Citations

Antisense oligonucleotides (ASOs) represent a promising therapeutic modality for RNA-based disease mechanisms, but identifying high-efficacy candidates from large sequence spaces remains a major bottleneck in drug development. Here, we present ASOFormer, a novel Transformer-based neural network that predicts the inhibition efficiency of RNase H-mediated ASOs from sequence, predicted secondary structure, and chemical modification features. ASOFormer was trained on a knockdown efficacy dataset spanning over 170,000 ASO-target pairs compiled largely from published patents. Ablation analysis confirmed that the addition of chemical modification features to the primary sequence drove the largest performance gains, with predicted secondary structure providing complementary benefit when combined with modification information. Further analysis highlighted the importance of the wings of gapmer ASOs and other features. As independent validation, ASOFormer was applied to ASOs targeting seven genes that were not in the training dataset gene list. For four out of the seven genes, the ground truth ASO inhibition efficiency was obtained from a public dataset, whereas the others were measured in-house via qPCR. Compared to two published state-of-the-art methods, ASOFormer achieved the best overall accuracy and prioritized the strongest inhibitors. Crucially, only ASOFormer consistently exceeded random expectation for top-candidate recovery across all seven test genes.

Background. Chronic kidney disease (CKD) is a common complication of nonrenal transplantation, influencing recipients’ morbidity and mortality. The incidence of CKD varies depending on the transplanted organ. Recipients of vascularized composite allotransplantation (VCA) need lifelong immunosuppression similar to that of solid organ transplantation. This study investigated renal function in VCA recipients. Methods. We studied 70 recipients from 17 centers (median follow-up: 8 y): 59 upper extremity, 10 face, and 1 penis transplant. Pretransplant and posttransplant clinical data and sera were collected to calculate the estimated glomerular filtration rate (eGFR) using the CKD Epidemiology collaboration 2021 formula. Results. Pretransplantation, the median eGFR was 105.36 mL/min (range, 71.39–155.39 mL/min). By 3 mo posttransplant, the eGFR had significantly decreased to 78.42 mL/min (range, 32.49–130.74 mL/min; P = 0.001) before stabilizing. Despite higher tacrolimus levels at 3 mo, no correlation with eGFR was found. During follow-up, 17.1% of patients experienced a ≥50% eGFR decline, 10.9% progressed to CKD, and 4.7% developed end-stage kidney disease. Some risk factors were identified, but their responsibility could not be established with certainty because of the low number of severe renal outcomes. Conclusions. Renal function declines in the majority of VCA recipients, but the function loss is lower compared with nonrenal solid organ transplantation recipients. Although no statistically significant correlation could be established between tacrolimus exposure and renal function decline, strategies aiming at minimizing calcineurin inhibitor exposure and avoiding other nephrotoxic causes are recommended to preserve long-term kidney function.

Abstract Background and Aims Stroke and systemic embolism are recognized but poorly characterized complications of atrial fibrillation (AF) and left atrial flutter (AFL) catheter ablation. Their incidence, outcomes, and procedural associations remain insufficiently defined. Methods Retrospective global registry was initiated by the European Heart Rhythm Association Scientific Committee. A secure database captured data from ablation centres worldwide on general AF and left AFL ablation activity, as well as detailed information on patients with symptomatic embolic events between 2017 and 2024. Results Overall, 204 centres reported 335 743 ablation procedures and 550 embolic events (incidence 0.16%). Centre-level rate ranged from 0% to 3.1%. Most events were cerebral (94%), followed by peripheral (5%), and combined (1%). Coronary air embolism accounted for 62% of all peripheral events. Intraprocedural events occurred in 93 patients (17%), within 72 h in 357 (78%) and beyond 72 h in 100 (22%); 128 cases (23%) were diagnosed after hospital discharge. Nonparoxysmal AF (0.23% vs 0.15%; P < .001), centre experience, and ablation energy source (radiofrequency, 0.16%; cryoballoon, 0.16%; pulsed field ablation, 0.25%; laserballoon, 0.88%; P < .001) were associated with embolic risk. Median NIHSS score after stroke was 4 (interquartile range, 2–9). Three-month follow-up was available for 518 patients with embolism (95%): 181 (35%) had sequelae and 16 (3%) died. Conclusions Symptomatic embolic events are infrequent but associated with significant morbidity and mortality. Most events occur early, though a substantial proportion arise later. Higher event rates were observed with laser and possibly with pulsed field ablation. A greater centre experience was associated with a lower embolism incidence.

Introduction Patient dehumanization when caring for critically ill patients in the intensive care unit (ICU) is commonplace. The Get to Know Me board (GTKMB) has been identified as a valuable tool for promoting humanized care and communication in the ICU; however, its implementation is highly variable. This study aimed to identify the barriers and facilitators influencing consistent implementation of the GTKMB in the ICU. Materials and methods We conducted multidisciplinary focus groups via teleconference with ICU clinicians at a large quaternary care academic medical center. A thematic content analysis was performed to identify key themes and concepts related to GTKMB implementation. Results Thirty-eight clinicians participated across six focus groups, including 10 nurses, 7 physicians, 6 advanced practice providers, 5 rehabilitation therapists (allied health clinicians), 1 respiratory therapist and 1 social worker. Major themes related to barriers and facilitators of GTKMB implementation included, (1) identifying appropriate patients and optimal timing for completing the GTKMB, (2) logistical challenges such as access to the board, visibility and unclear responsibility for completion, (3) the importance of clinician role modeling supporting the board completion, and (4) opportunities to improve the design and functionality of the board itself. Conclusion Although clinicians recognize the GTKMB as a meaningful tool to support humanized care in the ICU, inconsistent implementation limits its potential impact. This study highlights several key barriers and facilitators to its use. Future efforts should focus on targeted quality improvement initiatives to enhance consistent completion and integration of the GTKMB into ICU practice.

Abstract Background More than 85% of Alzheimer's disease (AD) donor brains exhibit some degree of cerebral amyloid angiopathy (CAA), which is characterized predominantly by Aβ40 deposits in the brain vasculature and can cause blood brain barrier (BBB) leakage. CAA is associated with risk of infarcts, cerebral hemorrhages, and cognitive decline. Brain cell type‐specific mechanisms that influence CAA pathology, as well as related measures such as Aβ40 biochemical measures and tight junction proteins, an indicator of BBB integrity, are still elusive. Method We performed single‐nucleus RNA sequencing (snRNAseq) of temporal cortex tissue from 79 AD donors, with varying levels of CAA co‐pathology. Measurements of soluble, insoluble and membrane‐associated fractions of brain Aβ40 and tight junction protein (claudin‐5, occludin) levels were available for these donors. We correlated these measures with cell proportions. We also conducted differential gene expression and analyses for intercellular communications and regulatory networks. Result We identified 25 clusters and annotated them with major brain cell types. There were 3 oligodendrocyte, 7 excitatory and 7 inhibitory neuronal, 2 microglial, 2 astrocytic, and 1 each for endothelial, pericytic, fibroblast, and oligodendrocyte precursor cell clusters. We found that higher CAA and Aβ40 levels correlate with reduced neuronal and increased microglial, astrocytic and vascular cell proportions. Increased tight junction proteins correlate with increased inhibitory neuronal and reduced microglial, astrocytic and vascular cell proportions. Among all clusters, one microglial, both astrocytic and the pericytic clusters have greater number of genes significantly associated with CAA, Aβ40 and tight junction protein levels. Most significant genes have negative associations with CAA and membrane‐bound Aβ40 and positive with tight junction protein measures. Using these genes as input for intercellular communications analysis, we prioritized ligand‐target interactions from microglia or astrocytes to pericytes. Some of the prioritized targets are transcription factors in pericytes. Using regulon analysis, we identified genes downstream of these transcription factors that are also significantly associated with lower CAA and membrane‐bound Aβ40 and higher tight junction protein levels. Conclusion We revealed glia‐to‐pericytes intercellular interactions and pericytic gene regulatory networks that are potentially perturbed by CAA and BBB breakdown. Future directions include analyzing external snRNAseq datasets and performing functional validations.

Abstract Background Alzheimer's disease (AD) affects all brain cells and has complex genomic and immunological alterations. Previous research discovered missense AD risk or protective variants in microglial genes ABI3 and PLCG2, respectively. Expression levels of these genes are altered in AD and can influence microglial function. This study aims to uncover protective, and risk microglial molecular signatures associated with these variants to determine their role in microglial subtypes and states in AD by single cell expression and functional studies. Method We generated microglia‐enriched snRNAseq data from donors harboring either AD protective PLCG2 or AD risk ABI3 missense mutations, or neither mutation. After standard snRNAseq QC, we performed differential expressed gene (DEG) analysis of all microglial cells between variant carriers and non‐carriers using MAST. We defined protective signature as genes that are both down in ABI3 and up in PLCG2 mutation‐carriers. In contrast, risk signature genes are up in ABI3 and down in PLCG2 mutation‐carriers. We investigated the conservation of protective and risk signatures across multiple datasets, including scRNAseq data from iPSC‐derived microglia cells carrying PLCG2 protective variant, snRNAseq data from AD‐resilient donors, and data from AD and other diagnostic groups across multiple brain regions sourced from external datasets. Result We obtained snRNAseq profiles of 35,000 microglia from AD variant carriers. Our DEG analysis among all microglia cells provided 227 microglial protective and 293 risk signature genes defined by these variants. Using integrated analysis of multiple internal and external datasets, we further narrowed down these signatures. We determined that these high‐confidence protective signature genes are downregulated in early AD, upregulated in late AD brains and positively‐correlated with protective variant load in in vitro models. In contrast, risk signature genes are upregulated in early AD, downregulated in late AD and resilient donors. Risk signature expression is decreased with protective PLCG2 variant load, and altered with Aβ treatment in in vitro models. Conclusion Our study uncovers microglia specific protective and risk signatures associated with AD using sn/scRNAseq datasets from multiple sources and models. These findings nominate novel immune targets and pathways with implications for microglial function in health and disease, and ultimately therapeutic potential.

Abstract Background Alzheimer's disease (AD) affects all brain cells and has complex genomic and immunological alterations. Previous research discovered missense AD risk or protective variants in microglial genes ABI3 and PLCG2 , respectively. Expression levels of these genes are altered in AD and can influence microglial function. This study aims to uncover protective, and risk microglial molecular signatures associated with these variants to determine their role in microglial subtypes and states in AD by single cell expression and functional studies. Method We generated microglia‐enriched snRNAseq data from donors harboring either AD protective PLCG2 or AD risk ABI3 missense mutations, or neither mutation. After standard snRNAseq QC, we performed differential expressed gene (DEG) analysis of all microglial cells between variant carriers and non‐carriers using MAST. We defined protective signature as genes that are both down in ABI3 and up in PLCG2 mutation‐carriers. In contrast, risk signature genes are up in ABI3 and down in PLCG2 mutation‐carriers. We investigated the conservation of protective and risk signatures across multiple datasets, including scRNAseq data from iPSC‐derived microglia cells carrying PLCG2 protective variant, snRNAseq data from AD‐resilient donors, and data from AD and other diagnostic groups across multiple brain regions sourced from external datasets. Result We obtained snRNAseq profiles of 35,000 microglia from AD variant carriers. Our DEG analysis among all microglia cells provided 227 microglial protective and 293 risk signature genes defined by these variants. Using integrated analysis of multiple internal and external datasets, we further narrowed down these signatures. We determined that these high‐confidence protective signature genes are downregulated in early AD, upregulated in late AD brains and positively‐correlated with protective variant load in in vitro models. In contrast, risk signature genes are upregulated in early AD, downregulated in late AD and resilient donors. Risk signature expression is decreased with protective PLCG2 variant load, and altered with Aβ treatment in in vitro models. Conclusion Our study uncovers microglia specific protective and risk signatures associated with AD using sn/scRNAseq datasets from multiple sources and models. These findings nominate novel immune targets and pathways with implications for microglial function in health and disease, and ultimately therapeutic potential.

Abstract Blood-brain barrier (BBB) dysfunction is a key feature of Alzheimer’s disease (AD), particularly in individuals carrying the APOE-ε4 allele. This dysfunction worsens neuroinflammation and hinders the removal of toxic proteins, such as amyloid-beta (Aβ42), from the brain. In post-mortem brain tissues and in animal models, we previously reported that fibronectin accumulates at the BBB predominantly in APOE-ε4 carriers. Furthermore, we found a loss-of-function variant in the fibronectin 1 ( FN1 ) gene significantly reduces aggregated fibronectin levels and decreases AD risk among APOE-ε4 carriers. Yet, the molecular mechanisms downstream of fibronectin at the BBB remain unclear. The extracellular matrix (ECM) plays a crucial role in maintaining BBB homeostasis and orchestrating the interactions between BBB cell types, including endothelia and astrocytes. Understanding the mechanisms affecting the ECM and BBB cell types will be critical for developing effective therapies against AD, especially among APOE-ε4 carriers. Here, we demonstrate that APOE-ε4 , Aβ42, and inflammation drive the induction of FN1 expression in several models including zebrafish, mice, iPSC-derived human 3D astrocyte and 3D cerebrovascular cell cultures, and in human brains. Fibronectin accumulation disrupts astroglial-endothelial interactions and the signalling cascade between vascular endothelial growth factor (VEGF), heparin-binding epidermal growth factor (HBEGF) and Insulin-like growth factor 1 (IGF1). This accumulation of fibronectin in APOE-ε4- associated AD potentiates BBB dysfunction, which strongly implicates reducing fibronectin deposition as a potential therapeutic target for AD. Graphical abstract Accessibility text This image illustrates the effects of different APOE isoforms (ApoE-ε3 and ApoE-ε4) on blood-brain barrier (BBB) integrity, focusing on the molecular interactions between astrocytes and endothelial cells. This figure emphasizes the detrimental effects of ApoE-ε4 on BBB integrity via fibronectin accumulation and altered signaling pathways. The top section provides a schematic overview of the blood-brain barrier, highlighting astrocytes, endothelial cells, and their interface. The left panel represents the ApoE-ε3 condition: Normal fibronectin (FN1) levels support healthy interactions between astrocytes and endothelial cells. Growth factors, including VEGFA, HBEGF, and IGF1, maintain BBB integrity through their respective receptors (VEGFR and EGFR). Green arrows indicate activation of these signaling pathways. The right panel depicts the ApoE-ε4 condition: Elevated fibronectin (FN1) disrupts astrocyte-endothelium interactions. FN1 binds integrins and activates focal adhesion kinase (FAK), inhibiting VEGFA, which is required for endothelial HBEGF that in turn activates IGF1 signaling. Red symbols indicate inhibition of HBEGF, VEGFA, and IGF1 pathways, leading to BBB dysfunction. Highlights APOE-ε4 drives fibronectin deposition in Alzheimer’s, disrupting astrocyte-endothelia interactions. APOE-ε4 and fibronectin co-localize, forming aggregates at blood-brain barrier (BBB). Fibronectin alters the signaling between VEGF, IGF1, and HBEGF impairing BBB function. Reducing fibronectin restores BBB integrity and offsets APOE-ε4 pathology.

AbstractBackgroundA complex, multicellular disease with genetic and immunological elements, Alzheimer’s disease (AD) affects millions worldwide. There has been previous research linking AD to the missense variants ABI3‐rs616338‐T and PLCG2‐rs72824905‐G, and the altered expression of these genes has been shown to disrupt microglial function. In our understanding of AD risk and resilience, limited research has been conducted on how these variants affect microglial subtypes and states in AD.MethodsWe previously identified DOCK8 protein as a target for fluorescent activated nuclei sorting (FANS) to enrich microglia nuclei from frozen human brains. Using this enrichment strategy, we generated microglia enriched snRNAseq data from temporal cortex tissue of 30 donors harboring ABI3 or PLCG2 missense mutations, or neither mutation. We introduced PLCG2 variant in either homozygote, and heterozygote forms into an AD patient derived iPSC through CRISPR/Cas9 approach, differentiated these cells into microglia cells (IMGLs) following established protocols, and generated scRNAseq data after treatment with Amyloidβ. We applied standard alignment and quality control pipelines to the snRNAseq and scRNAseq datasets in parallel. Established markers of microglia states and subtypes were utilized to annotate the clusters; analyzed for their hub gene expression using comparisons between cluster differential profiling to determine signature pathways. Differential gene expression was assessed using pseudo bulk and MAST approaches.ResultsWe obtained single nuclei transcriptome profiles of 54,000 frozen human brain nuclei, of which 35,000 are microglia, through snRNAseq, and single cell profiles of 63,000 iMGLs through scRNAseq, using our standard alignment and quality control procedures. Our differential gene expression analysis identified novel genes and pathways which were identified as part of resilience or AD risk networks in microglia subtypes and states. These alterations were validated using orthogonal experimental methods and further explored for their conservation using in vivo external datasets. Pseudotime inference will be completed to probe microglia marker gene expression dynamics, treatment conditions, along with continuous cell‐state changes.ConclusionOur study uncovers microglia subtype specific resilience and risk factors provided by AD related genetic variants using snRNAseq from frozen human brain and scRNAseq from IPSC derived microglia cells. These findings nominate novel targets and pathways with therapeutic potential.

AbstractBackgroundProgressive supranuclear palsy (PSP) is a devastating primary tauopathy with rapid progression to death. Although several therapies currently in the development pipeline show promising safety profiles and robust target engagement, few demonstrated significant efficacy in patients, underscoring the need to interrogate additional targets with novel therapeutic modalities to expand the potential therapeutic arsenal. To diversify the therapeutic avenues for PSP and related tauopathies (e.g. Alzheimer’s disease), we systematically integrated multi‐omics data from human brains of PSP and control donors with cross‐species validation to nominate high‐confidence therapeutic targets. We plan to translate our findings into safe and effective treatments for PSP using antisense oligonucleotides (ASO).MethodWe analyzed brain gene expression profiles in PSP and control individuals at bulk tissue (N = 408) and single‐cell levels (snRNAseq, N = 36). Cell‐type‐specific expression perturbations were systematically prioritized using a cross‐species validation paradigm, including rTG4510 tau mice and tau‐overexpressing Drosophila models. We conducted in vitro screening of ASO candidates against our prioritized target genes. ASO efficacy and toxicity were measured. RNAseq experiments will be performed to interrogate off‐target effects. The safety and efficacy of the lead ASOs will be assessed in PSP‐patient‐derived iPSC models.ResultWe previously reported discovery of novel genes with significant differential expression in PSP brains, characterized their brain cell‐specificity and validated them using snRNAseq data. We prioritized 21 genes using gene expression data from a mouse tauopathy model. Validation of these 21 high‐priority genes using a Drosophila tau model nominated astrocytic STOM, KANK2, and DDR2 as potential therapeutic targets for PSP. Knocking down their expression in Drosophila significantly rescues tau‐mediated neurodegenerative pathology. In vitro screening identified ASO leads that reduced the target expressions at mRNA and protein levels with low cellular toxicity.ConclusionWe developed a systems biology pipeline and nominated STOM, KANK2, and DDR2 as candidate gene targets for PSP. We identified ASOs that modulate these targets’ expression without cellular toxicity, suggesting they may be suitable as potential therapeutic candidates. Importantly, the shared pathophysiology and molecular aberrations between PSP and other tauopathies, such as Alzheimer’s disease, suggests that such therapeutic candidates may be repurposed for multiple neurodegenerative diseases, further accelerating, and streamlining the therapeutic pipeline.