mechanisms remain unknown. Targeted duplex sequencing of hippocampal DNA from 462 surgical resections revealed significant enrichment of deleterious somatic variants in MTLE versus controls, with >40% of MTLE specimens harboring activating Ras-MAPK variants in PTPN11, NF1, BRAF, KRAS, and twelve genes not previously associated with focal epilepsy. Eight Ras-MAPK genes showed positive clonal selection in MTLE. Increased somatic variant burden predicted worse surgical outcome. Somatic Ras-MAPK variants at ultra-low (<0.5%) variant allele fractions were associated with older seizure onset and HS pathology, supporting a late prenatal or postnatal origin. Ras-MAPK variants in MTLE were enriched in cells derived from hippocampal progenitors--neurons, astrocytes, oligodendrocytes--in line with the known neuronal hyperexcitability and seizures induced by Ras-MAPK overactivation9,10; in contrast, Alzheimer disease hippocampi exhibited microglial enrichment of Ras-MAPK variants, consistent with prior reports11. Single-nucleus RNA sequencing showed increased expression of Ras-MAPK genes in neurons and upregulation of pathways mediating neurogenesis and neural development in MTLE. Functional validation of novel, recurrent PTPN11 variants confirmed gain-of-function, while cellular modeling in induced pluripotent stem cells demonstrated proliferative/survival advantages for mutant cells in mosaic culture. Overall, our data suggest that somatic Ras-MAPK variants and acquired risk factors may converge on clonal competition in the hippocampus to modulate epilepsy risk.

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

Thalidomide is teratogenic in humans but not in rodents due to species-specific differences in the sequence of Cereblon (CRBN), an E3 ubiquitin ligase targeted by thalidomide and its derivative molecular glue degraders (MGDs). This species divergence has hindered the accurate prediction of MGD-induced toxicities in standard laboratory animals. GSPT1 MGDs, such as CC-90009, have shown potent anticancer activities in preclinical models and leukemia patients; however, their clinical development was challenging due to severe adverse effects. This highlights the critical need to characterize on-target toxicities in relevant animal models to exploit the therapeutic safety of this class of MGDs. Here, we generated humanized CrbnV380E and CrbnV380E/I391V knock-in mouse strains, in combination with a degradation-resistant Gspt1G574N strain, to interrogate the in vivo on-target effects of CC-90009 and its analog CC-885. We found that targeted GSPT1 depletion in mice led to rapid mortality, preceded by multiple dysfunctions including intestinal obstruction, liver damage, splenic atrophy, and hematological abnormalities. Remarkably, these toxicities, along with the underlying transcriptional perturbations, were completely rescued by the undegradable Gspt1G574N mutant, establishing a definitive causal link between GSPT1 degradation and systemic injury. Induced proximity and degradation proteomic analyses revealed that GSPT1 loss triggered a secondary downregulation of many proteins, including MYC, PLK1 and CDK4, which were not directly recruited by these MGDs to CRBN. Collectively, our data define the in vivo on-target toxicities associated with endogenous GSPT1 degradation and provide a genetic framework to guide the preclinical safety evaluation of CRBN-based MGD therapeutics.

We conducted the first genome-wide association meta-analyses of global and sectoral peripapillary retinal nerve fibre layer (pRNFL) thickness and Bruchs membrane opening-minimum rim width (BMO-MRW), the major optic nerve head structural and neurodegeneration biomarkers, including up to 25,942 and 12,080 participants, respectively, from the International Glaucoma Genetics Consortium. We identified 9 global pRNFL thickness and 9 global BMO-MRW loci, along with 28 and 19 loci for pRNFL and BMO-MRW sectors, respectively, comprising both shared and sector-specific loci. To identify intraocular pressure (IOP)-independent drug targets, global pRNFL thickness and BMO-MRW were conditioned on IOP. IOP-independent loci were then prioritised to identify candidate causal genes using transcriptome-wide association study and colocalization analysis. Several genes, such as NMNAT2 and TRIOBP, had robust associations with both phenotypes, with potential IOP-independent therapeutic translation for glaucoma. Overall, we identified novel loci for pRNFL thickness and BMO-MRW, highlighting potential drug-target genes acting independently from IOP, and elucidating genetic differences among pRNFL sectors.